Insulin-oligomer conjugates, formulations and uses thereof
1 Related Applications
This application claims priority to and incorporates by reference the entire disclosures of U.S.
Patent Application Nos. 60/589,058 filed July 19, 2004, 60/619,153 filed October 15, 2004,
60/632,578 filed December 2, 2004, and 60/655,838 filed February 24, 2005, and 60/655,803
filed February 24, 2005. This application also incorporates by reference the following
applications filed herewith on 19-Jul-05 by Radhakrishnan et al.: U.S. Patent Application No.
11/184,668, entitled "Cation complexes of insulin compound conjugates, formulations and uses
thereof; U.S. Patent Application No. 11/184,594, entitled "Insulin-oligomer compound
conjugates, formulations and uses thereof; U.S. Patent Application No. 11/184,528, entitled
'Tatty acid formulations for oral delivery of proteins and peptides, and uses thereof."
2 Field
The invention relates to novel insulin compound conjugates in which an insulin or insulin analog
is coupled to a modifying moiety. The invention also relates to cation complexes of such insulin
compound conjugates and to pharmaceutical formulations including such insulin compound
conjugates and/or modifying moieties.
3 Background
Zinc complexed insulin compound is commercially available, for example, under the trade names
HUMULIN® and HUMALOG®. Zinc complexed insulin typically exists in a hexameric form.
Various methods have been described for the use of zinc in the crystallization of acylated insulin.
For example, U.S. Patent Publication 20010041786, published on 15-Nov-Ol, by Mark L. Brader
et al., entitled "Stabilized acylated insulin formulations" describes a formulation with an aqueous
solution for parenteral delivery, particularly as an injectable formulation, with a pH of 7.1 to 7.6,
containing a fatty acid-acylated insulin or a fatty acid-acylated insulin analog and stabilized using
zinc and preferably a phenolic compound. U.S. Patent 6,451,970, issued on 17-Sept-02 to
Schaffer et al., assigned to Novo Nordisk A/S, entitled "Peptide derivatives" describes derivatives
of insulin compound and insulin analogs where the N-terminal amino group of the B-chain and/or
the e-amino group of Lys in position B28, B29 or B30 is acylated using long chain hydrocarbon
group having from 12 to 22 carbon atoms and zinc complexes thereof.
Protamines and phenolic compounds have been described for use in the crystallization of acylated
insulin. U.S. Patents 6,268,335 (31-Jul-Ol) and 6,465,426 (10-Oct-02) to Brader, both entitled
"Insoluble insulin compositions," describe insoluble compositions comprised of acylated insulin a
protamine complexing compound, a hexamer-stabilizing phenolic compound, and a divalent
metal cation.
Existing approaches are especially tailored for crystallization of native insulin compound or
insulin compound analogs or for acylated insulin compounds having increased lipophilicity
relative to non-acylated insulin compounds. There is a need in the art for pharmaceutically
acceptable complexes including derivatized insulin compounds, other than acylated insulin
compound, such as hydrophilic and/or amphiphilic insulin compound derivatives, and for
stabilizing non-acylated lipophilic insulin compound analogs. There is also a need in the art for
new protein conjugates having increased bioavailability or other improved pharmaceutical
attributes relative to existing conjugates. There is a need in the art for new formulations that
facilitate oral delivery of proteins and protein conjugates. Finally, there is a need for a combined
approach to improving the oral bioavailability of a protein, such as insulin compound, which
incorporates an improved oral protein conjugate provided as a solid in an improved formulation
to maximize the benefits for the oral delivery of proteins.
4 Summary of the Invention
In general, the invention provides a complex including an insulin compound conjugate with an
insulin compound conjugated to a modifying moiety, and a cation, where the insulin compound
conjugate is complexed with the cation. The insulin compound may, for example, be a native
insulin or an insulin analogs. Examples of insulin compounds include human insulin, lyspro
insulin, des30 insulin, native proinsulin, artificial proinsulins, etc. The cation component may,
for example, be a divalent metal cation selected from the group consisting of Zn++, Mn++, Ca++,
Fe++, Ni++, Cu-H-, Co++ and Mg++.
The modifying moiety may be selected to render the insulin compound conjugate more, less or
equally soluble as compared to the corresponding unconjugated insulin compound. The
modifying moiety is preferably selected to render the insulin compound conjugate at least 1.05,
1.25,1.5,1.75, 2, 2.5, 3, 3.5,4,4.5, 5, 5.5, 6, 6.5,7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5,
13, 13.5, 14, 14.5, or 15 times more soluble than a corresponding unconjugated insulin compound
in an aqueous solution at a pH of about 7.4. Preferably the modifying moiety is selected to render
5 an insulin compound conjugate having an aqueous solubility that exceeds about 1 g/L, 2 g/L, 3
g/L, 4 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L, 50 g/L, 75 g/L, 100 g/L, 125 g/L, or 150 g/L at a
pH of about 7.4. Further, the the modifying moiety is selected to render the insulin compound
conjugate equally or more soluble than a corresponding unconjugated insulin compound, and the
water solubility of the insulin compound conjugate is decreased by the addition of zinc. In
10 another embodiment, the modifying moiety is selected to render the insulin compound conjugate
equally or more soluble than a corresponding unconjugated insulin compound; the water
solubility of the insulin compound conjugate is decreased by the addition of zinc; and water
solubility of the complex is greater than the water solubility of insulin compound. In still another
embodiment, the relative lipophilicity of the insulin compound conjugate as compared to
15 corresponding parent insulin compound (krcl) is 1 or less than 1.
The invention also provides novel insulin compound conjugates having an insulin compound
conjugated to a modifying moiety. 'For example, the invention provides insulin compounds
coupled to a modifying moiety having a formula:
-X-R1-Y-PAG-Z-R2 (Formula VI)
,
X, Y and Z are independently selected linking groups and each is optionally present, and X, when
present, is coupled to the insulin compound by a covalent bond,
at least one of R1 and R2 is present, and is lower alkyl and may optionally include a carbonyl
group,
25 R2 is a capping group, such as -CH3, -H, tosylate, or an activating group, and
PAG is a linear or branched carbon chain incorporating one or more alkalene glycol moieties (i.e.,
oxyalkalene moieties), and optionally incorporating one or more additional moieties selected
from the group consisting of -S-, -O-, -N-, and -C(O)-, and
where the modifying moiety has a maximum number of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16,17,18,19, 20,21, 22,23,24, or 25 heavy atoms.
In embodiments of the invention, any one or more of X, Y and Z may be absent. Further, when
present, X, Y and/or Z may be independently selected from -C(O)-, -O-, -S-, -C- and -N-. In one
embodiment, Z is -C(O)-. In another embodiment, Z is not present.
to some embodiments, R1 is lower alkyl, and R2 is not present. In other embodiments, R2 is lower
alkyl, and R1 is not present.
In another embodiment, the modifying moiety may include a linear or branched, substituted
carbon chain moiety having a backbone of 3,4, 5, 6, 7, 8,9, 10, 11, 12, 13,14,15, 16,17, 19, 19,
20, 21, 22, 23, 24 or 25 atoms selected from the group consisting of -C, -C-, -O-,
=O, -S-, -N-, -Si-. The heavy atoms will typically include one or more carbon atoms and one or
more non-carbon heavy atoms selected from the group consisting of -O-, -S-, -N-, and =O. The
carbon atoms and non-carbon heavy atoms are typically present in a ratio of at least 1 carbon
atom for every non-carbon heavy atom, preferably at least 2 carbon atoms for every non-carbon
heavy atom, more preferably at least 3 carbon atoms for every non-carbon heavy atom. The
carbon atoms and oxygen atoms are typically present in a ratio of at least 1 carbon atom for every
oxygen atom, preferably at least 2 carbon atoms for every oxygen atom, more preferably at least 3
carbon atoms for every oxygen atom. The modifying moiety may include one or more capping
groups, such as branched or linear Ci.6, branched or linear, or a carbonyl. The modifying moiety
will typically include hydrogens, and one or more of the hydrogens may be substituted with a
fluorine (which is a heavy atom but should not be counted as a heavy atom in the foregoing
formula). The modifying moiety may in some cases specifially exclude unsubstituted alkyl
moieties. The modifying moiety may, for example, be coupled to an available group on an amino
acid, such as an amino group, a hydroxyl group or a free carboxyllic acid group the polypeptide,
e.g., by a linking group, such as a carbamate, carbonate, ether, ester, amide, or secondary amine
group, or by a disulfide bond. The molecules in the linking group are counted as part of the
modifying moiety. In a preferred embodiment, the molecular weight of the modifying moiety is
less than the molecular weight of the HIM2 modifying moiety.
The invention includes includes insulin compound conjugates having modifying moieties with a
formula:
(Formula VII),
where n is 1, 2, 3 or 4, and m is 1, 2, 3, 4 or 5; and/or
(Formula VIII),
where n is 1, 2, 3,4 or 5, and m is 1,2, 3 or 4.
It will be appreciated that the novel modifying moieties, as well as the use of such moities to
modfy insulin and other polypeptides are themselves aspects of the invention.
The invention also provides novel formulations including the insulin compound conjugates and/or
cation-insulin compound conjugates of the invention. The inventors have surprisingly discovered
that certain fatty acid compositions are particularly useful, especially for oral delivery of the
polypeptides and polypeptide conjugates, such as insulin and insulin compound conjugates and/or
oral delivery of the cation-insulin compound conjugate complexes of the invention. In one aspect,
the invention provides fatty acid compositions with one or more saturated or unsaturated C*, Cj,
Cs, €7, Cg, Cg or Cio fatty acids and/or salts of such fatty acids. Preferred fatty acids are caprylic,
capric, myristic and lauric. Preferred fatty acid salts are sodium salts of caprylic, capric, myristic
and lauric acid. The fatty acid content of the composition is typically within a range having as a
lower limit of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 % w/w, and having as an upper limit
of about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9,
5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,
7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,
9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1,
11.2,11.3,11.4,11.5, 11.6,11.7,11.8,11.9, or 12.0 %w/w. In yet another embodiment, the fatty
acid content of the composition is within a range having as a lower limit about 0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,
2.7, 2.8, 2.9, or 3.0 % w/w, and having as an upper limit about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,
3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5,7, 5.8, 5.9,
6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1,
8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2,
10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, or
12.0 % w/w, and the fatty acid content of the composition is typically greater than about 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, or 99.9% w/w a single fatty acid, preferably
caprylic, capric, myristic or lauric, or a salt thereof.
The invention also provides method of treating insulin deficiencies or otherwise supplementing
insulin in a subject using the insulin compound conjugates, cation-insulin compound conjugate
complexes, and/or formulations of the invention. The methods generally include administering a
therapeutically effective amount of one or more of the the insulin compound conjugates, cationinsulin
compound conjugate complexes, and/or formulations of the invention to a subject in need
thereof.
5 Brief Description of the Figures
Figures 1-15B show photomicrographs of various crystalline solids of the invention. Figures 1
and 2 are photomicrographs taken using a Zeiss Axiovert microscope showing T-type Zn
complex of of HIM2 30 g/L concentration, crystals grown for 24 hours. Figure 3 is a
photomicrograph taken using a Zeiss Axiovert microscope showing T-type Zn complex of of
HIM2 30 g/L concentration, crystals grown for 5 days. Figure 4 is a photomicrograph taken
' using a Zeiss Axiovert microscope showing R-type Zn complex of HIM2 at 30 g/L crystals
grown for 4 days. Figure 5 shows photomicrograph of R-type crystalline Zn complex of IN 105
containing 30% organic. Figures 6A-10B show photomicrographs of various R-type Zn
complexes of HIM2 made using organic solvent. Figures 11A-14B show photomicrographs of
crystals of various R-type co-crystallized Zn complexes of HIM2 and IN105. Figures 15A-15B
show photomicrographs of crystals of various R-type co-crystallized Zn complexes of HIM2 and
human insulin. The invention includes crystals having the morphologies shown in any of Figures
MSB.
Figures 16-20 show Mouse Blood Glucose Assay results for HIM2 and various Zn-HIM2
complexes. Figure 16 shows MBGA biopotency profiles for H1M2. Figure 17 shows MBGA
biopotency profiles for Zn HIM2 insulin compound product R type. Figure 18 shows MBGA
biopotency profiles for Zn HIM2 insulin compound product T-type. Figure 19 shows MBGA
biopotency profiles for Zn HIM2 insulin compound product with protamine. Figure 20 shows
glucose lowering effect of R type protamine complex at 30 and 90 minutes post dose.
Figures 21-24 show MBGA biopotency profiles for IN-186, IN-192, IN-190, IN-191, IN-189, IN-
178, IN-193, IN-194, IN-185, IN-196 and IN-197.
Figures 25 and 26 show dog clamp study results for Zn-HIM2 complexes of the invention.
Figures 27 and 28 show dog clamp study results for Zn-IN105 complexes of the invention.
Figures 29 and 30 show dog clamp study results for dogs dosed with IN105 in 3% w/v capric acid
sodium salt in a phosphate buffer without additional excipients.
Figures 31-33 show show dog clamp study results for dogs dosed with tablets containing 6mg of
IN105 and 150mg Mannitol, 30mg Exlotab with 143mg Caparte with or with out 143 mg laurate.
Figures 34-37 show show dog clamp study results for dogs dosed with prototype tablet 150mg
and 280mg caprate tablets and with 140mg/140mg caprate/laurate tablets.
6 Definitions
The following are definitions of the terms as used throughout this specification and claims. The
definitions provided apply throughout the present specification unless otherwise indicated. Terms
not defined herein have the meaning commonly understood in the art to which the term pertains.
"Addition," when used in reference to an amino acid sequence, includes extensions of one or
more amino acids at either or both ends of the sequence as well as insertions within the sequence.
"Complex" refers to a molecular association in which one or more insulin compounds or insulin
compound conjugates form coordinate bonds with one or more metal atoms or ions. Complexes
may exist in solution or as a solid, such as a crystal, microcrystal, or an amorphous solid.
"Complex mixture" means a mixture having two or more different complexes, whether in
solution or in solid form. Complexes mixtures may, for example, include complexes with
different insulin compounds, different insulin compound conjugates, different hybrid complexes,
different cations, combinations of the foregoing, and the like. "Hybrid complex" means a cationinsulin
compound conjugate complex having two or more different insulin compounds and/or
insulin compound conjugates.
"Complexing agent" means a molecule that has a multiplicity of charges and that binds to or
complexes with insulin compound conjugates. Examples of complexing agents suitable for use in
the present invention include protamines, surfen, globin proteins, spermine, spermidine albumin,
amino acids, carboxylic acids, polycationic polymer compounds, cationic polypeptides, anionic
polypeptides, nucleotides, and antisense. See Brange, L, Galenics of Insulin compound,
Springer-Verlag, Berlin Heidelberg (1987), the entire disclosure of which is incorporated herein
by reference.
"Conservative" used in reference to an addition, deletion or substitution of an amino acid means
an addition, deletion or substitution in an amino acid chain that does not completely diminish the
therapeutic efficacy of the insulin compound, i.e., the efficacy may be reduced, the same, or
enhanced, relative to the therapeutic efficacy of scientifically acceptable control, such as a
corresponding native insulin compound.
"Hydrophilic" means exhibiting characteristics of water solubility, and the term "hydrophilic
moiety" refers to a moiety which is hydrophilic and/or which when attached to another chemical
entity, increases the hydrophilicity of such chemical entity. Examples include, but are not limited
to, sugars and polyalkylene moieties such as polyethylene glycol. "Lipophilic" means exhibiting
characteristics of fat solubility, such as accumulation in fat and fatty tissues, the ability to
dissolve in lipids and/or the ability to penetrate, interact' with and/or traverse biological
membranes, and the term, "lipophilic moiety" means a moiety which is lipophilic and/or which,
when attached to another chemical entity, increases the lipophilicity of such chemical entity.
"Amphiphilic" means exhibiting characteristics of hydropilicity and lipophilicity, and the term
"amphiphilic moiety" means a moiety which is amphiphilic and/or which, when attached to a
polypeptide or non-polypeptide drug, increases the amphiphilicity (i.e., increases both the
hydrophilicity and the amphiphilicity) of the resulting conjugate, e.g., certain PEG-fatty acidmodifying moieties, and sugar-fatty acid modifying moieties."Lower alkyP' means substituted or unsubstituted, linear or branched alkyl moieties having from
one to six carbon atoms, i.e., Cj, C2, C3, C4, Cs or C6. "Higher alkyf means substituted or
unsubstituted, linear or branched alkyl moieties having six or more carbon atoms, e.g., C7, C8, C9,
GIO, Cj ], Cj2, CB, Cj4, Cj5, CK;, On, Cjg, C)9, Cao, etc.
"Monodispersed" describes a mixture of compounds where about 100 percent of the compounds
in the mixture have the same molecular weight. "Substantially monodispevsed" describes a
mixture of compounds where at least about 95 percent of the compounds in the mixture have the
same molecular weight. "Purely monodispersef describes a mixture of compounds where about
100 percent of the compounds in the mixture have the same molecular weight and have the same
molecular structure. Thus, a purely monodispersed mixture is a monodispersed mixture, but a
monodispersed mixture is not necessarily a purely monodispersed mixture. "Substantially purely
monodispersed" describes a mixture of compounds where at least about 95 percent of the
compounds in the mixture have the same molecular weight and same molecular structure. Thus,
a substantially purely monodispersed mixture is a substantially monodispersed mixture, but a
substantially monodispersed mixture is not necessarily a substantially purely monodispersed
mixture. The insulin compound conjugate components of the cation-insulin compound conjugate
compositions are preferably monodispersed, substantially monodispersed, purely monodispersed
or substantially purely monodispersed, but may also be polydispersed. "Polydispersed" means
having a dispersity that is not monodispersed, substantially monodispersed, purely monodispersed
or substantially purely monodispersed.
"Native insulin compound" as specifically used herein means mammalian insulin compound
(e.g., human insulin, bovine insulin compound, porcine insulin compound or whale insulin
compound), provided by natural, synthetic, or genetically engineered sources. Human insulin is
comprised of a twenty-one amino acid A-chain and a thirty-amino acid B-chain which are
cross-linked by disulfide bonds. A properly cross-linked human insulin includes three disulfide
bridges: one between A7 and B7, a second between A20 and B19, and a third between A6 and
All. Human insulin possesses three free amino groups: Bl-Phenylalanine, Al-Glycine, and
B29-Lysine. The free amino groups at positions Al and Bl are cc-amino groups. The free amino
group at position B29 is an e-amino group. "Insulin analog" means a polypeptide exhibiting
some, all or enhanced activity relative to a corresponding native insulin or which is converted in
in vivo or in vitro into a polypeptide exhibiting ome, all or enhanced activity relative to a
corresponding native insulin, e.g., a polypeptide having the structure of a human insulin with one
or more conservative amino acid additions, deletions and/or substitutions. Insulin analogs can be
identified using known techniques, such as those described in U.S. Patent Publication No.
20030049654, "Protein design automation for protein libraries," filed 18-Mar-02 in the name of
Dahiyat et al. Proinsulins, pre-proinsulins, insulin precursors, single chain insulin precursors of
humans and non-human animals and analogs of any of the foregoing are also referred to herein as
insulin analogs, as are non-mammalian insulins. Many insulin analogs are known in the art (see
discussion below). Unless context specifically indicates otherwise (e.g., were a specific insulin is
•eferenced, such as "human insulin" or the like), the term "insulin compound" is used broadly to
nclude native insulins and insulin analogs.
'Polyalkylene glycor or PAG refers to substituted or unsubstituted, linear or branched
polyalkylene glycol polymers such as polyethylene glycol (PEG), polypropylene glycol (PPG),
ind polybutylene glycol (PEG), and combinations thereof (e.g., linear or branched polymers
including combinations of two or more different PAG subunits, such as two or more different
PAG units selected from PEG, PPG, PPG, and PEG subunits), and includes the monoalkylether
of the polyalkylene glycol. The term PAG subunit means a single PAG unit, e.g., "PEG subunif
refers to a single polyethylene glycol unit, e.g., -(CH2CH20)-, "PPG subunit" refers to a single
polypropylene glycol unit, e.g., -(CH2CH2CH2O)-, and "PBG subunif refers to a single
polypropylene glycol unit, e.g., -(CH2CH2CHjCH2O)-. PAGs and/or PAG subunits also include
substituted PAGs or PAG subunits, e.g., PAGs including alkyl side chains, such as methyl, ethyl
or propyl side chains, or carbonyl side chains, as well as PAGs including one or more branched
PAG subunits, such as iso-PPG or iso-PBG.
"Proinsulin compound?' means an insulin compound in which the C-terminus of the B-chain is
coupled to the N-terminus of the A-chain via a natural or artificial C-peptide having 5 or more
ammo acids. "Preproinsulin compound" means a proinsulin compound further including a
leader sequence coupled to the N-terminus of the B-chain, such as a sequence selected to promote
excretion as a soluble protein, or a sequence selected to prevent conjugation of the N-terminus, or
a sequence selected to enhance purification (e.g., a sequence with binding affinity to a
purification column). "Single chain insulin compound precursor" or "miniproinsulin
compound" means an insulin compound in which the C-terminus of the B-chain (or a truncated
B-chain having 1, 2, 3 or 4 amino acids removed from the C-terminus) is coupled to the
N-terminus of the A-chain or a truncated A-chain shortened at the N-terminus by 1, 2, 3 or 4
amino acids, without an intervening C-peptide, or via a shortened C-peptide having 1, 2, 3 or 4
amino acids.
"Protamine" refers to a mixture of strongly basic proteins obtained from natural (e.g., fish sperm)
or recombinant sources. See Hoffmann, J. A., et al., Protein Expression and Purification,
1:127-133 (1990). The Protamine composition can be provided in a relatively salt-free
preparation of the proteins, often called "protamine base" or in a preparation including salts of the
proteins.
10
"Protein" "peptide" and "polypeptide" are used interchangeably herein to refer to compounds
laving amino acid sequences of at least two and up to any length.
'R-type" means a complex conformation formed in the presence of insulin compound conjugate,
a cation and a stabilizing compound, such as phenol. "T-type" means a complex conformation
formed in the presence of insulin compound conjugate and a cation without a stabilizing
compound, such as phenol. A T-type or R-type complex may include or exclude protamine.
"Scientifically acceptable controf means an experimental control that is acceptable to a person
of ordinary skill in the art of the subject matter of the experiment.
"Solid" means a state of matter in which there is three-dimensional regularity of structure; the
term is used broadly herein to refer to both crystalline solids, amorphous solids, and combinations
of crystalline solids and amorphous solids. "Cation-insulin compound conjugate solid," refers
to a solid that includes a cation-insulin compound conjugate, preferably coordinated with a
monovalent or multivalent cation. "Ciystal" means a solid with a regular polyhedral shape.
"Crystalline" refers to solids having the characteristics of crystals. "Microcrystar means a solid
that is comprised primarily of matter in a crystalline state that is microscopic in size, typically of
longest dimension within the range 1 micron to 100 microns. In some cases, the individual
crystals of a microcrystalline composition are predominantly of a single crystallographic
composition. In some embodiments, the crystals of the invention are not microcrystals. The term
"microcrystalline" refers to the state of being a microcrystal. "Amorphous" refers to a solid
material that is not crystalline in form. The person of ordinary skill in the art can distinguish
crystals from amorphous materials using standard techniques, e.g., using x-ray crystallographic
techniques, scanning electron microscopy or optical microscopy. "Solid mixture" means a
mixture of two different solids. "Crystal mixture" means a mixture of two different crystals.
"Co-crystaF means a crystal having two or more different insulin compounds and/or insulin
compound conjugates. The cation-insulin compound conjugate complexes of the invention may
be provided in any of the foregoing forms or in mixtures of two or more of such forms.
"Substitution" means replacement of one or more amino acid residues within the insulin
compound sequence with another amino acid. In some cases, the substituted amino acid acts as a
functional equivalent, resulting in a silent alteration. Substitutions may be conservative; for
example, conservative substitutions may be selected from other members of the class to which the
substituted amino acid belongs. Examples of nonpolar (hydrophobia) amino acids include
alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Examples
of polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine. Examples of positively charged (basic) amino acids include arginine, lysine and
histidine. Examples of negatively charged (acidic) amino acids include aspartic acid and
glutamic acid.
'Water solubility" or "aqueous solubility" unless otherwise indicated, is determined in an
aqueous buffer solution at a pH of 7.4.The invention provides cation-insulin compound conjugate complexes and various compositions
including such complexes, as well as methods of making and using such complexes and
compositions. The complexes are useful for administering insulin compound for the treatment of
various medical conditions, such as conditions characterized by insulin compound deficiency.
The complexes generally include a cation component and an insulin compound conjugate
component. The insulin compound conjugate component generally includes an insulin compound
coupled to a modifying moiety. Examples of other suitable components of the complexes and/or
compositions include stabilizing agents, complexing agents, and other components known in the
art for use in preparing cation-protein complexes. The invention also provides novel insulin
compound conjugates and fatty acid formulations including such insulin compound conjugates
and/or cation-insulin compound conjugate complexes.
7.1 Insulin Compound
The cation-insulin compound conjugate includes an insulin compound component. The insulin
compound may, for example, be a mammalian insulin compound, such as human insulin, or an
insulin compound analog.
A wide variety of insulin compound analogs are known in the art. Preferred insulin compound
analogs are those which include a lysine, preferably a lysine within 5 amino acids of the
C-terminus of the B chain, e.g., at position B26, B27, B28, B29 and/or B30. A set of suitable
analogs is described in EP-A 1227000107 (the entire disclosure of which is incorporated herein
by reference), having the sequence of insulin compound, except that the amino acid residue at
position B28 is Asp, Lys, Leu, Val, or Ala; the amino acid residue at position B29 is Lys or Pro;
the amino acid residue at position BIO is His or Asp; the amino acid residue at position Bl is Phe,
Asp, or deleted alone or in combination with a deletion of the residue at position B2; the amino
12
acid residue at position B30 is Thr, Ala, or deleted; and the amino acid residue at position B9 is
Ser or Asp; provided that either position B28 or B29 is Lys.
Other examples of suitable insulin compound analogs include Asp828 human insulin, LysB2S
human insulin, Leu828 human insulin, ValB28 human insulin, Ala828 human insulin, AspB28ProB29
human insulin, LysB28ProB29 human insulin, LeuB28ProB29 human insulin, ValB28ProB29 human
insulin, AlaB28Pro829 human insulin, as well as analogs provided using the substitution guidelines
described above. Insulin compound fragments include, but are not limited to, B22-B30 human
insulin, B23-B30 human insulin, B25-B30 human insulin, B26-B30 human insulin, B27-B30
human insulin, B29-B30 human insulin, B1-B2 human insulin, B1-B3 human insulin, B1-B4
human insulin, B1-B5 human insulin, the A chain of human insulin, and the B chain of human
insulin.
Still other examples of suitable insulin compound analogs can be found in U.S. Patent
Publication No. 20030144181A1, entitled "Insoluble compositions for controlling blood
glucose," 31-M-03; U.S. Patent Publication No. 20030104983A1, entitled "Stable insulin
formulations," 5-Jun-03; U.S. Patent Publication No. 20030040601 Al, entitled "Method for
making insulin precursors and insulin analog precursors," 27-Feb-03; U.S. Patent Publication
No. 20030004096A1, entitled "Zinc-free and low-zinc insulin preparations having improved
stability," 2-Jan-03; U.S. Patent 6,551,992B1, entitled "Stable insulin formulations," 22-Apr-03;
U.S. Patent 6,534,28881, entitled "C peptide for improved preparation of insulin and insulin
analogs," 18-Mar-03; U.S. Patent 6,531,44861, entitled "Insoluble compositions for controlling
blood glucose," ll-Mar-03; U.S. Patent RE37,971E, entitled "Selective acylation of
epsilon-amino groups," 28-Jan-03; U.S. Patent Publication No. 20020198140A1, entitled
"Pulmonary insulin crystals," 26-Dec-02; U.S. Patent 6,465,426B2, entitled "Insoluble insulin
compositions," 15-Oct-02; U.S. Patent 6,444,641B1, entitled "Fatty acid-acylated insulin
analogs," 3-Sep-02; U.S. Patent Publication No. 20020137144A1, entitled "Method for making
insulin precursors and insulin precursor analogues having improved fermentation yield in yeast,"
26-Sep-02; U.S. Patent Publication No. 20020132760A1, entitled "Stabilized insulin
formulations," 19-Sep-02; U.S. Patent Publication No. 20020082199A1, entitled "Insoluble
insulin compositions," 27-Jun-02; U.S. Patent 6,335,316B1, entitled "Method for administering
acylated insulin," l-Jan-02; U.S. Patent 6,268,33561, entitled "Insoluble insulin compositions,"
31-Jul-Ol; U.S. Patent Publication No, 20010041787A1, entitled "Method for making insulinprecursors and insulin precursor analogues having improved fermentation yield in yeast,"
15-Nov-Ol; U.S. Patent Publication No. 20010041786Al, entitled "Stabilized acylated insulin
13
formulations," 15-Nov-Ol; U.S. Patent Publication No. 20010039260A1, entitled "Pulmonary
insulin crystals," 8-Nov-Ol; U.S. Patent Publication No. 20010036916A1, entitled "Insoluble
insulin compositions," l-Nov-01; U.S. Patent Publication No. 20010007853A1, entitled
"Method for administering monomeric insulin analogs," 12-Jul-Ol; U.S. Patent 6,051,551 A,
entitled "Method for administering acylated insulin," 18-Apr-OO; U.S. Patent 6,034,054A,
entitled "Stable insulin formulations," 7-Mar-OO; U.S. Patent 5,970,973A, entitled "Method of
delivering insulin lispro," 26-Oct-99; U.S. Patent 5,952,297A, entitled "Monomeric insulin
analog formulations," 14-Sep-99; U.S. Patent 5,922,615A, entitled "Acylated Insulin Analogs,"
13-M-99; U.S. Patent 5,888,477A, entitled "Use of monomeric insulin as a means for improving
the bioavailability of inhaled insulin," 30-Mar-99; U.S. Patent 5,873,358A, entitled "Method of
maintaining a diabetic patient's blood glucose level in a desired range," 23-Feb-99; U.S. Patent
5,747,642A, entitled "Monomeric insulin analog formulations," 5-May-98; U.S. Patent
5,693,609A, entitled "Acylated insulin compound analogs," 2-Dec-97; U.S. Patent 5,650,486A,
entitled "Monomeric insulin analog formulations," 22-M-97; U.S. Patent 5,646,242A, entitled
"Selective acylation of epsilon-amino groups," 8-M-97; U.S. Patent 5,597,893A, entitled
"Preparation of stable insulin analog crystals," 28-Jan-97; U.S. Patent 5,547,929A, entitled
"Insulin analog formulations," 20-Aug-96; U.S. 5,504,188A, entitled "Preparation of stable zinc
insulin compound analog crystals," 2-Apr-96; U.S. 5,474,978A, entitled "Insulin analog
formulations," 12-Dec-95; U.S. Patent 5,461,031A, entitled "Monomeric insulin analog
formulations," 24-Oct-95; U.S. Patent 4,421,685A, entitled "Process for producing an insulin,"
20-Dec-83; U.S. Patent 6,221,837, entitled "Insulin derivatives with increased zinc binding"
24-Apr-Ol; U.S. Patent 5,177,058, entitled "Pharmaceutical formulation for the treatment of
diabetes mellitus" 5-Jan-93 (describes pharmaceutical formulations including an insulin
compound derivative modified with a base at B31 and having an isoelectric point between 5.8 and
8.5 and/or at least one of its physiologically tolerated salts in a pharmaceutically acceptable
excipient, and a relatively high zinc ion content in the range from above 1 ug to about 200 (j.g of
zinc/IU, including insulin compound-B31-Arg-OH and human insulin-B31-Arg-B32-Arg-OH).
The entire disclosure of each of the foregoing patent documents is incorporated herein by
reference, particularly for teaching about the making, using and compositions of various insulin
compound analogs.
Insulin compound used to prepare the cation-insulin compound conjugates can be prepared by
any of a variety of recognized peptide synthesis techniques, e.g., classical (solution) methods,
solid phase methods, semi-synthetic methods, and recombinant DNA methods. For example,
14
Chance et al., U.S. Patent Application No. 07/388,201, EPO383472, Brange et al., EPO214826,
and Belagaje et al., U.S. Patent 5,304,473 disclose the preparation of various proinsulin
compound and insulin compound analogs and are herein incorporated by reference. The A and B
chains of the insulin compound analogs may also be prepared via a proinsulin compound-like
precursor molecule or single chain insulin compound precursor molecule using recombinant
DNA techniques. See Frank at al., "Peptides: Synthesis-Structure-Function," Proc. Seventh Am.
Pept. Syrnp., Eds. D. Rich and E. Gross (1981); Bernd Gutte, Peptides : Synthesis, Structures,
and Applications, Academic Press (October 19, 1995); Chan, Weng and White, Peter (Eds.),
Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press (March
2000); the entire disclosures of which are incorporated herein by reference for their teachings
concerning peptide synthesis, recombinant production and manufacture.7.2 Modifying Moiety
The cation-insulin compound conjugate complexes include a modifying moiety coupled (e.g.,
covalently or iom'cally) to the insulin compound to provide the insulin compound conjugate.
Modifying moieties are moieties coupled to the insulin compound that provide the insulin
compound with desired properties as described herein. For example, the modifying moiety can
reduce the rate of degradation of the insulin compound in various environments (such as the GI
tract, and/or the bloodstream), such that less of the insulin compound is degraded in the modified
form than would be degraded in the absence of the modifying moiety in such environments.
Preferred modifying moieties are those which permit the insulin compound conjugate to retain a
therapeutically significant percentage of the biological activity of the parent insulin compound.
Further, preferred modifying moieties are those which are amphiphilic or hydrophilic, and/or
which render the insulin compound conjugate amphiphilic or hydrophilic or less lipophilic than a
scientifically acceptable control, such as a corresponding insulin compound, or a corresponding
unconjugated insulin compound.
Examples of suitable modifying moieties and insulin compound conjugates useful in the
cation-insulin compound conjugate compositions can be found in the following patents, the entire
disclosures of which are incorporated herein by reference: U.S. Patent 6,303,569, entitled
"Trialkyl-lock-facilitated polymeric prodrugs of amino-containing bioactive agents," 16-Oct-Ol;
U.S. Patent 6,214,330, "Coumarin and related aromatic-based polymeric prodrugs," 10-Apr-Ol;
U.S. Patent 6,113,906, entitled "Water-soluble non-antigenic polymer linkable to biologically
active material," 05-Sep-OO; U.S. Patent 5,985,263, entitled "Substantially pure histidine-linked
15
protein polymer conjugates," 16-Nov-99; U.S. Patent 5,900,402, entitled "Method of reducing
side effects associated with administration of oxygen-carrying proteins," 04-05-99; U.S. Patent
5,681,811, "Conjugation-stabilized therapeutic agent compositions, delivery and diagnostic
formulations comprising same, and method of making and using the same" 28-Oct-97; U.S.
Patent 5,637,749, entitled "Aryl imidate activated polyalkylene oxides," 10-Jun-97; U.S. Patent
5,612,460, entitled "Active carbonates of polyalkylene oxides for modification of polypeptides,"
18-Mar-97; U.S. Patent 5,567,422, entitled "Azlactone activated polyalkylene oxides conjugated
to biologically active nucleophiles," 22-Oct-96; U.S. Patent 5,405,877, entitled "Cyclic imide
thione activated polyalkylene oxides," ll-Apr-95; and U.S. Patent 5,359,030, entitled
"Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic
formulations comprising same, and method of making and using the same," 25-Oct-94.
Additional examples of conjugated polypeptides useful in the formulations of the instant
invention can be found in the following U.S. patent applications, the entire specifications of
which are incorporated herein by reference: U.S. Patent Application Nos. 09/134,803, filed
14-Aug-98; 10/018,879, filed 19-Dec-Ol; 10/235,381, filed 05-Sep-02; 10/235,284, filed
05-Sep-02; and 09/873,797, filed 04-Jun-Ol. The entire disclosure of each of the foregoing
patents and patent applications is incorporated herein by reference for their teachings concerning
moieties used to modify polypeptides.
The modifying moieties may include weak or degradable linkages in their backbones. For
example, the PAGs can include hydrolytically unstable linkages, such as lactide, glycolide,
carbonate, ester, carbamate and the like, which are susceptible to hydrolysis. This approach
allows the polymers to be cleaved into lower molecular weight fragments. Examples of such
polymers are described, for example, in U.S. Patent 6,153,211, entitled, to Hubbell et al., the
entire disclosure of which is incorporated herein by reference. See also U.S. Patent 6,309,633, to
Ekwuribe et al., the entire disclosure of which is incorporated herein by reference.
The modifying moiety can include any hydrophilic moieties, lipophilic moieties, amphiphilic
moieties, salt-forming moieties, and combinations thereof. Representative hydrophilic,
amphiphilic, and lipophilic polymers and modifying moieties are described in more detail below.
7.2.1 Hydrophilic Moieties
Examples of suitable hydrophilic moieties include PAG moieties, other hydrophilic polymers,
sugar moieties, polysorbate moieties, and combinations thereof.
16
7.2.2 Polyalkylene Glycol Moieties
PAGs are compounds with repeat alkylene glycol units. In some embodiments, the units are all
identical (e.g., PEG or PPG). In other embodiments, the alkylene units are different (e.g.,
polyetbylene-co-propylene glycol, or PLURONICS®). The polymers can be random copolymers
(for example, where ethylene oxide and propylene oxide are co-polymerized) or branched or graft
copolymers.
PEG is a preferred PAG, and is useful in biological applications because it has highly desirable
properties and is generally regarded as safe (GRAS) by the Food and Drug Administration. PEG
generally has the formula H-(CH2CH2O)n-H, where n can range from about 2 to about 4000 ormore, though the capping moieties may vary, e.g., mono-methoxy or di-hydroxy. PEG typically
is colorless, odorless, water-soluble or water-miscible (depending on molecular weight), heat
stable, chemically inert, hydrolytically stable, and generally nontoxic. PEG is also
biocompatible, and typically does not produce an immune response in the body. Preferred PEG
moieties include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41,42, 43, 44,45,46, 47, 48, 49, 50,
or more PEG subunits.
The PEG may be monodispersed, substantially monodispersed, purely monodispersed or
substantially purely monodispersed (e.g., as previously described by the applicants in U.S. Patent
09/873,731 and U.S. Patent 09/873,797, both filed 04-Jun-Ol, the entire disclosures of which are
incorporated herein by reference) or polydispersed. One advantage of using the relatively low
molecular weight, monodispersed polymers is that they form easily defined conjugate molecules,
which can facilitate both reproducible synthesis and FDA approval.
The PEG can be linear with a hydroxyl group at each terminus (before being conjugated to the
remainder of the insulin compound). The PEG can also be an alkoxy PEG, such as methoxy-PEG
(or mPEG), where one terminus is a relatively inert alkoxy group (e.g., linear or branched OCj^),
while the other terminus is a hydroxyl group (that is coupled to the insulin compound).
The PEG can also be branched, which can in one embodiment be represented as R(-PEG-nOH)m
in which R represents a central (typically polyhydric) core agent such as pentaerythritol, sugar,
lysine or glycerol, n represents the number of PEG subunits and can vary for each arm and is
typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 and
17
m represents the number of arms, and ranges from 2 to the maximum number of attachment sitesz
on the core agent. Each branch can be the same or different and can be terminated, for example,
with ethers and/or esters. The number of arms m can range from three to a hundred or more, and
one or more of the terminal hydroxyl groups can be coupled to the remainder of the insulin
compound, or otherwise subject to chemical modification.
Other branched PEGs include those represented by the formula (CH3O-PEG-)pR-Z, where p
equals 2 or 3, R represents a central core such as lysine or glycerol, and Z represents a group such
as carboxyl that is subject to ready chemical activation. Still another branched form, the pendant
PEG, has reactive groups, such as carboxyls, along the PEG backbone rather than, or in addition
to, the end of the PEG chains. Forked PEG can be represented by the formula PEG(-LCHX2)n,
where L is a linking group and X is an activated terminal group.
7.2.3 Sugar Moieties
The modifying moieties described herein can include sugar moieties. In general, the sugar moiety
is a carbohydrate product of at least one saccharose group. Representative sugar moieties
include, but are not limited to, glycerol moieties, mono-, di-, tri-, and oligosaccharides, and
polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific
monosaccharides include C6 and above (preferably C6 to C8) sugars such as glucose, fructose,
mannose, galactose, ribose, and sedoheptulose; di- and trisaccharides include moieties having two
or three monosaccharide units (preferably C5 to C8) such as sucrose, cellobiose, maltose, lactose,
and raffinose. Conjugation using sugar moieties is described in US Patents 5,681,811, 5,438,040,
and 5,359,030, the entire disclosures of which are incorporated herein by reference.
7.2.4 Polysorbate Moieties
The modifying moieties may include one or more polysorbate. moieties. Examples include
sorbitan esters, and polysorbate derivatized with polyoxyethylene. Conjugation using polysorbate
moieties is described in US Patents 5,681,811, 5,438,040, and 5,359,030, the entire disclosures of
which are incorporated herein by reference.
7.2.5 Biocompatible Water-soluble Polycationic Moieties
In some embodiments, biocompatible water-soluble polycationic polymers can be used.
Biocompatible water-soluble polycationic polymers include, for example, any modifying moiety
having protonated heterocycles attached as pendant groups. "Water soluble" in this context
means that the entire modifying moiety is soluble in aqueous solutions, such as buffered saline or
buffered saline with small amounts of added organic solvents as cosolvents, at a temperature
between 20 and 37°C. In some embodiments, the modifying moiety itself is not sufficiently
soluble in aqueous solutions per se but is brought into solution by grafting with water-soluble
polymers such as PEG chains. Examples include polyamines having amine groups on either the
modifying moiety backbone or the modifying moiety side chains, such as poly-L-Lys and other
positively charged polyamino acids of natural or synthetic amino acids or mixtures of amino
acids, including poly(D-Lys), poly(omithine), poly(Arg), and poly(histidine), and nonpeptide
polyamines such as poly(aminostyrene), poly(aminoacrylate), poly (N-methyl aminoacrylate),
poly (N-ethylaminoacrylate), poly(N,N-dimethyl aminoacrylate),
poly(N,N-diethylaminoacrylate), poly(aminomethacrylate), poly(N-methyl amino-methacrylate),
poly(N-ethyl aminomethacrylate), poly(N,N-dimethyl aminomethacrylate), poly(N,N-diethyl
aminomethacrylate), poly(ethyleneimine), polymers of quaternary amines, such as
poly(N,N,N-trimethylaminoacrylate chloride), poly(uietb.yacrylamidopropyltrimethyl ammonium
chloride), and natural or synthetic polysaccharides such as chitosan.
7.2.6 Other Hydrophilic Moieties
The modifying moieties may also include other hydrophilic polymers. Examples include
poly(oxyethylated polyols) such as poly(oxyethylated glycerol), poly(oxyethylated sorbitol), and
poly(oxyethylated glucose); poly(vinyl alcohol) ("PVA"); dextran; carbohydrate-based polymers
and the like. The polymers can be homopolymers or random or block copolymers and
terpolymers based on the monomers of the above polymers, linear chain or branched.
Specific examples of suitable additional polymers include, but are not limited to, poly(oxazoline),
difunctional poly(acryloylmorpholine) ("PAcM"), and poly(vinylpyrrolidone)("PVP"). PVP and
poly(oxazoline) are well known polymers in the art and their preparation will be readily apparent
to the skilled artisan. PAcM and its synthesis and use are described in U.S. Patent 5,629,384 and
U.S. Patent 5,631,322, the disclosures of which are incorporated herein by reference in their
entirety.
7.2.7 Bioadhesive Polyanionic Moieties
Certain hydrophilic polymers appear to have potentially useful bioadhesive properties. Examples
of such polymers are found, for example, in U.S. Patent 6,197,346, to Mathiowitz, et al. Those
polymers containing carboxylic groups (e.g., poly(acrylic acid)) exhibit bioadhesive properties,
19
and are also readily conjugated with the insulin compounds described herein. Rapidly
bioerodible polymers that expose carboxylic acid groups on degradation, such as
poly(lactide-co-glycolide), polyanhydrides, and polyorthoesters, are also bioadhesive polymers.
These polymers can be used to deliver the insulin compounds to the gastrointestinal tract. As the
polymers degrade, they can expose carboxylic acid groups to enable them to adhere strongly to
the gastrointestinal tract, and can aid in the delivery of the insulin compound conjugates.
7.2.8 Lipophilic Moieties
In some embodiments, the modifying moieties include one or more lipophilic moieties. The
lipophilic moiety may be various lipophilic moieties as will be understood by those skilled in the
art including, but not limited to, alkyl moieties, alkenyl moieties, alkynyl moieties, aryl moieties,
arylalkyl moieties, alkylaryl moieties, fatty acid moieties, adamantantyl, and cholesteryl, as well
as lipophilic polymers and/or oligomers.
The alkyl moiety can be a saturated or unsaturated, linear, branched, or cyclic hydrocarbon chain.
In some embodiments, the alkyl moiety has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43,
44, 45, 46, 47, 48, 49, 50 or more carbon atoms. Examples include saturated, linear alkyl
moieties such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,
dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; saturated,
branched alkyl moieties such as isopropyl, sec-butyl, /ert-butyl, 2-methylbutyl, /er/-pentyl,
2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl; and unsaturated alkyl moieties
derived from the above saturated alkyl moieties including, but not limited to, vinyl, allyl,
1-butenyl, 2-butenyl, ethynyl, 1-propynyl, and 2-propynyl. In other embodiments, the alkyl
moiety is a lower alkyl moiety. In still other embodiments, the alkyl moiety is a Ci to Ca lower
alkyl moiety. In some embodiments, the modifying moiety specifically does not consist of an
alkyl moiety, or specifically does not consist of a lower alkyl moiety, or specifically does not
consist of an alkane moiety, or specifically does not consist of a lower alkane moiety.
The alkyl groups can either be unsubstituted or substituted with one or more substituents, and
such substituents preferably either do not interfere with the methods of synthesis of the
conjugates or eliminate the biological activity of the conjugates. Potentially interfering
functionality can be suitably blocked with a protecting group so as to render the functionality
non-interfering. Each substituent may be optionally substituted with additional non-interfering
20
substituents. The term "non-interfering" characterizes the substituents as not eliminating the
feasibility of any reactions to be performed in accordance with the process of this invention.
The lipophilic moiety may be a fatty acid moiety, such as a natural or synthetic, saturated or
unsaturated, linear or branched fatty acid moiety. In some embodiments, the fatty acid moiety
has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more carbon
atoms. In some embodiments, the modifying moiety specifically does not consist of a fatty acid
moiety; or specifically does not consist of a fatty acid moiety having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12,13,14,15,16,17, 18,19, 20, 21, 22, 23, 24 or more carbon atoms.
When the modifying moiety includes an aryl ring, the ring can be functionalized with a
nucleophilic functional group (such as OH, or SH) that is positioned so that it can react in an
intramolecular fashion with the carbamate moiety and assist in its hydrolysis. In some
embodiments, the nucleophilic group is protected with a protecting group capable of being
hydrolyzed or otherwise degraded in vivo, with the result being that when the protecting group is
deprotected, hydrolysis of the conjugate, and resultant release of the parent insulin compound, is
facilitated.
Other examples of suitable modifying moieties include -C(CH2OH)3; - CH(CH2OH)2; -C(CH3)3; -
CH(CH3)2.
7.2.9 Amphiphilic Moieties
In some embodiments, the modifying moiety includes an amphiphilic moiety. Many polymers
and oligomers are amphiphilic. These are often block co-polymers, branched copolymers or graft
co-polymers that include hydrophilic and lipophilic moieties, which can be in the form of
oligomers and/or polymers, such as linear chain, branched, or graft polymers or co-polymers.
The amphiphilic modifying moieties may include combinations of any of the lipophilic and
hydrophilic moieties described herein. Such modifying moieties typically include at least one
reactive functional group, for example, halo, hydroxyl, amine, thiol, sulfonic acid, carboxylic
acid, isocyanate, epoxy, ester, and the like, which is often at a terminal end of the modifying
moiety. These reactive functional groups can be used to attach a lipophilic linear or branched
chain alkyl, alkenyl, alkynyl, arylalkyl, or alkylaryl group, or a lipophilic polymer or oligomer,
thereby increasing the lipophilicity of the modifying moiety (and thereby rendering them
generally amphiphilic).
21
The lipophilic groups can, for example, be derived from mono- or di-carboxylic acids, or where
appropriate, reactive equivalents of carboxylic acids such as anhydrides or acid chlorides.
Examples of suitable precursors for the lipophilic groups are acetic acid, propionic acid, butyric
acid, valeric acid, isobutyric acid, trimethylacetic acid, caproic acid, caprylic acid, heptanoic acid,
capric acid, pelargonic acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid,
lignoceric acid, ceratic acid, montanoic acid, isostearic acid, isononanoic acid, 2-ethylhexanoic
acid, oleic acid, ricinoleic acid, linoleic acid, linolenic acid, erucic acid, soybean fatty acid,
linseed fatty acid, dehydrated castor fatty acid, tall oil fatty acid, tung oil fatty acid, sunflower
fatty acid, safflower fatty acid, acrylic acid, methacrylic acid, maleic anhydride, orthophthalic
anhydride, terephthalic acid, isophthalic acid, adipic acid, azelaic acid, sebacic acid,
tetrahydrophthalic anhydride, hexahydrophthalic anhydride, succinic acid and polyolefin
carboxylic acids.
The terminal lipophilic groups need not be equivalent, i.e., the resulting copolymers can include
terminal lipophilic groups that are the same or different. The lipophilic groups can be derived
from more than one mono or di-functional alkyl, alkenyl, alkynyl, cycloalkyl, arylalkyl or
alkylaryl group as defined above.
7.2.10 PAG-alkyl Modifying Moieties
The modifying moiety may be a linear or branched polymeric moiety having one or more linear
or branched PAG moieties and/or one or more linear or branched, substituted or unsubstituted
alkyl moieties. In certain cases, such moieties are considered amphiphilic; however, the PAG and
alkyl moieties may be varied to render such moieties more lipophilic or more hydrophilic. In
certain embodiments, the modifying moiety specifically does not consist of an alkyl moiety and
in other embodiments, the modifying moiety specifically does not consist of an alkane moiety.
The PAG moieties in some embodiments include 1, 2, 3,4, 5, 6, 7, 8, 9,10, 11,12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, or 25 PAG subunits arranged in linear or branched form. The PAG
moieties in some embodiments include PEG, PPG and/or PBG subunits. The alkyl moieties in
some embodiments preferably have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or
20 carbon atoms. The alkyl moieties are preferably alkane moieties. The modifying moiety may
include a capping moiety, such as -OCH3. Further, the modifying moiety may include a
hydrophobia group, such as a pivaloyl group.
In one embodiment, the modifying moiety has a formula:
22
-<-(CH2)0-X-(CH2CH20)p-Y-(CH2)q-Z-R
^ (Formula I)
where o, p and q are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18,19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45,
46, 47, 48, 49, or 50, and at least one of o, p and q is at least 2. X, Y and Z are independently
selected from -C-, -O-, -C(O)-, -C(O)O-, -OC(O)-, -NH-, -NHC(O)-, and -C(O)NH-, and R is H
or an alkyl, preferably a lower alkyl, more preferably methyl. The variables o, p and q are
preferably selected to yield a hydrophilic or amphiphilic modifying moiety, and are preferably
selected in relation to the insulin compound to yield a hydrophilic or amphiphilic insulin
compound conjugate, preferably a monoconjugate, diconjugate or triconjugate. In one preferred
embodiment for an insulin compound conjugate which is to be used for basal insulin compound
maintenance, o, p and q are selected to yield a PAG which is proximal to the insulin compound
and an alkyl moiety which is distal to the insulin compound. Alternatively, O, P and Q may be
selected to yield a PAG which is distal to the insulin compound and an alkyl which is proximal to
the insulin. In an alternative embodiment, R is a pivaloyl group or an alkyl-pivaloyl group.
In a related embodiment, the modifying moiety has a formula:
where mis 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12,13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25
and n is from 2 to 100, preferably 2 to 50, more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14,
15, 16,17,18,19, 20, 21, 22, 23, 24 or 25, X is -C-, -O-, -C(O)-, -NH-, -NHC(O)-, or -C(O)NH-,
and Y is lower alkyl or -H. X is preferably 0 and Y is preferably -CH3. In some cases the
carbonyl group (-C(O)-) may be absent, and the -(CH2)- moiety may be coupled to an available
group on an amino acid, such as a hydroxyl group or a free carboxyllic acid group.
In a preferred embodiment, the modifying moiety has a structure selected from the following:
(when the immediately preceding modifying moiety is coupled to human insulin at B29, the
resulting monoconjugate is referred to as IN 105).
O
(when the immediately preceding modifying moiety is coupled to human insulin at B29, the
resulting monoconjugate is referred to as HIM2). Any of the foregoing moieties may, for
example, be coupled to human insulin at a nucleophilic residue, e.g., Al, Bl or B29. In some
cases the carbonyl group (-C(O)-) may be absent or replaced with an alkyl moiety, preferably a
lower alkyl moiety, and the — (CHz)- moiety may be coupled to an available group on an amino
acid, such as a hydroxyl group or a free carboxyllic acid group.
In another embodiment, the modifying moiety has a formula:
(Formula III),
where each C is independently selected and is an alkyl moiety having m carbons and m is 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and each PAG is independently
selected and is a PAG moiety having n subunits and n is 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; each X is independently selected and is a linking
moiety coupling PAG to C, and is preferably -C-, -O-, -C(O)-, -NH-, -NHC(O)-, or -C(O)NH-. In
some embodiments the Cm-X moiety is absent, and the PAGn moiety is terminated with an -OH
moiety or an -OCH3 moiety. For example, the PAG may be methoxy-terminated or
hydroxy-terminated PAG, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or
20 PAG subunits, including PEG, PPG, and/or PBG subunits. In some cases the carbonyl group (-
C(O)-) may be replaced with an alkyl moiety, preferably a lower alkyl moiety, which may be
coupled to an available group on an amino acid, such as a hydroxyl group or a free carboxyllic
acid group.
The modifying moiety may, for example, have a formula:
(Formula IV),
where each C is independently selected and is an alkyl moiety having m carbons and m is 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; and each PAG is independently
selected and is a PAG moiety having n subunits and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25; X is -O-, or -NH-; each o is independently selected
and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14 or 15. For example, the PAG may have 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 PAG subunits, including PEG, PPG,
and/or PEG subunits. In some cases the carbonyl group (-C(O)-) proximal to the point of
attachment may be absent or replaced with an alkyl moiety, preferably a lower alkyl moiety, and
the -(CH2)- moiety may be coupled to an available group on an amino acid, such as a hydroxyl
group or a free carboxyllic acid group.
The modifying moiety may, for example, have a formula:
O
Cm-X-PAGn .A. PAGn-X-Cm
\^ \^ (Formula V),
where each C is independently selected and is an alkyl moiety having m carbons and m is 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; and each PAG is independently
selected and is a PAG moiety having n subunits and n is 1,2, 3, 4, 5, 6, 7, 8, 9,10,11,12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25; each X is independently selected and is a linking
moiety coupling PAG to C, and is preferably -C-, -O-, -C(O)-, -NH-, -NHC(O)-, or -C(O)NH-;
each o is independently selected and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In some
embodiments the Cm-X moiety is absent, and the PAGn moiety is terminated with an -OH moiety
or an -OCH3 moiety. For example, the PAG may be methoxy-terminated or hydroxy-terminated
PAG, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 PAG submits,
including PEG, PPG, and/or PEG subunits. In some cases the carbonyl group (-C(O)-) proximal
to the point of attachment may be absent, and the -(CH2)- moiety may be coupled to an available
group on an amino acid, such as a hydroxyl group or a free carboxyllic acid group.
In another embodiment, the modifying moiety may have a formula:
-X-R1-Y-PAG-Z-R2 (Formula VI)
where,
X, Y and Z are independently selected linking groups and each is optionally present, and X, when
present, is coupled to the insulin compound by a covalent bond,
at least one of R1 and R2 is present, and is lower alkyl and may optionally include a carbonyl
group,
R2 is a capping group, such as -CH3, -H, tosylate, or an activating group, and
PAG is a linear or branched carbon chain incorporating one or more alkalene glycol moieties (i.e.,
oxyalkalene moieties), and optionally incorporating one or more additional moieties selected
from the group consisting of-S-, -O-, -N-, and -C(O)-, and
where the modifying moiety has a maximum number of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16,17,18,19, 20,21, 22, 23, 24, or 25 heavy atoms.
In embodiments of the invention, any one or more of X, Y and Z may be absent. Further, when
present, X, Y and/or Z may be independently selected from -C(O)-, -O-, -S-, -C- and -N-. In one
embodiment, Z is -C(O)-. In another embodiment, Z is not present.
In some embodiments, R1 is lower alkyl, and R2 is not present. In other embodiments, R2 is lower
alkyl, and R1 is not present.
In another embodiment, the modifying moiety may include a linear or branched, substituted
carbon chain moiety having a backbone of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19,
20, 21, 22, 23, 24 or 25 atoms selected from the group consisting of -C, -C-, -O-,
=O, -S-, -N-, -Si-. The heavy atoms will typically include one or more carbon atoms and one or
more non-carbon heavy atoms selected from the group consisting of -O-, -S-, -N-, and =O. The
carbon atoms and non-carbon heavy atoms are typically present in a ratio of at least 1 carbon
atom for every non-carbon heavy atom, preferably at least 2 carbon atoms for every non-carbon
heavy atom, more preferably at least 3 carbon atoms for every non-carbon heavy atom. The
carbon atoms and oxygen atoms are typically present in a ratio of at least 1 carbon atom for every
oxygen atom, preferably at least 2 carbon atoms for every oxygen atom, more preferably at least 3
carbon atoms for every oxygen atom. The modifying moiety may include one or more capping
groups, such as branched or linear Ci.6) branched or linear, or a carbonyl. The modifying moiety
will typically include hydrogens, and one or more of the hydrogens may be substituted with a
fluorine (which is a heavy atom but should not be counted as a heavy atom in the foregoing
formula). The modifying moiety may in some cases specifially exclude unsubstituted alkyl
moieties. The modifying moiety may, for example, be coupled to an available group on an amino
acid, such as an amino group, a hydroxyl group or a free carboxyllic acid group the polypeptide,
e.g., by a linking group, such as a carbamate, carbonate, ether, ester, amide, or secondary amine
group, or by a disulfide bond. The molecules in the linking group are counted as part of the
modifying moiety. In a preferred embodiment, the molecular weight of the modifying moiety is
less than the molecular weight of the HIM2 modifying moiety.
The invention includes modifying moieties having a formula:
O
(Formula VII),
where n is 1, 2, 3 or 4, and m is 1, 2, 3, 4 or 5.
The invention includes modifying moieties having a formula:
O
(Formula VIII),
where n is 1, 2, 3,4 or 5, and m is 1,2, 3 or 4.
The invention includes modifying moieties having a formula:
(Formula IX),
where m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15,16, 17,18, 19 or 20 and n is 1, 2, 3, 4, 5,
6,7,8,9, 10, 11,12, 13, 14, 15,16,17, 18,19 or 20.
The invention also includes modifying moieties having a formula:
O O
HO O(PAG)^,
(Formula X),
where PAG is a PAG moiety having m sutmnits and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14,15,16,17,18,19 or 20 and n is 1, 2, 3,4, 5, 6,7, 8, 9, 10,11, 12,13, 14,15,16,17,18, 19 or
20.
Other preferred modifying moieties include:
'EH3-O-zHO-zHO-zHO-zHO-zHO-zHO-zHO-zHO-y

R-CHrCH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-0-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-CHrO-CH2-CH2-O-CH2-CH2-CH3>
R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CHrO-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2"Cn2"CH2~CH2~CH2~CH2-O-CH2~CH2-Cn2~CH2"CH2~CH2"
R-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CHrCH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-0-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-0-CH2-CH2-CH3,
R-CH2-CHrCH2-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-0-CH2-CH2-0-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-0-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-0-CH2-CH2-CH2-O-,
R-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-0-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-0-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CHrO-CH2-CH2-CH2-CH3>
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-0-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH3>
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH3,
32
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CHrCH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-0-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-0-CHrCH2-O-CH2-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-0-CH2-CHrO-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-CHrCH2-CH2-CH2-O-CH2-CHrO-CH2-CH3,
R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-CH2-O-CHZ-CH2-CH2-CH2-CH2-CH3,
R-CHrCH2-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CHrCH2-CH3,
R-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-0-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-0-CH2-CH2-CH2-0-CH2-CH2-0-CH2-CH3,
R-CHrCH2-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH3>
R-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CHrCH3,
R-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3l
R-CHrCH2-CH2-CH2-O-CH2-CH2-O-CH2-CHrO-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-CHrCH2-O-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CHrCH2-0-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
33
R-CH2-CH2-CH2-O-CH2-CH2-CHrCH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-,
R-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CHZ-CH2-O-CH2-CH2-CH2-CH2-CH2-CH3,
R-CHrCH2-CH2-O-CH2-CHrCH2-O-CH2-CH2-CH2-CH2-CH3>
R-CH2-CH2-CH2-O-CH2-CHZ-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-CHrO-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CHrCH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CHrCH2-CHrO-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2*CH2~CH2~O-CH2~CH2~O-CH2"CH2~Cn2~CH2~Cn2~CH2~Cn3,
R-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-CHrCH2-O-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-0-CH2-CH2-O-CH2-CHrCH2-O-CH2-CH2-CH2-CH3l
R-CH2-CHrCH2-O-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH3,
R-CHrCH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CHrCH2-O-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3)
R-CH2-CH2-CH2-O-CHrCH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-0-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-CH2-0-CH2-CH2-0-CH3,
R-CH2-CH2-CH2-0-CH2-CH3,
R-CH2-CH2-CH2-O-CH3,
R-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CHrCH2-CH2-CH3,
R-CH2-CHrO-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3,
R-CH2-CHrO-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-CH2-CHrO-CH2-CH2-CH2-O-CH3,
34
R-CH2-CHrO-CH2-CH2-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CHrO-CH2-CH3,
R-CH2-CHrO-CH2-CH2-CH2-CH2-O-CH2-CH2-0-CH3,
R-CH2-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-0-CHZ-CH2-CH2-CH2-O-CH3,
R-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CHrCH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH3l
R-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-CH2-0-CH2-CH2-CH2-O-CH2-CH2-CH3r
R-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CHrCH2-O-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-0-CH2-CH2-0-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-0-CH2-CH2-0-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3,
R-CHrCH2-O-CH2-CH2-O-CH2-CH2-CH2-0-CH2-CH2-CH2-CH3,
R-CH2-CHrO-CH2-CHrO-CH2-CH2-CH2-0-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-0-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-0-CH2-CH2-O-CH3,
R-CH2-CH2-O-CH2-CH2-0-CH2-CH2-CH2-0-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CHrCH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-0-CH2-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CHrO-CH2-CHz-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-CH2-O-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-0-CH2-CH2-0-CH3,
R-CH2-CH2-O-CH2-CH3,
R-CH2-CH2-O-CH3,
R~CH2-O-CH2-CH2-CH2"Cn2~CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2~CH3,
R-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3l
R-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CHZ-CH3,
35
R-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CHrCH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH3>
R-CH2-O-CH2-CHrCH2-CH2-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-0-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-O-CH3,
R-CH2-O-CHrCH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-CH2-O-CH2-CHrCH2-CH3,
R-CH2-O-CHrCH2-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH3,
R-CHrO-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-CH2-O-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CHrCH3,
R-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-O-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-CH2-O-CH3,
R-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CHrCH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-CHrO-CH2-CH2-O-CH2-CHrO-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-O-CH2-CHrCH2-O-CH2-CH2-0-CH2-CH3,
R-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-O-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-,
R-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
36
R-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3,
R-CH2-0-CH2-CH2-0-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-0-CH2-CH2-CH2-CH3,
R-CH2-O-CHrCH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-CH2-0-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CHZ-CH2-O-CH2-CH2-O-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-CH2-0-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-CH2-O-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3>
R-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-0-CH2-CH2-CH2-CH3.
R-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-0-CH2-CH2-O-CH3,
R-CH2-O-CH2-CH2-0-CH2-CH2-O-CH2-CH2-0-CH2-CH3,
R-CH2-0-CH2-CH2-0-CH2-CH2-0-CH2-CH2-0-CH3,
R-CH2-0-CH2-CH2-0-CH2-CH2-0-CH2-CH3,
R-CH2-O-CH2-CH2-O-CH2-CH2-O-CH3,
R-CH2-O-CH2-CH2-O-CH3,
R-CH2-O-CH2-CH3,
R-CH2-O-CH3,
where R is -H, -OH, -CH2OH, -CH(OH)2, -C(O)OH, -CH2C(O)OH, or an activating moiety, such
as a carbodiimide, a mixed anhydride, or an N-hydroxysuccinimide, or a capping group. The
invention also includes such moieties attached to a protein or peptide, preferably to an insulin
compound. Specific conjugation strategies are discussed in more detail below. Of these
modifying moieties, prefered moieties are those which render the insulin compound less
lipophilic and/or more hydrophilic than the corresponding unconjugated insulin compound. The
invention includes such modifying moieties further including one or more carbonyl groups,
preferably 1, 2, 3, 4, or 5 carbonyl groups; the carbonyl groups may be inserted into the
modifying moiety, or an -O- or -CH2- may be replaced with a carbonyl. Further, any of the -CH2-
or -CH3 moieties may be substituted, e.g., with a lower alkyl or an -OH or a PAG chain having 1,
2, 3, 4, or 5 PAG subunits, which may be the same or different. Preferably R is selected so that
each -0- is separated from the nearest -O- by at least 2 carbons. The invention also includes
branched modifying moieties in which two or more of the moieties are attached to a branching
moiety, such as a lysine.
37
The pharmaceutical characteristics, such as hydrophilicity/lipophilicity of the conjugates
according to embodiments of the invention, can be varied by, for example, adjusting the
lipophilic and hydrophilic portions of the modifying moieties, e.g., by increasing or decreasing
the number of PAG monomers, the type and length of alkyl chain, the nature of the PAG-peptide
linkage, and the number of conjugation sites. The exact nature of the modifying moiety-peptide
linkage can be varied such that it is stable and/or sensitive to hydrolysis at physiological pH or in
plasma. The invention also includes any of the foregoing modifying moieties coupled to a
polypeptide, preferably to insulin compound. Preferably, the modifying moiety renders the
polypeptide more soluble than a corresponding unconjugated polypeptide, e.g., by a multiplier of
at least 1.05, 1.25. 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11,
11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15. A modifying moiety of the invention may be coupled,
for example, to an insulin compound, such as a human insulin, at any available point of
attachment. A preferred point of attachment is a nucleophilic residue, e.g., Al, Bl and/or B29.
Moreover, it will be appreciated that one aspect of the invention includes novel modifying
moieties, such as but not limited to the moieties of Formulas VII and VIII, in a carboxylic acid
form. Further, where the modifying moiety includes a carboxyl group, it can be converted to a
mixed anhydride and reacted with an amino group of a peptide to create a conjugate containing an
amide bond. In another procedure, the carboxyl group can be treated with water-soluble
carbodiimide and reacted with the peptide to produce conjugates containing amide bonds.
Consequently, the invention includes activated forms of the novel moieties presented herein, such
as activated forms of the modifying moieties of Formulas VII and VIII and other novel oligomers
of the invention, such as carbodiimides, mixed anhydrides, or N-hydroxysuccinimides.
hi some cases, the modifying moiety may be coupled to the polypeptide via an amino acid or
series of 2 or more amino acids coupled to the C-terminus, or a side chain of the polypeptide. For
example, in one embodiment, the modifying moiety is coupled at the —OH or -C(O)OH of Thr,
and the mm-modified Thr is coupled to a polypeptide at the carboxy terminus. For example, in
one embodiment, the modifying moiety is coupled at the -OH or -C(O)OH of Thr, and the
modified Thr is coupled to the B29 amino acid (e.g., a B29 Lys for human insulin) of des-Thr
insulin compound. In another example, the mm is coupled at the -OH or -C(O)OH of Thr of a
terminal octapeptide from the insulin compound B-chain, and the mm-modified octapeptide is
coupled to the B22 amino acid of des-octa insulin compound. Other variations will be apparent
to one skilled in the art in light of this specification.
38
7.2.11 Salt-forming Moieties
In some embodiments, the modifying moiety comprises a salt-forming moiety. The salt-forming
moiety may be various suitable salt-forming moieties as will be understood by those skilled in the
art including, but not limited to, carboxylate and ammonium. In some embodiments where the
modifying moiety includes a salt forming moiety, the insulin compound conjugate is provided n
salt form. In these embodiments, the insulin compound conjugate is associated with a suitable
pharmaceutically acceptable counterion as will be understood by those skilled in the art including,
but not limited to, negative ions such as chloro, bromo, iodo, phosphate, acetate, carbonate,
sulfate, tosylate, and mesylate, or positive ions such as sodium, potassium, calcium, lithium, and
ammonium.
The foregoing examples of modifying moieties are intended as illustrative and should not be
taken as limiting in any way. One skilled in the art will recognize that suitable moieties for
conjugation to achieve particular functionality will be possible within the bounds of the chemical
conjugation mechanisms disclosed and claimed herein. Accordingly, additional moieties can be
selected and used according to the principles as disclosed herein.
7.3 Conjugation Strategies
Factors such as the degree of conjugation with modifying moieties, selection of conjugation sites
on the molecule and selection of modifying moieties may be varied to produce a conjugate which,
for example, is less susceptible to in vivo degradation, and thus, has an increased plasma half life.
For example, the insulin compounds may be modified to include a modifying moiety at one, two,
three, four, five, or more sites on the insulin compound structure at appropriate attachment (i.e.,
modifying moiety conjugation) sites suitable for facilitating the association of a modifying moiety
thereon. By way of example, such suitable conjugation sites may comprise an amino acid
residue, such as a lysine amino acid residue.
In some embodiments, the insulin compound conjugates are monoconjugates. In other
embodiments, the insulin compound conjugates are multi-conjugates, such as di-conjugates,
tri-conjugates, terra-conjugates, penta-conjugates and the like. The number of modifying
moieties on the insulin compound is limited only by the number of conjugation sites on the
insulin compound. In still other embodiments, the insulin compound conjugates are a mixture of
mono-conjugates, di-conjugates, tri-conjugates, terra-conjugates, and/or penta-conjugates.
Preferred conjugation strategies are those which yield a conjugate relating some or all of the
bioactivity of the parent insulin compound.
Preferred attachment sites include Al N-terminus, Bl N-terminus, and B29 lysine side chain.
The B29 monoconjugate and Bl, B29 diconjugates are highly preferred. Another preferred point
of attachment is an amino functionality on a C-peptide component or a leader peptide component
of the insulin compound.
One or more modifying moieties (i.e., a single or a plurality of modifying moiety structures) may
be coupled to the insulin compound. The modifying moieties in the plurality are preferably the
same. However, it is to be understood that the modifying moieties in the plurality may be
different from one another, or, alternatively, some of the modifying moieties in the plurality may
be the same and some may be different. When a plurality of modifying moieties are coupled to
the insulin compound, it may be preferable to couple one or more of the modifying moieties to
the insulin compound with hydrolyzable bonds and couple one or more of the modifying moieties
to the insulin compound with non-hydrolyzable bonds. Alternatively, all of the bonds coupling
the plurality of modifying moieties to the insulin compound may be hydrolyzable but have
varying degrees of hydrolyzability such that, for example, one or more of the modifying moieties
may be relatively rapidly removed from the insulin compound by hydrolysis in the body and one
or more of the modifying moieties is more slowly removed from the insulin compound by
hydrolysis in the body.
7.3.1 Coupling of Modifying Moiety to Insulin Compound
The modifying moiety is preferably covalently coupled to the insulin compound. More than one
moiety on the modifying moiety may be covalently coupled to the insulin compound. Coupling
may employ hydrolyzable or non-hydrolyzable bonds or mixtures of the two (i.e., different bonds
at different conjugation sites).
In some embodiments, the insulin compound is coupled to the modifying moiety using a
hydrolyzable bond (e.g., an ester, carbonate or hydrolyzable carbamate bond). Use of a
hydrolyzable coupling will provide an insulin compound conjugate that acts as a prodrug. A
prodrug approach may be desirable where the insulin compound-modifying moiety conjugate is
inactive (i.e., the conjugate lacks the ability to affect the body through the insulin compound's
primary mechanism of action), such as when the modifying moiety conjugation site is in a
binding region of insulin compound. Use of a hydrolyzable coupling can also provide for a
time-release or controlled-release effect, administering the insulin compound over a given time
period as one or more modifying moieties are cleaved from their respective insulin
compound-modifying moiety conjugates to provide the active drug.
In other embodiments, the insulin compound is coupled to the modifying moiety utilizing a
non-hydrolyzable bond (e.g., a non-hydrolyzable carbamate, amide, or ether bond). Use of a
non-hydrolyzable bond may be preferable when it is desirable to allow therapeutically significant
amounts of the insulin compound conjugate to circulate in the bloodstream for an extended period
of time, e.g., at least 2 hours post administration. Bonds used to covalently couple the insulin
compound to the modifying moiety in a non-hydrolyzable fashion are typically selected from the
group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties,
amide moieties and secondary amine moieties.
The modifying moiety may be coupled to the insulin compound at various nucleophilic residues,
including, but not limited to, nucleophilic hydroxyl functions and/or amino functions.
Nucleophilic hydroxyl functions may be found, for example, at serine and/or tyrosine residues,
and nucleophilic amino functions may be found, for example, at histidine and/or Lys residues,
and/or at the one or more N-terminus of the A or B chains of the insulin compound. When a
modifying moiety is coupled to the N-terminus of the natriuretic peptide, coupling preferably
forms a secondary amine.
The modifying moiety may be coupled to the insulin compound at a free -SH group, e.g., by
forming a thioester, thioether or sulfonate bond.
The modifying moiety may be coupled to the insulin compound via one or more amino groups.
Examples in human insulin include the amino groups at Al, Bl and B29. In one embodiment, a
single modifying moiety is coupled to a single amino group on the insulin compound. In another
embodiment, two modifying moieties are each connected to a different amino group on the
insulin compound. Where there are two modifying moieties coupled to two amino groups, a
preferred arrangement is coupling of at Bl and B29. Where there are multiple polymers, the
polymers may all be the same or or one or more of the polymers may be different from the others.
Various methods and types of coupling of polymers to insulin compounds are described in U.S.
Patent Application No. 09/873,899, entitled "Mixtures of insulin compound conjugates
comprising polyalkylene glycol, uses thereof, and methods of making same," filed on 04-Jun-Ol,
the entire disclosure of which is incorporated herein by reference.
In still other embodiments, a partial prodrug approach may be used, in which a portion of the
modifying moiety is hydrolyzed. For example, see U.S. Patent 6,309,633 to Ekwuribe et al. (the
entire disclosure of which is incorporated herein by reference), which describes modifying
moieties having hydrophilic and lipophilic components in which the lipophilic components
hydrolyze in vivo to yield a micropegylated conjugate.
7.3.2 Selection of Modifying Moiety and Properties of the Insulin- Compound
Conjugate and Complexes Thereof
The modifying moiety may be selected to provide desired attributes to the insulin compound
conjugate and complexes thereof. Preferred modifying moieties are selected to render the insulin
compound more soluble in an aqueous solution than the aqueous solubility of the insulin
compound in the absence of the modifying moiety, preferably at least 1.05, 1.25, 1.5, 1.75, 2,
2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,
14.5, or 15 times more soluble than the parent insulin compound (i.e., the corresponding
unconjugated insulin compound) in an aqueous solution. For example, uncomplexed native
human insulin has a solubility of ~18mg/ml at a pH of about 7.4. The inventors have surprisingly
discovered a method of complexing human insulin conjugates that are more soluble than human
insulin by a multiplier of at least 1.05, 1.25, 1.5, 1.75,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,
8.5, 9, 9.5,10, 10.5, 11, 11.5,12,12.5,13, 13.5,14,14.5, or 15.
In certain embodiments, the modifying moiety is selected to render an insulin compound
conjugate having an aqueous solubility that exceeds 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 20 g/L, 50
g/L, 100 g/L, or even 150 g/L at a pH ranging from about 4 to about 8, preferably preferably a pH
ranging from about 5 to about 7.5, ideally pH of about 7.4.
The insulin compound conjugate can be more orally bioavalable in a mammal than a scientifically
acceptable control, such as a corresponding unconjugated insulin compound. In other
embodiments, the insulin compound conjugate is more orally bioavalable in a human than a
scientifically acceptable control, such as a corresponding unconjugated insulin compound. In
certain embodiments, absorption of the insulin compound conjugate, e.g., as measured by plasma
levels of the conjugate, is at least 1.5, 2, 2.5, 3, 3.5, or 4 times greater that the absorption of an
unconjugated insulin compound control.
It will be appreciated that while in some aspects of the invention the modifying moiety is selected
to render the insulin compound conjugate more soluble than a corresponding unconjugated
42
insulin compound, in other aspects the modifying moiety may also or alternatively be selected to
render the insulin compound conjugate equally or more hydrophilic than a corresponding
unconjugated insulin compound. Further, the modifying moiety may be selected to render the
insulin compound conjugate more amphiphilic than a corresponding unconjugated insulin
compound.
In some embodiments, the cation-insulin compound conjugate complex is equally as water
soluble or more water soluble than .(a) a corresponding uncomplexed insulin compound
conjugate, (b) a corresponding uncomplexed and unconjugated insulin compound, and/or (c) a
corresponding complexed but unconjugated insulin compound.
In a preferred embodiment, the water solubility of the insulin compound conjugate is decreased
by the addition of Zn'1"1'. hi some embodiments, the modifying moiety is selected to render the
insulin compound conjugate equally or more soluble than a corresponding unconjugated insulin
compound, and the water solubility of the insulin compound conjugate is decreased by the
addition of zinc. In other embodiments, the modifying moiety is selected to render the insulin
compound conjugate equally or more soluble than a corresponding unconjugated insulin
compound, the water solubility of the insulin compound conjugate is decreased by the addition of
zinc, and the water solubility of the cation complex is greater than the water solubility of insulin
compound. In another aspect, the insulin compound conjugate is a fatty acid acylated insulin
compound, the cation is zinc, and the water solubility of the insulin compound conjugate is
decreased by the addition of the zinc, hi still another embodiment, the insulin compound
conjugate is a fatty acid acylated insulin compound that is equally or more water soluble than a
corresponding unconjugated insulin compound, the cation is zinc, and the water solubility of the
insulin compound conjugate is decreased by the addition of the zinc.
In certain preferred embodiments, the lipophilicity of the insulin compound conjugate relative to
the corrsesponding parent insulin compound is 1 or less than 1. The relative lipophilicity of the
insulin compound conjugate as compared to corrsesponding parent insulin compound (krei) can,
for example, be determined as follows: krel = (tconjuga,e - t0)/(thuman - to), where relative
lipophilicity is measured on an LiChroSorb RP18 (Sum, 250 X 4 mm) high performance liquid
chromatography column by isocratic elution at 40° C. The following mixtures can be used as
eluents: 0.1M sodium phosphate buffer at pH 7.3 containing 10% acetonitrile, and 50%
acetonitrile in water. Void time (to) is identified by injecting 0.1 mM sodium nitrate. Retention
time for human insulin is adjusted to at least 2t0 by varying the ration between the mixtures of
43
(c)(i) and (c)(ii). Preferably, in these embodiments, the relative lipophilicity is about equal to 1
or is less than 1 or substantially less than 1. In a preferred embodiment, the insulin compound is
human insulin, and the relative lipophilicity is less than 1. Preferably the relative lipophilicity is
less than about 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.92, 0.91, or 0.90. Discussion of
techniques for determining solubility and/or lipophilicity of insulin and insulin conjugates are set
forth in the U.S. Patent 5,750,499 entitled "Acylated insulin" issued to Harelund et al., on 12-
May-98, the entire disclosure of which is incorporated herein by reference.
In one embodiment, the relative lipophilicity is as described above and the modifying moiety is a
carbon chain having 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15,16, 17 or 18 carbons, wherein the carbon
chain comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 oxy groups inserted therein. In another embodiment,
the relative lipophilicity is as described above and the modifying moiety is a carbon chain having
5, 6, 7, 8, 9 or 10 carbons, wherein the carbon chain comprises 2, 3 or 4 oxy groups inserted
therein. In a related embodiment, the relative lipophilicity is as described above and the
modifying moiety comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 polyalkalene glycol units. In another
related embodiment, relative lipophilicity is as described above and the modifying moiety
comprises 1, 2 or 3 polyethylene glycol units and 1,2 or 3 polypropylene glycol units.
7.4 Metal Cation Component and Characteristics of Complexes
The cation-insulin compound conjugate complexes include a metal cation. Suitable metal cations
for use as the cation component include any metal cation capable of complexing, aggregating, or
crystallizing with the insulin compound conjugate. It is preferred that the metal cation be
complexed to the insulin compound conjugate. Single or multiple cations can be used. The
cation is preferably not significantly oxidizing to the insulin compound conjugate, i.e., not
oxidizing to the extent that the complexes are rendered useless for their intended purpose.
In some embodiments, the metal cation is biocompatible. A metal cation is biocompatible if the
cation presents no unduly significant deleterious effects on the recipient's body, such as a
significant immunological reaction at the injection site. However, it will be appreciated that in
some circumstances, the risks of toxity and other deleterious effects may be outweighed by the
benefits of the cation-insulin compound conjugate composition, and therefore may be acceptable
under such circumstances.
The suitability of metal cations for stabilizing biologically active agents and the ratio of metal
performing a variety of tests for stability such as polyacrylamide gel electrophoresis, isoelectric
focusing, reverse phase chromatography, and HPLC analysis on particles of metal
cation-stabilized biologically active agents prior to and following particle size reduction and/or
encapsulation.
The metal cation component suitably includes one or more monovalent, divalent, or trivalent
metal cations, or combinations thereof. In a preferred embodiment, the metal cation is a Group II
or transition metal cation. Examples of suitable divalent cations include Zn44, Mn44, Ca44", Fe44",
Ni44, Cu44, Co44 and/or Mg44. Where a monovalent cation is included, it is preferably Na4, Li4, or
K+. The cation is preferably added as a salt, such as a chloride or acetate salt, most preferred are
ZnCl2 and ZnAc.
The molar ratio of insulin compound conjugate to cation is typically between about 1:1 and about
1:100, preferably between about 1:2 and about 1:12, and more preferably between about 1:2 and
about 1: 7 or about 1:2, 1:3, 1:4, 1:5, 1:6, or 1:7. In a particular embodiment, Zn44" is used as the
cation component, it is provided at a zinc cation component to insulin compound conjugate molar
ratio of about 1:1 and about 1:100, preferably between about 1:2' and about 1:12, and more
preferably between about 1:2 and about 1:7 or about 1:2,1:3,1:4,1:5,1:6, or 1:7.
The cation component is preferably greater than about 90% a single cation, such as Zn4"1".
Preferably, the cation is greater than about 95%, 99%, or 99.9% Zn44.
Preferably resistance of the complexed insulin compound conjugate to chymotrypsin degradation
is greater than the chymotrypsin degradation of the corresponding uncomplexed insulin
compound conjugate. Preferably resistance of the complexed insulin compound conjugate to
chymotrypsin degradation is greater than the chymotrypsin degradation of the corresponding
complexed but unconjugated insulin compound.
The complexed insulin compound conjugate can be more orally bioavalable in a mammal than a
scientifically acceptable control, such as a corresponding uncomplexed insulin compound
conjugate. In other embodiments, the complexed insulin compound conjugate is more orally
bioavalable in a human than a scientifically acceptable control, such as a corresponding
uncomplexed insulin compound conjugate, hi certain embodiments, absorption of the complexed
insulin compound conjugate, e.g., as measured by plasma levels of the conjugate, is at least 1.5, 2,
2.5,3, 3.5, or 4 times greater that the absorption of an uncomplexed insulin compound conjugate.
45
The complexed insulin compound conjugate can be more orally bioavalable in a mammal than a
scientifically acceptable control, such as a corresponding complexed but itnconjugated insulin
compound. In other embodiments, the complexed insulin compound conjugate is more orally
bioavalable in a human than a scientifically acceptable control, such as a corresponding
complexed but unconjugated insulin compound.. In certain embodiments, absorption of the
complexed insulin compound conjugate, e.g., as measured by plasma levels of the conjugate, is at
least 1.5, 2, 2.5, 3, 3.5, or 4 times greater that the absorption of an complexed but unconjugated
insulin compound..
7.5 Complexing agents
In some embodiments, the cation-insulin compound conjugatecompositions include one or more
complexing agents. Examples of cornplexing agents suitable for use in the present invention
include protamines, surfen, globin proteins, spermine, spermidine albumin, amino acids,
carboxyllic acids, polycationic polymer compounds, cationic polypeptides, am'onic polypeptides,
nucleotides, and antisense. See Brange, J., Galenics of Insulin compound, Springer-Verlag,
Berlin Heidelberg (1987), the entire disclosure of which is incorporated herein by reference. The
suitability of complexing agents for stabilizing the compositions can be determined by one of
ordinary skill in the art in the light of the present disclosure, hi some embodiments, the
cation-insulin compound conjugatecompositions specifically exclude or are substantially devoid
of a complexing agent.
A preferred complexing agent is protamine. In a solid form, the protamine will preferably be
present in about 3:1 to about 1:3 molar ratio of insulin compound to protamine, more preferably
about 2:1 to about 1:2 molar ratio, ideally about 1:1 molar ratio. In some embodiments, the
cation-insulin compound conjugatecompositions specifically exclude or are substantially devoid
of protamine.
Amino acids may also be used as complexing agents, e.g., glycine, alanine, valine, leucine,
isoleucine, serine threonine, phenyl alanine, proline, tryptophan, asparagine, glutamic acid, and
histidine, and oligopeptides, such as diglycine.
Carboxylic acids are also suitable for use as complexing agents; examples include acetic acid, and
hydroxycarboxylic acids, such as citric acid, 3-hydroxybutyric acid, and lactic acid.
7.6 Stabilizing agents
In some embodiments, the cation-insulin compound conjugate compositions include one or more
stabilizing agents. Preferred stabilizing agents include phenolic compounds and aromatic
compounds. Preferred phenolic compounds are phenol, m-cresol and m-paraben or mixtures
thereof. The stabilizing agent may be provided in any amount that improves stability of the
cation-insulin compound conjugate compositions relative to a scientifically acceptable control,
such as a corresponding cation-insulin compound conjugate composition in the absence of the
stabilizing agent.
7.7 Presentation of Complexes
The complexes may be provided as a dry solid, such as a substantially pure powder of
cation-insulin compound conjugate, or a powder including a cation-insulin compound conjugate
solid along with other pharmaceutically acceptable components. The complexes may also be
provided in a dissolved state in aqueous or organic medium, and/or as undissolved solids in such
mediums.
7.7.1 Solid Compositions
The cation-insulin compound conjugate complexe may be provided as as a solid. The solid may,
for example be in a dried state or in an undissolved state in an aqueous solution, organic solvent,
. emulsion, microemulsion, or oher non-dried form.
hi one embodiment, the cation-insulin compound conjugate complexe is provided as a pure
processed solid composition. In a pure processed solid compostion, the molar ratio of insulin
compound conjugate to cation is typically about 3:4 to about 3:0.5 (insulin compound
conjugate:cation), about 3:3.5 to about 3:1, or ideally about 3:1.
In a processed pure solid T-type compostion (with cation, insulin compound conjugate and
without protamine), the molar ratio of insulin compound conjugate to cation is typically about is
typically about 3:4 to about 3:0.5 (insulin compound conjugatexation), about 3:3.5 to about 3:1,
or ideally about 3:1. In a processed pure solid T-type protamine compostion (with cation, insulin
compound conjugate and protamine), the molar ratio of insulin compound conjugate to cation is
typically about 3:6 to about 3:0.5 (insulin compound conjugate:cation), about 3:5 to about 3:1, or
ideally about 3:2.
47
In a processed pure solid R-type (lente) compostion (with cation, insulin compound and
stabilizing compound (e.g., a phenolic compound), and without protamine), the molar ratio of
insulin compound conjugate to cation can typically range from about 3:4.5 to about 3:0.9,
preferably about 3:3.9 to about 3:2.4. In a processed pure solid R-type (ultralente) compostion
(with cation, insulin compound and stabilizing compound (e.g., a phenolic compound), and
without protamine), the molar ratio of insulin compound conjugate to cation can typically range
from about 3:12 to greater than about 3:4.5, preferably about 3:9 to about 3:4.8, more preferably
about 3:6 to about 3:5.4. hi a processed pure solid R-type protamine compostion (with cation,
insulin compound and stabilizing compound (e.g., a phenolic compound), and protamine), the
molar ratio of insulin compound conjugate to cation can typically range from about 3:12 to about
3:3, preferably about 3:9 to about 3:4.5, more preferably about 3:6.9 to about 3:5.4.
For a monovalent cation, such as Na+, the solid would be expected to have an insulin compound
conjugate to cation ratio of about 3:6 to about 3:3.
Solid compositions of the invention may, for example, include compositions, such as powders,
including insulin compound conjugates and/or cation-insulin compound conjugate complexes of
the invention. Preferably the solid compositions are provided at a pharmaceutically acceptable
level of purity, i.e., free of contaminants which would unacceptably diminish the suitability of the
compositions for use in humans.
In some embodiments, compositions are provided in which the cation-insulin compound
conjugate component is greater than about 90% crystalline, preferably greater than about 95%
crystalline, more preferably greater than about 99% crystalline. In other embodiments,
compositions are provided in which the cation-insulin compound conjugate component is greater
than about 90% amorphous solids, preferably greater than about 95% amorphous solids, more
preferably greater than about 99% amorphous solids.
In still other embodiments, compositions are provided in which the cation-insulin compound
conjugate component is present in a mixture of amorphous solids and crystalline solids. For
example, the ratio of amorphous solid to crystalline solid maybe from about 1:10 to about 10:1,
or about 1:9 to about 9:1, or about 1:8 to about 8:1, or about 1:7 to about 7:1, or about 1:6 to
about 6:1, or about 1:5 to about 5:1, or about 1:4 to about 4:1, or about 1:3 to about 3:1, or about
1:2 to about 2:1, or about 1:1.
Furthermore, compositions can be provided using mixtures of cation-insulin compound solids
having different insulin compounds, such as a solid including native insulin compound with a
solid including insulin compound conjugates, or solids including one insulin compound conjugate
with a solid including a different insulin compound conjugate.
Moreover, the solid type and insulin compound/insulin compound conjugate component may all
vary. For example, compositions can be provided which include Zn-insulin compound crystals
using native insulin compound and amorphous insulin compound conjugates, or compositions can
be provided which include amorphous Zn-insulin compound solids using native insulin
compound and crystalline Zn-insulin compound conjugates. Such mixtures may be used to
achieve variations in physical characteristics, such as dissolution profile and/or variations in
pharmacokinetic profile.
The average particle size of the solids are preferably in the range of about 0.1 to about 100
microns, more preferably 1-50 microns, still more preferably 1-25 microns, ideally 1-15 microns.
Small particle sizes may be obtained by microcrystallization conditions, spray drying, milling,
vacuum drying, freeze drying and the like.
In one embodiment the composition, when dried, contains greater than about 96% w/w insulin
compound conjugate and from about 0.05, 0.1, 0.15, or 0.2 to about 4% w/w zinc. In another
embodiment the composition, when dried, contains greater than about 91% w/w insulin
compound conjugate, from about 0.05, 0.1, 0.15, or 0.2 to about 4% w/w zinc, and from about 0.2
to about 5% w/w phenol. In yet another embodiment the composition, when dried, contains
greater than about 82% w/w insulin compound conjugate, from about 0.05, 0.1, 0.15, or 0.2 to
about 4% w/w zinc, from about 0.2 to about 14 % w/w protamine. In yet another embodiment the
composition, when dried, contains greater than about 71% w/w insulin compound conjugate,
from about 0.05, 0.1, 0.15, or 0.2 to about 4% w/w zinc, from about 0.2 to about 14 % w/w
protamine, and from about 0.2 to about 15% w/w phenol.
hi another embodiment the composition, when dried, includes from about 0.1 to about 2% w/w
Zn**, and from about 0.08 to about 1% w/w phenol, preferably from about 0.5 to about 1.3%
w/w Zn"14, andfrom about 0.1 to about 0.7% w/w phenol, more preferably from greater than or
equal to 1 to about 3.5% w/w Zn4*, and from about 0.1 to about 3% w/w phenol, and still more
preferably from greater than or equal to 1.3 to about 2.2% w/w Zn"1"1", and from about 0.4 to about
2% w/w phenol.
49
The complexes can be provided in a lente-type preparation. For example, in a preferred dried
lente-type preparation, Zn is provided in an amount ranging from about 0.1 to about 2% w/w and
phenol is present in an amount ranging from about 0.08 to about 1% w/w, with the remaining %
w/w being insulin compound conjugate. Ideally, for a dried lente-type preparation, Zn is
provided in an amount ranging from about 0.5 to about 1.3% w/w and phenol is present in an
amount ranging from about 0.1 to about 0.7% w/w, with the remaining % w/w being insulin
compound conjugate.
The complexes can be provided in an ultralente-type preparation. For example, in a preferred
dried ultralente-type preparation, Zn is provided in an amount ranging from greater than or equal
to 1 to about 3.5% w/w, and phenol is present in an amount ranging from about 0.1 to about 3%
w/w, with the remaining % w/w being insulin compound conjugate. Ideally, for a dried
ultralente-type preparation, Zn is provided in an amount ranging from greater than or equal to 1.3
to about 2.2% w/w, and phenol is present in an amount ranging from about 0.4 to about 2% w/w,
with the remaining % w/w being insulin compound conjugate.
7.7.2 Liquid Compositions
The cation-insulin compound conjugate complexes may be provided as components undissolved
components of a liquid. For example, the liquid may be an aqueous solution including a
cation-insulin compound conjugate as a precipitate, or the cation-insulin compound conjugate
may be provided as a component of a suspension, emulsion or microemulsion. The liquid may
also include dissolved components or complexes, along with the undissolved components.
7.7.3 Mixtures and Co-crystals
The compositions of the invention may, for example, include complex mixtures, solid mixtures,
hybrid complexes and co-crystals.
Thus, for example, the invention provides compositions which include two or more insulin
compound conjugates and/or unconjugated insulin compounds. Further, where the compositions
include solids, the solids may have different forms. Thus, for example, on solid may be
crystalline and another solid may be an amorphous solid. As noted elsewhere, the solids may be
provided in a dried form or may be provided as solid components of a liquid mixture. In a
preferred embodiment, the mixture of the invention includes two or more different insulin
compound conjugates, and the different insulin compound conjugates have different solubilities.
In one embodiment, one of the complexes comprises a lipophilic insulin compound conjugate and
the other comprises a hydrophilic insulin compound conjugate. In still another embodiment, the
complexes may include different insulin compound conjugates, where one ore more of the
complexes has a circulation half-life of from about 1 to about 4 hours, and one or more the
complexes has a circulation half-life that is significantly greater than the circulation half-life of
the first complex. In a related embodiment, one of the complexes has a rapid-acting profile and
another of the complexes has a medium-to-long acting profile. Furthemore, one of the complexes
may have profile suitable for basal insulin compound control while another has a profile suitable
for post-prandial glucose control. Preferred mixtures are mixtures of HIM2 and insulin, mixtures
of HIM2 and IN105, mixtures of IN105 and insulin compound, mixtures of IN105 and fatty acid
acylated insulin, mixtures of HIM2 and fatty acid acylated insulin. Suitable fatty acid acylated
insulins are described in the following U.S. patents, the entire disclosures of which are
incoiporated herein by reference: U.S. Patent 6,531,448, entitled "Insoluble compositions for
controlling blood glucose," issued ll-Mar-03; U.S. Patent RE37,971, entitled "Selective
acylation of epsilon-amino groups," issued 28-Jan-03; U.S. Patent 6,465,426, entitled "Insoluble
insulin compositions," issued 15-Oct-02; U.S. Patent 6,444,641, entitled "Fatty acid-acylated
insulin analogs." issued 03-Sep-02; U.S. Patent 6,335,316, entitled "Method for administering
acylated insulin," issued Ol-Jan-02; U.S. Patent 6,268,335, entitled "Insoluble insulin
compositions," issued 31-Jul-Ol; U.S. Patent 6,051,551, entitled "Method for administering
acylated insulin," issued 18-Apr-OO; U.S. Patent 5,922,675, entitled "Acylated Insulin Analogs,"
issued 13-M-99; U.S. Patent 5,700,904, entitled "Preparation of an acylated protein powder,"
issued 23-Dec-97; U.S. Patent 5,693,609, entitled "Acylated insulin analogs Granted," issued 02-
Dec97; U.S. Patent 5,646,242, entitled "Selective acylation of epsilon-amino groups," issue 08-
Jul-97; U.S. Patent 5,631,347, entitled "Reducing gelation of a fatty acid-acylated protein," issued
20-May-97; U.S. Patent.6,451,974, entitled "Method of acylating peptides and novel acylating
agents," issued 17-Sep-02; U.S. Patent 6,011,007, entitled "Acylated insulin," issued 04-Jan-OO;
U.S. Patent 5,750,497, entitled "Acylated insulin Granted: 12-May-98; U.S. Patent 5,905,140,
entitled "Selective acylation method," issued May 18, 1999; U.S. Patent 6,620,780, entitled
"Insulin derivatives," issued Sep. 16, 2003; U.S. Patent 6,251,856, entitled "Insulin derivatives,"
issued Jun. 26, 2001; U.S. Patent 6,211,144, entitled "Stable concentrated insulin preparations for
pulmonary delivery," issued Apr. 3, 2001; U.S. Patent 6,310,038, entitled "Pulmonary insulin
crystals," issued Oct. 30, 2001; and U.S. Patent 6,174,856, entitled "Stabilized insulin
compositions," issued Jan. 16, 2001. Especially preferred mono-fatty acid acylated insulins
having 12,13,14, 15, or 16-carbon fatty acids covalently bound to Lys(B29) of human insulin.
51
In one embodiment, the invention provides a co-crystal having two different insulin compounds
and/or insulin compound conjugates. Preferably the co-crystal exhibits one or more of the
following characteristics: substantially homogenous dissolution, a single in vivo dissolution
curve, and/or a single peak pharmacodynamic profile. Preferred co-crystals are co-crystals of
HIM2 and insulin, co-crystals of HIM2 and IN105, co-crystals of IN105 and insulin compound.
In one embodiment, the co-crystal includes human insulin, and co-crystallization with human
insulin reduces the solubility of the crystal relative to the solubility of a corresponding crystal of
the insulin compound conjugate. In another embodiment, the co-crystal includes human insulin,
and co-crystallization with human insulin decreases the solubility of the crystal relative to the
solubility of a corresponding crystal of the insulin compound conjugate.
In another embodiment, the co-crystal includes a rapid acting, rapid clearing, and/or highly potent
insulin compound conjugate, and a long-acting, slow clearing, and/or poorly potent insulin
compound conjugate. Preferably the co-crystal has a PK/PD profile suitable for post-prandial
glucose control or for overnight basal insulin compound control.
In another embodiment, the invention provides a mixture or co-crystal in which an insulin
compound conjugate is included with human insulin or lyspro insulin. The mixtures of the
invention may include two different insulin compound conjugates. The mixtures may include an
insulin compound conjugate and an unconjugated insulin compound. The mixtures may include
different insulin compound conjugates with different insulin compounds.
Further, the invention provides complexes having two different insulin compound conjugates
and/or an insulin compound conjugate and an unconjugated insulin compound. The invention
provides hybrid co-crystals of two, three or more different insulin compound conjugates. The
invention provides a complex having an insulin compound conjugate with an unconjugated
insulin compound. The invention provides a co-crustal with two or more different hydrophilic
insulin compound conjugates; two or more different hydrophobic insulin compound conjugates;
two or more different amphiphilic insulin compound conjugates; a hydrophilic insulin compound
conjugate and a lipophilic insulin compound conjugate; a. hydrophilic insulin compound
conjugate and an unconjugated insulin compound; HIM2 together with an unconjugated insulin
compound; IN105 together with an unconjugated insulin compound; HIM2 together with IN105;
HIM2 together with insulin compound and IN105; and other combinations of the foregiong
elements. As mentioned elsewhere, the complexes may be provided as dried solids, as dissolved
complexes in solution and/or as undissolved complexes in solution. Various combinations may,
for example, be employed to provide a complex or co-crystal having an extended profile.
7.8 Solubility of Complexes of the Invention
Preferably the aqueous solubility of the cation-insulin compound conjugate complex at a pH of
about 7.4 is from about 1/15, 1/14, 1/13, 1/12, 1/11, 1/10, 1/9, 1/8, 1/7, 1/6, 1/5 up to about 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, or <10 times the aqueous solubility of the uncomplexed insulin compound
conjugate. Any combination of the foregoing upper and lower limits is within the scope of the
invention. However, a preferred range is from about 1/15 to <5, more preferred is about 1/10 to
about 2, ideal is about 1/10 to <0. In a particularly surprising aspect of the invention, the aqueous
solubility of the cation-insulin compound conjugate in solution at a pH of about 7.4 is often
substantially less than the aqueous solubility of the insulin compound conjugate in solution at a
pH of about 7.4. However, it will be appreciated that in certain embodiments, the aqueous
solubility of the cation-insulin compound conjugate in solution at a pH of about 7.4 may be the
same as, greater than, or substantially greater than, the aqueous solubility of the insulin
compound conjugate in solution at a pH of about 7.4.
In one surprising embodiment, the aqueous solubility of the cation-insulin compound conjugate
complex at a pH of about 7.4 is substantially less than the solubility of the corresponding
uncomplexed insulin compound conjugate in solution at a pH of about 7.4, and the cation-insulin
compound conjugate complex remains soluble at greater than about 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120 or 130 g/L in aqueous
solution across a pH range beginning at about 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.1, or 6.9 and ending at about 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, or
8.9. In yet another embodiment, the aqueous solubility of the cation-insulin compound conjugate
complex at a pH of about 7.4 is substantially less than the solubility of the corresponding insulin
compound conjugate in solution at a pH of about 7.4, and the cation-insulin compound conjugate
complex remains soluble at greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or 130 g/L in aqueous solution across a
pH range from about 5.8 to about 8.5, preferably across a pH range from about 6.5 to about 8,
more preferably across a pH range from about 6.9 to about 7.8. [QUESTION: Is the g/L
solubility measured using the weight of the complex or the weight of the insulin compound
conjugate?]
53
Preferably the insulin compound conjugates of the invention are selected to produce crystals in
aqueous solution at a pH which is equal to pi +/-anout 2.5, where the concentration of insulin
compound conjugate is from about 0.5 mg/ml to about 50 mg/ml, preferably about 5 mg/ml to
about 30 mg/ml, more preferably about 15 mg/ml to about 30 mg/ml, and the crystal formulation
begins to occur at about 3, 4 or 5% w/w/ cation to insulin compound conjugate, where the cation
is preferable Z**. Preferably crystals are present for a monoconjugate without protamine in an
aqueous solution at a pH ranging from about 4, 4.1, 4.2, 4.3 or 4.4 to about 5.2., 5.3, 5.4, 5.5, 5.6,
5.7 or 5.8, preferably at pH of about 4 to <6.5, preferably about 4 to <5.8, preferably about 4.2 to
about 5.5, more preferably about 4.4 to about 5.2. Preferably crystals are present for a
diconjugate without protamine at pH of about 3.5 to <5.8, preferably about 3.8 to about 5.5, more
preferably about 4.0 to about 5.2. Preferably crystals are present for a triconjugate without
protamine at pH of about 3 to <5.5, preferably about 3.3 to about 5, more preferably about 3.8 to
about 4.8.
7.8.1 R-type Complexes
Preferably the aqueous solubility of the R type Zn complex of the insulin compound conjugate at
a pH of about 7.4 has a range of about 10 to about 150 g/L, more preferably about 20 to about
130 g/L, more preferably about 30 to about 110 g/L, more preferably about 35 to about 60 g/L.
Preferably the aqueous solubility of the R type Zn complex of the insulin compound conjugate
with protamine at a pH of about 7.4 has a range of about 10 to about 110 g/L, more preferably
about 20 to about 85 g/L, more preferably about 30 to about 70 g/L.
7.8.2 T-type Complexes
Preferably the aqueous solubility of the T-type Zn complex of the insulin compound conjugate at
a pH of about 7.4 has a range of about 30 to about 175 g/L, more preferably about 50 to about
160 g/L, more preferably about 70 to about 150 g/L.
Preferably the aqueous solubility of the T-type Zn complex of the insulin compound conjugate
with protamine at a pH of about 7.4 has a range of about 10 to about 150 g/L, more preferably
about 20 to about 130 g/L, more preferably about 30 to about 110 g/L, more preferably about 35
7.8.3 NPH-Type Complexes
Preferably the aqueous solubility of the NPH-type complex, of the insulin compound conjugate at
a pH of about 7.4 has a range of about about 1 to about 150 g/L, more preferably about 5 to about
120 g/L, still more preferably about 10 to about 90 g/L.
7.9 Pharmaceutical Properties
Complexation of the insulin compound conjugate with cation generally results in improved
pharmaceutical properties of the insulin compound conjugate, relative to a scientifically
acceptable control, such as a corresponding uncomplexed insulin compound conjugate.
In some cases, the complexed insulin compound conjugate will exhibit an extended or otherwise
altered pK profile relative to a scientifically acceptable control, such as a corresponding
uncomplexed insulin compound conjugate. In certain cases, the pK. profile will exhibit a
lispro-like profile. pK profile can be assessed using standard in vivo experiments, e.g., in mice,
rats, dogs, or humans. Assays described herein for assessing the attributes of cation-insulin
compound conjugate complexes are an aspect of the invention.
The complexes may exhibit improved chemical stability. Various attributes of stability can be
assessed by exposing the complex to various assay conditions such as the presence of plasma, the
presence of proteases, the presence of liver homogenate, the presence of acidic conditions, and
the presence of basic conditions. Stability is improved relative to uncomplexed insulin compound
conjugate when stability of the complexed insulin compound conjugate in any one or more of
these assay conditions is greater than stability of the uncomplexed insulin compound conjugate in
the same conditions. A preferred assay for determining stability in an acidic environment
involves exposing the complexed insulin compound conjugate to a solution having a pH of 2 for
at least 2 hours, where decreased degradation of the complexed insulin compound conjugate
relative to a scientifically acceptable control, such as a corresponding uncomplexed insulin
compound conjugate, is indicative of improved stability. In vivo assays can also be used to test
stability. For example, stability of the complexed insulin compound conjugate can be tested by
exposure to the gastrointestinal tract of a subject and comparison with an appropriate control.
7.10 Method of Making
The invention also provides a method of making cation-insulin compound conjugate
compositions described herein. The method generally involves contacting one or more insulin
compound conjugates, as described herein, with one or more cations, as described herein, to form
a solid.
For a divalent cation, such as Zn4"4", the molar ratio of insulin compound conjugate to cation used
to make the composition in an aqueous solution with an insulin compound concentration ranging
from about 2 mg/ml to about 50 mg/ml can typically range from about 1:15 (insulin compound
conjugatercation) to about 1:0.4, preferably about 1:9 to about 1:2.
To make T-type solid (with cation and insulin compound conjugate and without protamine) in the
aqueous solution conditions described above, the molar ratio of insulin compound conjugate to
cation is preferably about 1:1.5 to 1:3, ideally about 1:2. To make R-type solid (with cation,
insulin compound and stabilizing compound (e.g., a phenolic compound), and without protamine)
in the aqueous solution conditions described above, the molar ratio of insulin compound
conjugate to cation is preferably about 1:4 to 1:9, preferably about 1:7 to about 1:9 ideally about
1:8.
To make T-type protamine solid (with cation and insulin compound conjugate and protamine) in
the aqueous solution conditions described above, the molar ratio of insulin compound conjugate
to cation is preferably about 1:1.5 to 1:9, ideally about 1:2. To make R-type protamine solid
(with cation, insulin compound and stabilizing compound (e.g., a phenolic compound), and
protamine) in the aqueous solution conditions described above, the molar ratio of insulin
compound conjugate to cation is preferably about 1:2 to 1:15, preferably about 1:7 to about 1:9
ideally about 1:8.
The insulin compound conjugate is preferably added to the buffer in an amount which is
calculated to achieve a concentration in the range of from greater than 2 to about 100 g/L,
preferably from about 3, 4, 5, 6, 7, 8, 9 or 10 to about 40 g/L, more preferably from about 10 to
about 30 g/L.
Where the cation is divalent (e.g., ZnH~,Ca'H'), it is preferably added in an amount which
calculated to achieve a concentration in the range of from about 0.04 to about 10 g/L, preferably
from about 0.1 to about 5 g/L, more preferably from about 0.2 to about 4 g/L. For T-type crystals
or T-type protamine crystals, the cation concentration is preferably in the range of from about
0.04 to about 1 g/L, more preferably about 0.1 to about 0.3 g/L. For R-type crystals or R-type
protamine crystals, the cation concentration is preferably in the range of from about 1 to about 5
g/L, more preferably about 1.5 to about 4 g/L.
56
Where the cation is monovalent, it is preferably added in an amount which calculated to achieve a
:oncentration in the range of from about 0.08 to about 40 g/L, preferably from about 0.4 to about
20 g/L, more preferably from about 0.8 to about 16 g/L.
The method may further include combining a stablizing agent with the cation and insulin
compound conjugate. Preferred stablizing agents are described above. When used, the
stabilizing agent is added in an amount sufficient to provide a greater degree of solid formation
than is achieved using the same reagents and reaction conditions in the absence of the stabilizing
agent. Where the stabilizing agent is a phenolic compound (e.g., phenol, m-cresol, m-paraben),
can be added in an amount ranging from about 10 to about 50% w/w, more preferably from about
20 to about 40% w/w, still more preferably from about 25 to about 35% w/w. In a more preferred
embodiment, the stabilizing agent is a phenolic compound (e.g., phenol, m-cresol, m-paraben),
can be added in an amount ranging from about 0.01 to about 10% w/w, more preferably 0.01 to
about 5% w/w, still more preferably 0.01 to about 1% w/w. Thus, in one embodiment, the
method involves combining insulin compound conjugate, a cation and a stabilizing agent in an
aqueous solution to yield the cation-insulin compound conjugate composition, where the
combination may yield solublized complexes and/or crystalline or non-crystalline solids.
The method may further include the use of a complexing agent, such as protamine, which is
combined with the cation and insulin compound conjugate, and optionally also includes a
stabilizing agent.
To prepare a solid in an aqueous solution having a pH in the range of about 5 to about 8,
protamine is preferably provided in an amount relative to insulin compound conjugate of about 4
to about 45% w/w (protamine/insulin compound), preferably about 8 to about 25% w/w, more
preferably about 9 to about 20% w/w, ideally about 10 to about 12% w/w. For T-type solids, a
preferred pH range is from about 5 to about 6, more preferably about 5 to about 5.5, still more
preferably about 5.1 to about 5.3, ideally about 5.2. For R-type solids, a preferred pH range is
from about 6 to about 7, more preferably about 6.2 to about 6.8, still more preferably about 6.4 to
about 6.6, ideally about 6.5.
The inventors have surprisingly discovered that T-type complexes can be converted to protamine
T-type complexes in the absence of a stabilizing agent, such as phenol. The T-type complex is
made by complexing Zn with the insulin compound molecule in aqueous solution in the absence
of phenol. Protamine is then added to convert the T-type complex into a protamine T-type
complex. Amounts and pH ranges are as described above.
Thus, in one embodiment, the method involves combining insulin compound conjugate, a cation
and a complexing agent in an aqueous solution to yield the cation-insulin compound conjugate
composition, where the combination may yield solublized complexes and/or crystalline or
amorphous solids. In another embodiment, the method involves combining insulin compound
conjugate, a cation, a complexing agent, and a stabilizing agent in an aqueous solution to yield
the cation-insulin compound conjugate composition, where the combination may yield solublized
complexes and/or crystalline or amorphous solids.
In some embodiments, the compositions can include preservatives. Examples of suitable
preservatives include benzyl alcohol, p-hydroxybenzoic acid esters, glycerol. Stabilizing agents,
such as phenol, m-cresol, and m-paraben, can also be used as preservatives. Glycerol and phenol
are suitably added together to enhance antimicrobial effectiveness.
Other components useful in preparing the solids include isotonic agents, such as NaCl, glycerol,
and monosaccharides.
The cation insulin compound conjugate solids can typically be formed relatively quickly. For
example, solid formation is typically complete within three days, often within 24 hours. It may
be desirable in some instances to slow the reaction down in order to improve crystal formation.
In one embodiment of the invention, the solids are formed at room temperature (25° C) without
requiring temperature reduction for inducing precipitation of solids. For example, room
temperature is effective for R-type and T-type crystals. The temperature for solid formation is
preferably about 0 to about 40° C, preferably about 17 to about 30° C, and more preferably about
22 to about 27° C, ideally about 25° C.
In one embodiment, the method includes combining in an aqueous solution an insulin compound
conjugate and a metal cation to provide a crystalline or amorphous solid. The aqueous solution
containing the insulin compound conjugate to which the cation will be added is preferably a
buffered solution having a pH in the range of pi of the insulin compound conjugate +/- about 1.5,
preferably pi +/- about 1, more preferably pi +/- about 0.75. These ranges also apply to T-type,
R-type and protamine complexes. However, for neutral protamine complexes (NPH-type), the
preferred pH is about 7 to about 8.5, more preferably about 7.5 to about 8. Once the metal cation
58
is added, the pH may change slightly, and the pH may be adjusted to target the pH ranges
described above. With phenolic compounds, there may be a minor pH change, and an acid or
base can be used to adjust to the preferred ranges.
pi values for insulin compound conjugates typically require a pH of less than about 7, preferably
less than about 6, more preferably less than about 5.5. Human insulin monoconjugates with
neutral modifying moieties typically have a pi range of about 4.75 +I-.25. For human insulin
diconjugates, the pi range is typically 4.25 +/-.2S. For human insulin triconjugates, the pi range
is typically 3.5 +/-.2S.
Examples of suitable buffer systems include ammonium acetate buffer, sodium phosphate buffer,
tris buffer, mixture of sodium phosphate and ammonium acetate, sodium acetate buffer, mixture
of sodium acetate and ammonium acetate, and citric acid buffer, and any of the foregoing buffer
systems [A] also containing ethanol and/or acetonitrile [B] (e.g., at percent ratio of A:B of about
1:1 to about 10:1). It is a surprising aspect of the invention that the cation-insulin compound
conjugate solid can be formed in an aqueous mixture containing an organic solvent, such as
ethanol or acetonitrile.
One unique feature of the invention is that in addition to providing useful cation-insulin
compound conjugate complexes, the invention also provides a method of separating
cation-insulin compound conjugates from unconjugated insulin compound in the manufacturing
process. In this process, the cation-insulin compound conjugates can be precipitated out of
solution and the solubilized unconjugated insulin compound can be removed by filtration, for
example. This feature eliminates 2 steps in the manufacture of insulin compound conjugates: the
concentration step and the lyophilization step.
Processed pure solid composition may be formed using standard techniques, such as
centrifugation and/or filtration, followed by washing (e.g., with ethanol/water), and lyophilization
or vacuum drying. Multiple washings may be used to adjust phenol and/or cation content.
7.11 Formulation
The complexes may be formulated for administration in a pharmaceutical carrier in accordance
with known techniques. See, e.g., Alfonso R. Gennaro, Remington: The Science and Practice of
Pharmacy, Lippincott Williams & Wilkins Publishers (June 2003), and Howard C. Ansel,
Pharmaceutical Dosage Forms and Drug Delivery Systems, Lippincott Williams & Wilkins
59
Publishers, 7th ed. (October 1999), the entire disclosures of which are incorporated herein by
reference for their teachings concerning the selection, making and using of pharmaceutical
dosage forms.
The complexes, typically in the form of an amorphous or crystalline solid, can be combined with
a pharmaceutically acceptable carrier. The carrier must be acceptable in the sense of being
compatible with any other ingredients in the pharmaceutical composition and should not be
unduly deleterious to the subject, relative to the benefit provided by the active ingredient(s). The
carrier may be a solid or a liquid, or both. It is preferably formulated as a unit-dose formulation,
for example, a tablet. The unit dosage form may, for example, contain from about 0.01 or 0.5%
to about 95% or 99% by weight of the cation-insulin compound complex. The pharmaceutical
compositions may be prepared by any of the well known techniques of pharmacy including, but
not limited to, admixing the components, optionally including one or more accessory ingredients.
Examples of suitable pharmaceutical compositions include those made for oral, rectal, inhalation
(e.g., via an aerosol) buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous,
intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, inracerebral, intraarterial,
or intravenous), topical, mucosal surfaces (including airway surfaces), nasal surfaces, and
transdermal administration. The most suitable route in any given case will depend on the nature
and severity of the condition being treated and on the nature of the particular cation-insulin
compound complexes being used. Preferred oral compositions are compositions prepared for
ingestion by the subject. Ideally, the oral compositions are prepared to survive or substantially
survive passage through the stomach and to completely or substantially completely dissolve in
the intestine for delivery of the active ingredient. Examples of suitable transdermal systems
include ultrasonic, iontophoretic, and patch delivery systems.
In one aspect, the invention provides fatty acid compositions comprising one or more saturated or
unsaturated C4, Cs, Cs, C7, C8, C9 or do fatty acids and/or salts of such fatty acids. Preferred fatty
acids are caprylic, capric, myristic and lauric. Preferred fatty acid salts are sodium salts of
caprylic, capric, myristic and lauric acid.
Preferred fatty acid compositions include a single fatty acid or a single fatty acid salt and do not
include substantial amounts of other fatty acids or fatty acid salts, hi one aspect of the invention,
the fatty acid content of the composition is greater than about 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, 99.5, 99.6, 99.7, 99.8, or 99.9% w/w a single fatty acid. In another embodiment, the fatty acid
content of the composition is within a range having as a lower limit of about 0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,
2.7, 2.8, 2.9, or 3.0 % w/w, and having as an upper limit of about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,
3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,
5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0,
8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1,
10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8,
11.9, or 12.0 % w/w. In yet another embodiment, the fatty acid content of the composition is
within a range having as a lower limit about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 % w/w, and
having as an upper limit about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4,
4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,
8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9,10.0,10.1,10.2, 10.3,10.4, 10.5, 10.6, 10.7, 10.8,
10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, or 12.0 % w/w, and the fatty acid
content of the composition is greater than about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6,
99.7, 99.8, or 99.9% w/w a single fatty acid.
Active components of these formulations may include conjugated or unconjugated, complexed or
uncomplexed proteins and/or peptides. Preferred proteins and/or peptides are those described
herein. Preferred conjugates are those described herein. Preferred complexes are those described
herein. Preferred oral compositions are compositions prepared for ingestion by the subject.
Ideally, the oral compositions are prepared to survive or substantially survive passage through the
stomach and to completely or substantially completely dissolve in the intestine for delivery of the
active ingredient. The formulation may in some cases include an enteric coating, and in some
cases, the formulation will specifically exclude an enteric coating. The composition is preferably
provided as a tablet, powder, hard gelatin capsule, or soft gelatin capsule, though other forms
described herein are suitable as well.
The fatty acid compositions of the invention may include fatty acid acylated insulins. Examples
of suitable fatty acid acylated insulins are described in the following U.S. patents, the entire
disclosures of which are incorporated herein by reference: U.S. Patent 6,531,448, entitled
"Insoluble compositions for controlling blood glucose," issued 1 l-Mar-03; U.S. Patent RES7,971,
entitled "Selective acylation of epsilon-amino groups," issued 28-Jan-03; U.S. Patent 6,465,426,
entitled "Insoluble insulin compositions," issued 15-Oct-02; U.S. Patent 6,444,641, entitled
Fatty acid-acylated insulin analogs." issued 03-Sep-02; U.S. Patent 6,335,316, entitled "Method
for administering acylated insulin," issued Ol-Jan-02; U.S. Patent 6,268,335, entitled "Insoluble
insulin compositions," issued 31-Jul-Ol; U.S. Patent 6,051,551, entitled "Method for
administering acylated insulin," issued 18-Apr-OO; U.S. Patent 5,922,675, entitled "Acylated
Insulin Analogs," issued 13-M-99; U.S. Patent 5,700,904, entitled "Preparation of an acylated
protein powder," issued 23-Dec-97; U.S. Patent 5,693,609, entitled "Acylated insulin analogs
Granted," issued 02-Dec97; U.S. Patent 5,646,242, entitled "Selective acylation of epsilon-amino
groups," issue 08-Jul-97; U.S. Patent 5,631,347, entitled "Reducing gelation of a fatty acidacylated
protein," issued 20-May-97; U.S. Patent 6,451,974, entitled "Method of acylating
peptides and novel acylating agents," issued 17-Sep-02; U.S. Patent 6,011,007, entitled "Acylated
insulin," issued 04-Jan-OO; U.S. Patent 5,750,497, entitled "Acylated insulin Granted: 12-May-98;
U.S. Patent 5,905,140, entitled "Selective acylation method," issued May 18, 1999; U.S. Patent
6,620,780, entitled "Insulin derivatives," issued Sep. 16, 2003; U.S. Patent 6,251,856, entitled
"Insulin derivatives," issued Jun. 26, 2001; U.S. Patent 6,211,144, entitled "Stable concentrated
insulin preparations for pulmonary delivery," issued Apr. 3, 2001; U.S. Patent 6,310,038, entitled
"Pulmonary insulin crystals," issued Oct. 30, 2001; and U.S. Patent 6,174,856, entitled
"Stabilized insulin compositions," issued Jan. 16, 2001. Especially preferred are mono-fatty acid
acylated insulins having 12, 13, 14, 15, or 16-carbon fatty acids covalently bound to Lys(B29) of
human insulin.
Pharmaceutical compositions suitable for oral administration may be presented in discrete units,
such as capsules, cachets, lozenges, or tables, each containing a predetermined amount of the
mixture of insulin compound conjugates; as a powder or granules; as a solution or a suspension in
an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such
formulations may be prepared by any suitable method of pharmacy which includes the step of
bringing into association the mixture of insulin compound conjugates and a suitable carrier
(which may contain one or more accessory ingredients as noted above). Formulations may
include suspensions of solids, complexed cation-insulin compound conjugates, uncomplexed
active ingredient (e.g., native insulin compound, insulin compound conjugates), and mixtures of
the foregoing.
In general, the pharmaceutical compositions of the invention are prepared by uniformly and
intimately admixing the complexes with a liquid or solid carrier, or both, and then, if necessary,
shaping the resulting mixture. For example, a tablet may be prepared by compressing or molding
a powder or granules containing the mixture of insulin compound conjugates, optionally with one
or more accessory ingredients. Compressed tablets may be prepared by compressing, in a
suitable machine, the mixture in a free-flowing form, such as a powder or granules optionally
mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded
tablets may be made by molding, in a suitable machine, the powdered composition moistened
with an inert liquid binder.
Pharmaceutical compositions suitable for buccal (sub-lingual) administration include lozenges
comprising the mixture of insulin compound conjugates in a flavoured base, usually sucrose and
acacia or tragacanth; and pastilles comprising the mixture of insulin compound conjugates in an
inert base such as gelatin and glycerin or sucrose and acacia. Examples of suitable formulations
can be found in U.S. Patent Publication Nos. 20030229022 ("Pharmaceutical formulation");
20030236192 ("Method of modifying the release profile of sustained release compositions");
20030096011 ("Method of producing submicron particles of a labile agent and use thereof);
20020037309 ("Process for the preparation of polymer-based sustained release compositions");
20030118660 ("Residual solvent extraction method and microparticles produced thereby"); as
well as U.S. Patents 6,180,141 ("Composite gel microparticles as active principle carriers");
6,737,045 ("Methods and compositions for the pulmonary delivery insulin compound");
6,730,334 ("Multi-arm block copolymers as drug delivery vehicles"); 6,685,967 ("Methods and
compositions for pulmonary delivery of insulin compound"); 6,630,169 ("Particulate delivery
systems and methods of use"); 6,589,560 ("Stable glassy state powder formulations; 6,592,904
("Dispersible macromolecule compositions and methods for their preparation and use");
6,582,728 ("Spray drying of macromolecules to produce inhaleable dry powders"); 6,565,885
("Methods of spray drying pharmaceutical compositions"); 6,546,929 ("Dry powder dispersing
apparatus and methods for their use"); 6,543,448 ("Apparatus and methods for dispersing dry
powder medicaments"); 6,518,239 ("Dry powder compositions having improved dispersivity");
6,514,496 ("Dispersible antibody compositions and methods for their preparation and use");
6,509,006 ("Devices compositions and methods for the pulmonary delivery of aerosolized
medicaments"); 6,433,040 ("Stabilized bioactive preparations and methods of use"); 6,423,344
("Dispersible macromolecule compositions and methods for their preparation and use");
6,372,258 ("Methods of spray-drying a drug and a hydrophobic amino acid"); 6,309,671 ("Stable
glassy state powder formulations"); 6,309,623 ("Stabilized preparations for use in metered dose
inhalers"); 6,294,204 ("Method of producing morphologically uniform microcapsules and
microcapsules produced by this method"); 6,267,155 ("Powder filling systems, apparatus and
methods"); 6,258,341 ("Stable glassy state powder formulations"); 6,182,712 ("Power filling
63
apparatus and methods for their use"); 6,165,463 ("Dispersible antibody compositions and
methods for their preparation and use"); 6,138,668 ("Method and device for delivering
aerosolized medicaments"); 6,103,270 ("Methods and system for processing dispersible fine
powders"); 6,089,228 ("Apparatus and methods for dispersing dry powder medicaments");
6,080,721 ("Pulmonary delivery of active fragments of parathyroid hormone"); 6,051,256
("Dispersible macromolecule compositions and methods for their preparation and use");
6,019,968 ("Dispersible antibody compositions and methods for their preparation and use");
5,997,848 ("Methods and compositions for pulmonary delivery of insulin compound"); 5,993,783
("Method and apparatus for pulmonary administration of dry powder.alpha.l-antitrypsin");
5,922,354 ("Methods and system for processing dispersible fine powders"); 5,826,633 ("Powder
filling systems, apparatus and methods"); 5,814,607 ("Pulmonary delivery of active fragments of
parathyroid hormone"); 5,785,049 ("Method and apparatus for dispersion of dry powder
medicaments"); 5,780,014 ("Method and apparatus for pulmonary administration of dry powder
alpha 1-antitrypsin"); 5,775,320 ("Method and device for delivering aerosolized medicaments");
5,740,794 ("Apparatus and methods for dispersing dry powder medicaments"); 5,654,007
("Methods and system for processing dispersible fine powders"); 5,607,915 ("Pulmonary delivery
of active fragments of parathyroid hormone"); 5,458,135 ("Method and device for delivering
aerosolized medicaments"); 6,602,952 ("Hydrogels derived from chitosan and poly(ethylene
glycol) or related polymers"); and 5,932,462 ("Multiarmed, monofunctional, polymer for
coupling to molecules and surfaces"). Further, Suitable sustained release formulations are
described in Cardinal Health's US Patent 5,968,554, entitled "A sustained release pharmaceutical
preparation," issued 19-Oct-99, the entire disclosure of which is incorporated herein by reference.
Suitable microparticle formulations are described in Spherics, Inc.'s International Patent
Publication WO/2003-049,701, entitled "Methods and products useful in the formation and
isolation of microparticles," published 30-Oct-03. Suitable bioadhesive formulations are
described in Spherics, Inc.'s International Patent Publication WO/2003-051,304, entitled
"Bioadhesive drug delivery system with enhanced gastric retention", published 06-May-04.
Pharmaceutical compositions according to embodiments of the invention suitable for parenteral
administration comprise sterile aqueous and non-aqueous injection solutions of the complexes,
which preparations are preferably isotonic with the blood of the intended recipient. These
preparations may contain anti-oxidants, buffers, bacteriostats and solutes which render the
composition isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile
suspensions may include suspending agents and thickening agents. The compositions may be
presented in unitXdose or multi-dose containers, for example sealed ampoules and vials, and may
be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid
carrier, for example, saline or water-for-injection immediately prior to use. Extemporaneous
injection solutions and suspensions may be prepared from sterile powders, granules and tablets of
the kind previously described. For example, an injectable, stable, sterile composition with a
mixture of complexes in a unit dosage form in a sealed container may be provided. The mixture
of complexes can be provided in the form of a lyophilizate which is capable of being
reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition
suitable for injection into a subject. The parenteral unit dosage form typically comprises from
about 1 microgram to about 10 mg of the mixture of complexes. When the complexes are
substantially water-insoluble, a sufficient amount of emulsifying agent which is physiologically
acceptable may be employed in sufficient quantity to emulsify the complexes in an aqueous
carrier. One such useful emulsifying agent is phosphatidyl choline.
A solid dosage form for oral administration typically includes from about 2 mg to about 500 mg,
preferably about 10 mg to about 250 mg, ideally about 20 mg to about 110 mg of the complexes.
Pharmaceutical compositions suitable for rectal administration are preferably presented as unit
dose suppositories. These may be prepared by admixing the complexes with one or more
conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
Pharmaceutical compositions suitable for topical application to the skin preferably take the form
of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used
include petroleum jelly, lanoline, PEGs, alcohols, transdermal enhancers, and combinations of
two or more thereof.
Pharmaceutical compositions suitable for transdermal administration may be presented as discrete
patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged
period of time. Compositions suitable for transdermal administration may also be delivered by
iontophoresis (see, for example, Pharmaceutical Research 3 (6):318 (1986)) and typically take
the form of an optionally buffered aqueous solution of the mixture of insulin compound
conjugates. Suitable formulations comprise citrate or bis/tris buffer (pH 6) or ethanol/water and
contain from 0.1 to 0.2M active ingredient.
65
In a preferred embodiment, the complexes are administered as components of solid fatty acid
formulations as described in U.S. Patent Application No. 60/494,821, filed on 13-Aug-03, by
Opawale et al., the entire disclosure of which is incorporated herein by reference.
In certain embodiments, the insulin compound conjugate may be provided separately from the
cation and/or other components needed to form the solids. For example, the insulin compound
conjugate may be provided as a dried solid, and the buffer solution including the cation,
stabilizing agent, preservative and/or other component may be provided separately, so that the
user may combine the separate components to produce the cation-insulin compound conjugate
complexes.
7.12 Methods of treatment
The cation-insulin compound conjugate compositions and formulations thereof are useful in the
treatment of conditions in which increasing the amount of circulating insulin compound (relative
to the amount provided by the subject in the absence of administration of insulin compound from
an exogenous source) yields a desirable therapeutic or physiological effect. For example, the
condition treated may be Type I oR-type II diabetes, prediabetes and/or metabolic syndrome. In
one embodiment, the compositions are administered to alleviate symptoms of diabetes. In
another embodiment, the compositions are administered to a prediabetic subject in order to
prevent or delay the onset of diabetes.
The effective amount of the cation-insulin compound conjugate composition for administration
according to the methods of the invention will vary somewhat from mixture to mixture, and
subject to subject, and will depend upon factors such as the age and condition of the subject, the
route of delivery and the condition being treated. Such dosages can be determined in accordance
with routine pharmacological procedures known to those skilled in the art.
As a general proposition, an oral dosage from about 0.025 to about 10 mg/kg of active ingredient
(i.e., the conjugate) will have therapeutic efficacy, with all weights being calculated based upon
the weight of the mixture of insulin compound conjugates. A more preferred range is about 0.06
to about 1 mg/kg, and an even more preferred range is about 0.125 to about 0.5 mg/kg
A parenteral dosage typically ranges from about 0.5 ng/kg to about 0.5 mg/kg, with all weights
being calculated based upon the weight of the mixture of insulin compound conjugates. A more
preferred range is about 1 ng/kg to about 100 ug/kg.
The frequency of administration is usually one, two, or three times per day or as necessary to
control the condition. Alternatively, the cation-insulin compound conjugate compositions may be
administered by continuous infusion. The duration of treatment depends on the type of insulin
compound deficiency being treated and may be for as long as the life of the subject. The
complexes may, for example, be administered within 0 to 30 minutes prior to a meal. The
complexes may, for example, be administered within 0 to 2 hours prior to bedtime.
8 Synthesis Examples
The following examples are presented to illustrate and explain the invention.
8.1 Synthesis of protected MPEG6C3 oligomer (3-{2-[2-(2-{2-[2-(2-Methoxyethoxy)-
ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionic acid tert-butyl
ester)
O O
O Na
THF
4 h, 58%
O
Methyl hexaethylene glycol (1.0 g, 3.37 mmol) and terf-butyl acrylate (0.216 g, 1.69 mmol) were
dissolved in dry THF (10 mL). Sodium metal 0.4 mg, 0.016 mmol) was added to the solution.
After stirring for 4 h at room temperature, the reaction mixture was quenched by the addition of 1
M HC1 (15 mL). The quenched reaction mixture was then extracted with CH2C12 (1 x 50 mL, 1 x
25 mL). The organic layer was dried (MgS04) and concentrated. After purification by silica gel
chromatography (ethyl acetate as eluent), the product was obtained as an oil (0.832 g, 58%).
1.2 Synthesis of the MPEG6C3 oligomer acid (3-{2-[2-(2-{2-[2-(2-Methoxyethoxy)-
ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionicacid)
TFA
45 min, 87%
The tert-butyl ester (0.165 g, 0.0389 mmol) was deprotected by stirring at room temperature in
trifluoroacetic acid (2.0 mL). The contents were then concentrated to a constant weight (0.125 g,
87%).
8.3 Synthesis of the activated MPEG6C3 oligomer (3-{2-[2-(2-{2-[2-(2-Methoxyethoxy)-
ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionic acid 2,5-dioxopyrrolidin-
1-yl ester)
DCM
The acid (0.660 g, 1.79 mmol) and N-hydroxysuccinimide (0.2278 g, 1.97 mmol) were dissolved
in dry CH2C12 (15 mL). Ethyl dimethylaminopropyl carbodiimide hydrochloride (EDC, 0.343 g,
1.79 mmol) was added. After stirring at room temperature overnight, the reaction mixture was
diluted with CH2C12 and was washed with water (2 x 45 mL). The organic layer was dried
(MgSO4) and concentrated to a constant weight. The product was an oil (0.441 g, 53%).
68
8.4 Synthesis of the protected MPEG4C3 oligomer (3-(2-{2-[2-(2-Methoxyethoxy)-
ethoxy]-ethoxy}-ethoxy)-propionic acid tert-butyl ester)
Methyl tetraethylene glycol (1.0 g, 4.80 mmol) and tert-butyl acrylate (0.308 g, 2.40 mmol) were
dissolved in dry THF (10 mL). Sodium metal 0.6 mg, 0.024 mmol) was added to the solution.
After stirring for 4 h at room temperature, the reaction mixture was quenched by the addition of 1
M HC1 (15 mL). The quenched reaction mixture was then extracted with CH2C12 (1 x 50 mL, 1 x
25 mL). The organic layer was dried (MgSO4) and concentrated. After purification by silica gel
chromatography (ethyl acetate as eluent), the product was obtained as an oil (1.28 g, 79%).
8.5 Synthesis of the MPEG6C3 oligomer acid (3-(2-{2-[2-(2-Methoxy-ethoxy)-
ethoxy] -ethoxy}-ethoxy)-propionic acid)
The ter/-butyl ester (1 g, 3.42 mmol) was deprotected by stirring at room temperature in
trifluoroacetic acid (6.0 mL). The contents were then concentrated to a constant weight (0.87 g,
69
8.6 Synthesis of the activated MPEG4C3 oligomer (3-(2-{2-[2-(2-Methoxyethoxy)-
ethoxy]-ethoxy}-ethoxy)-propionic acid 2,5-dioxo-pyrrolidin-l-yl
ester)
wn // EDC
'Ovx-~-Q^xxX-Osxx^-o'~x_x-O^^-x^OH
The acid (0.6 g, 2.14 mmol) and A'-hydroxysuccinimide (0.271 g, 2.35 mmol) were dissolved in
dry CH2C12 (20 mL). Ethyl dimethylaminopropyl carbodiimide hydrochloride (EDC, 0.409 g,
2.14 mmol) was added. After stirring at room temperature overnight, the reaction mixture was
diluted with Cl^Ck and was washed with water (2 x 45 mL). The organic layer was dried
(MgSO4) and concentrated to a constant weight. The product was an oil (0.563 g, 69%).
8.7 Synthesis of the protected MPEG4C3 oligomer (3-(2-Methoxy-ethoxy)-
propionic acid tert-butyl ester)
Methyl tetraethylene glycol (5.0 g, 41.6 mmol) and tert-butyl acrylate (2.66 g, 20.8 mmol) were
dissolved in dry THF (20 mL). Sodium metal 0.47mg, 20.8 mmol) was added to the solution.
After stirring for 4 h at room temperature, the reaction mixture was quenched by the addition of 1
M HC1 (30 mL). The quenched reaction mixture was then extracted with CH2C12 (1 x 100 mL, 1
x 50 mL). The organic layer was dried (MgS04) and concentrated. After purification by silica
gel chromatography (ethyl acetate as eluent), the product was obtained as an oil (7.5 g, 89%).
70
8.8 Synthesis of the MPEG6C3 oligomer acid (3-[2-(2-Methoxy-ethoxy)-ethoxy]-
propionic acid)
The tert-butyl ester (1 g, 4.90 mmol) was deprotected by stirring at room temperature in
trifluoroacetic acid (6.0 mL). The contents were then concentrated to a constant weight (0.652 g,
89%).
8.9 Synthesis of 2-[2-(2-Propoxy-ethoxy)-ethoxy]-ethanol (1)
Triethylene glycol (19.5 g, 0.13 mol) was dissolved in tetrahydrofuran (150 mL) and sodium
hydride (2.60 g, 0.065 mol) was added portion wise over 0.5 h and the reaction was stirred for an
additional 1 h. Then 1-bromopropanol (8.0 g, 0.065 mol) dissolved in tetrahydrofuran (30 mL)
was added dropwise via addition funnel and the reaction was stirred overnight at room
temperature. Crude reaction mixture was filtered through Celite, washed CH2C12, and evaporated
to dryness. The resultant oil was dissolved in CH2C12 (250 rnL), washed sat. NaCl (250 mL),
H2O (250 mL), dried MgSO4, and evaporated to dryness. Column chromatography (Silica, ethyl
acetate) afforded 1 a yellowish oil (2.24 g, 18% yield).
8.10 Syntheis of carbonic acid 4-nitro-phenyl ester 2-[2-(2-propoxy-ethoxy)-
ethoxy] -ethyl ester
4-Nitrochloroformate (3.45 g, 17.1 mmol) and 1 (2.2 g, 11.4 mmol) were dissolved in CH2C12 (20
mL). After stirring for 10 min, TEA (2.1 mL, 15 mmol) was added and reaction stirred overnight
at room temperature. Crude reaction was diluted with CH2C12 (50 mL), washed 1M HC1 (50 mL),
71
H2O (50 mL), dried MgSO4, and evaporated to dryness. Column chromatography (silica, ethyl
acetate / hexanes, 3:2) afforded 2 a yellowish oil (2.57 g, 63% yield).
8.11 Synthesis of carbonic acid 2,5-dioxo-pyrrolidin-l-yI ester 2-[2-(2-methoxyethoxy)-
ethoxy]-ethyl ester
Triethylene glycol monomethyl ether (1.0 g, 6.1 mmol) and N,N'-disuccinimidyl carbonate (1.87
g, 7.3 mmol) were dissolved in acetonitrile (10 mL). Then triethylamine (1.3 mL, 9.15 mmol)
was added and the reaction stirred overnight at room temperature. Crude reaction was evaporated
to dryness, dissolved in sat. NaHCO3 (50 mL), washed ethyl acetate (2 x 50 mL), dried MgSC-4,
and evaporated to dryness. Column chromatography (Silica, ethyl acetate) afforded 1 a clear oil
(0.367 g, 20% yield).
8.12 Synthesis of carbonic acid 2,5-dioxo-pyrrolidin-l-yl ester 2-{2-[2-(2-methoxyethoxy)-
ethoxy]-ethoxy}-ethyl ester (1).
Tetraethylene glycol monomethyl ether (1.0 g, 4.8 mmol) and N,N'-disuccinimidyl carbonate
(1.48 g, 5.8 mmol) were dissolved in acetonitrile (10 mL). Then triethylamine (1.0 mL, 7.2
mmol) was added and the reaction stirred overnight at room temperature. Crude reaction was
evaporated to dryness, dissolved in sat. NaHCO3 (30 mL), washed ethyl acetate (2 x 30 mL),
dried MgSC-4, and evaporated to dryness. Column chromatography (Silica, ethyl acetate / MeOH,
20:1) afforded 1 a clear oil (0.462 g, 28% yield).
8.13 Synthesis of but-3-enoic acid ethyl ester
o
Vinylacetic acid (10.0 g, 0.12 mol) was dissolved in ethanol (200 mL) and cone, sulfuric acid
(0.75 mL, 0.014 mol) was added. The reaction was heated to reflux for 4 h. Crude reaction was
diluted with ethyl acetate (200 mL), washed H2O (200mL), sat. NaHCO3 (200 mL), dried MgSO4,
and evaporated to dryness to afford 1 a clear oil (3.17 g, 23%).
8.14 Synthesis of 4-{2-[2-(2-Methoxy-ethoxy)-ethoxyJ-ethoxy}-butyric acid ethyl
ester
Triethylene glycol monomethyl ether (4.27 g, 0.026 mol) and But-3-enoic acid ethyl ester (1.5 g,
0.013 mol) were dissolved in tetrahydrofuran (10 mL). Then lump Na° (0.030 g, 0.013 mol) was
added and the reaction was stirred for 4 h. Crude reaction was quenched with IM HCI (20 mL),
washed ethyl acetate (3 x 20 mL). Organic layers were combined and washed with H2O (2 x 10
mL), dried MgSO4, and evaporated to dryness to afford 2 a yellowish oil (1.07 g, 30% yield).
8.15 Synthesis of 4-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-butyric acid
o
4-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-butyric acid ethyl ester (1.07 g, 4.0 mmol) was
dissolved in IM NaOH (10 mL) and the reaction was stirred for 2 h. Crude reaction was diluted
with sat. NaCl (40 mL), acidified to pH ~2 with cone. HCI, washed CH2C12 (2 x 50 mL), dried
MgSO4, and evaporated to dryness to afford 3 a clear oil (0.945 g, 94% yield).
8.16 Synthesis of 4-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-butyric acid 2,5-
dioxo-pyrrolidin-1-yl ester
N-hydroxysuccinimide (0.55 g, 4.8 mmol) and EDCI (1.15 g, 6.0 mmol) were dissolved in
CH2C12 (7 mL). Then 4-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-butyric acid (0.940 g, 3.8
mmol), dissolved in CH2C12 (2 mL), was added. Reaction stirred overnight at room temperature.
Crude reaction was diluted with CH2C12 (21 mL), washed IM HCI (30 mL), H2O (30 mL), dried
MgSO4, and evaporated to dryness. Column chromatography (Silica, ethyl acetate) afforded 4, a
clear oil (0.556 g, 43% yield).
9 Preparation of Complexes
Methods were investigated for the preparation of zinc complexes of insulin compound
conjugates. New methods, exceptional to published methods used for
complexation/crystallization of insulin compound and insulin compound analogs, were developed
to make zinc complex of HIM2. HIM2 is a human insulin monoconjugate with a modifying
moiety coupled at B29, where the modifying moiety has the following structure:
Further complexes were prepared using INI 05, a human insulin monoconjugate with a modifying
moiety coupled at B29, where the modifying moiety has the following structure:
The methods provided three main types, "T-type" and "R-type" and "protamine" cation
complexes of insulin compound conjugate solids.
9.1 Preparation and Analysis of T-type Solids
9.1.1 Attempted Preparation of T-type Zn Complex of HIM2 (2 g/L)
A HM2 solution at approximately 2 g/L was prepared having a final pH ~3 with 10% HC1.
Glacial acetic acid was added to a lOmL aliquot (20mg protein) of the above solution to a final
concentration of 0.25M. Twenty (or forty) uL of a 2% w/w ZnCl2 solution was added to the
sample. The pH was adjusted to 5.1 (or 5.5) with concentrated ammonium hydroxide. The
solution stirred for 15 minutes at room temperature (or +5°C) and then stood for one day at room
temperature (or +5°C) to allow solid formation. No crystals or precipitation formed after
allowing the reaction to stand one day at room temperature (or at +5°C). See Example 2 of U.S.
Patent 5,504,188, entitled "Preparation of stable zinc insulin compound analog crystals."
9.1.2 T-type Zn Complex of HIM2 (10 g/L Concentration)
A HIM2 solution at approximately 10 g/L was prepared having a final pH ~3 with 10% HC1.
Glacial acetic acid was added to a 10 mL (lOOmg protein) aliquot of the above solution to a final
concentration of 0.25M. Forty uL of a 10% w/w ZnC12 solution was added to the sample. The
pH was adjusted to 5.20 with concentrated ammonium hydroxide. The solution was stirred for 15
minutes at +5°C and then allowed to stand for five days at +5°C to allow solid formation.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2000 RPM for
lOminutes. The solution was decanted and the solid was washed with 5 mL cold DI water. This
solution was centrifuged at 2000 RPM for 10 minutes before the water was decanted and the
solids were washed with another 5 mL cold DI water. Again, the sample was centrifuged at about
2000 RPM for about 10 minutes before the H2O was decanted. The sample was washed with 5
mL 200 proof cold EtOH and centrifuged at 2000 RPM for 10 minutes before the EtOH was
decanted. The sample was dried in a lyophilizer to provide white solid.
9.1.3 T-type Zn Complex of HIM2 (20 g/L Concentration)
A HIM2 solution at approximately 20 g/L was prepared having a final pH -3 with 10% HC1.
Glacial acetic acid was added to a 10 mL (200mg protein) aliquot of the above solution to a final
concentration of 0.25 M. Eighty uL of a 10% w/w ZnCI2 solution was added to the sample. The
pH was adjusted to 5.37 with concentrated ammonium hydroxide. The solution stirred for 15
minutes at +5°C and then stood for four days at +5°C to allow solid formation.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for
20minutes. The solution was decanted and the solid was washed with 5 mL cold DI water. This
solution was centrifuged at 2600 RPM for 20 minutes before the water was decanted and the
solids were washed with another 5mL cold DI water. Again, the sample was centrifuged at about
2600 RPM for about 20 minutes before the H2O was decanted. The sample was washed with 5
mL 200 proof cold EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was
decanted. The sample was dried in a lyophilizer to provide white solid.
9.1.4 T-type Zn Complex of of HIM2 (30 g/L Concentration)
A HIM2 solution at approximately 30 g/L is prepared having a final pH ~3 with 10% HC1.
Glacial acetic acid was added to a 50 mL (1.5g protein) aliquot of the above solution to a final
concentration of 0.25 M. Six hundred uL of a 10% w/w ZnCl2 solution was added to the sample.
The pH was adjusted to 5.34 with concentrated ammonium hydroxide. The solution stood at
+5°C for five days to allow solid formation.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2800 RPM for 15
minutes. The solution was decanted and the solid was washed three times with 10 mL cold DI
water, centrifuging and decanting the H2O each wash. The sample was then washed three times
with 10 mL 200 proof cold EtOH. It was centrifuged at 2800 RPM for 15 minutes and decanted
after each wash. The sample was dried in a lyophilizer to provide white solid.
Figures 1 and 2 are photomicrographs taken using a Zeiss Axiovert microscope showing crystals
grown for 24 hours. In Figure 1. the crystal size is approximately 11.3 pM in length and
approximately 5.3 joM in diameter. In figure 2. the size of the crystal on the left is approximately
15.1 uM in length and approximately 5.9 uM in diameter, and the size of the crystal on the right
is approximately 9.1 uM in length and approximately 5.3 uM in diameter. Figure 3 is a
photomicrograph taken using a Zeiss Axiovert microscope showing crystals grown for 5 days. In
one aspect, the invention includes crystals having a morphology as shown in Figure 1, 2 or 3.
9.1.5 T-type Zn Complex of of HIM2 (50 g/L Concentration)
A HIM2 solution at approximately 50 g/L was prepared to a final pH ~3 with 10% HC1. Glacial
acetic acid was added to a 10 mL aliquot of the above solution to a final concentration of 0.25 M.
Two hundred uL of a 10% ZnCl2 solution was added to the sample. The pH was adjusted to 5.23
with concentrated ammonium hydroxide. The solution was stirred at +5 °C for 15 minutes and
then stood at +5 °C for four days to allow solid formation to occur.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20
minutes. The solution was decanted and the solid was washed with 5mL cold DI H2O. This
solution was centrifuged at 2600 RPM for 20 minutes before the H2O was decanted and the solid
was washed with another 5 mL cold DI H2O. Again, the sample was centrifuged at 2600 RPM
for 20 minutes before the H2O was decanted. The sample was washed with 5 mL 200 proof cold
EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted. The sample
was dried in a lyophilizer for three days.
9.1.6 T-type Zn Complex of of HIM2 (1 g Scale)
A HIM2 solution at approximately 10 g/L was prepared to a final pH ~3 with 10% HC1. Glacial
acetic acid was added to a 50 mL (SOOmg protein) aliquot of the above solution to a final
concentration of 0.25 M. Two hundred pL of a 10% ZnCl2 solution was added to the sample.
The pH was adjusted to 5.49 with concentrated ammonium hydroxide. The solution was stirred
at +5 °C for 15 minutes and then stood at +5 °C for seven days to allow solid formation to occur.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20
minutes. The solution was decanted and the solid was washed with 10 mL cold DI H2O. This
solution was centrifuged at 2600 RPM for 20 minutes before the H2O was decanted. The water
washes were repeated two additional times. The sample was then washed with 10 mL 200 proof
cold EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted. Two
more EtOH washes were carried out the same way before the sample was placed on the
lyophilizer to dry for four days.
9.1.7 T-type Zn Complex of of HIM2 at Neutral pH (5 g scale)
A HIM2 solution at approximately 10 g/L was prepared to a final pH ~3 with 10% HC1. Two
mililiters of a 10% ZnC^ solution was added to the sample. The pH was adjusted to 7.05 with
concentrated ammonium hydroxide. The solution was stirred at room temperature overnight to
allow solid formation to occur.
The milky Zn-HIM2 reaction mixture (SOOmL) was added, in parts, to a 350mL fine-fritted
(4.5-5um) disc'funnel (ChemGlass CG1402-28, 90mm diameter). The filtrate was collected in a
side-arm flask while applying vacuum for about 4-6 hours. As an option, the cake may be
washed with 100 mL cold 1% ZnC^ and the filtrate collected separately. The cake was washed
with 100 mL ice-cold water, and the filtrate was again collected. The cake was also washed with
an additional 100 mL ice-cold 100% ethanol and the filtrate was collected once again. The final
wash of the cake was lOOmL of fresh ice-cold water and the final filtrate collected. The cake was
dried under vacuum and/or air-dried over 12-18 hours. After drying, the cake was scraped off the
funnel, weighed, and moisture/protein contents were measured via HPLC. The collected filtrates
from the various wash steps were also analyzed using HPLC to determine the concentration of the
lost Zn-HM2 during the process. The filtration yielded a 2.5% w/w Zn content with an overall
yield of 98%.
9.1.8 T-type Zn Complex of HIM2 at neutral pH (SOOmg scale)
A HIM2 solution at approximately 10 g/L was prepared to a final pH ~3 with 10% HC1. Two
hundred uL of a 10% ZnCl2 solution was added to the sample. The pH was adjusted to 7.06 with
77
concentrated ammonium hydroxide. The solution was stirred at +5 for 15 minutes and then stood
at +5 for two days to allow solid formation to occur.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2800 RPM for 15
minutes. The solution was decanted and the solid was washed with 10 mL cold DI H20. This
solution was centrifuged at 2800 RPM for 15 minutes before the H2O was decanted. The water
washes were repeated two additional times. The sample was then washed with 10 mL 200 proof
cold EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted. Two
more EtOH washes were carried out the same way before the sample was placed on the
lyophilizer to dry for two days.
9.2 Preparation and Analysis of R-type Solids
9.2.1 R-type Zn complex of HIM2 with Phenol at 2 g/L
A HIM2 solution at approximately 2 g/L was prepared to a final pH ~3 with glacial acetic acid.
Thirty three mircoliters of liquefied phenol was added to a 10 mL aliquot of the above solution.
ThepH was adjusted to 5.89 with concentrated ammonium hydroxide. One hundred sixty jiL of a
10% w/w ZnCl2 solution was added to the sample. The solution was stirred at room temperature
for 15 minutes and then stood at room temperature for three days to allow more precipitate to
form.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 3400 RPM for
ISminutes. The supernatant was decanter and the solid was washed with 5 mL cold Dl water.
This solution was centrifuged at 3200 RPM for 15 minutes before the H2O was decanted. The
78
sample was then washed with 5 mL 200 proof cold EtOH and centrifuged at 3200 RPM for 15
minutes before the EtOH was decanted. Again the sample was washed with 5 mL of cold EtOH,
however, it was not centrifuged. The solid was allowed to settle to the bottom of the tube and
then placed in the speed vacuum to dry.
9.2.2 Preparation of R-type Zn Complex of HIM2 at 20 g/L
A HIM2 solution at approximately 20 g/L was prepared to a final pH ~3 with 10% HC1. Sixty six
uL of liquefied phenol was added to a 10 mL aliquot of the above solution. The pH was adjusted
to 6.43 with concentrated ammonium hydroxide. Three hundred twenty uL of a 10% ZnCl2
solution was added to the sample. The solution was stirred at room temperature for 15 minutes
and then stood at room temperature for four days to allow more precipitate to form.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20
minutes. The solution was decanted and the solid was washed with 5 mL cold DI H2O. This
solution was centrifuged at 2600 RPM for 20 minutes before the H2O was decanted and the solid
was washed with another 5mL cold DI H2O. Again, the sample was centrifuged at 2600 RPM for
20 minutes before the H2O was decanted. The sample was washed with 5 mL 200 proof cold
EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted. The sample
was lyophilized for three days.
9.2.3 Preparation of R-type Zn Complex of HIM2 at 30 g/L
A HIM2 solution at approximately 30 g/L was prepared to a final pH ~3 with 10% HC1. Ninety
nine mircoliters of liquefied phenol was added to a 10 mL aliquot of the above solution. The pH
was adjusted to 6.47 with concentrated ammonium hydroxide. Then, 480 uL of a 10% ZnCl2
solution was added to the sample. The solution was stirred at room temperature for 15 minutes
and then stood at room temperature for four days to allow more precipitate to form.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for
20minutes. The solution was decanted and the solid was washed with 5 mL cold DI H2O. This
solution was centrifuged at 2600 RPM for 20 minutes before the H2O was decanted and the solid
was washed with another 5 mL cold DI H2O. Again, the sample was centrifuged at 2600 RPM
for 20 minutes before the H2O was decanted. The sample was washed with 5 mL 200 proof cold
EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted. The sample
was lyophilized for three days.
79
Figure 4 shows solid grown for 4 days. The picture was taken using a Zeiss Axiovert
microscope. The average length of the crystals is approximately 9.7uM.
9.2.4 Preparation of R-type Zn Complex of HIM2 at 50 g/L
A HIM2 solution at approximately 50 g/L was prepared to a final pH ~3 with 10% HC1. One
hundred sixty five mircoliters of liquefied phenol was added to a 10 mL aliquot of the above
solution. The pH was adjusted to 6.82 with concentrated ammonium hydroxide. Eight hundred
uL of a 10% ZnQ2 solution was added to the sample. The solution was stirred at room
temperature for 15 minutes and then stood at room temperature for four days to allow more
precipitate to form.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for
20minutes. The solution was decanted, and the solid was washed with 5 mL cold DI H2O. This
solution was centrifuged at 2600 RPM for 20 minutes before the H2O was decanted and the solid
was washed with another 5 mL cold DI H2O. Again, the sample was centrifuged at 2600 RPM
for 20 minutes before the H2O was decanted. The sample was washed with 5 mL 200 proof cold
EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted. The sample
was lyophilized for three days.
9.2.5 Preparation of R-type Zn Complex of HDLM2 at 1 g Scale
A HIM2 solution at approximately 10 g/L was prepared to a final pH ~3 with 10% HC1. One
hundred sixty five mircoliters of liquefied phenol was added to a 50 mL aliquot of the above
solution. The pH was adjusted to 6.42 with concentrated ammonium hydroxide. Eight hundred
uL of a 10% ZnClj solution was added to the sample. The solution was stirred at room
temperature for 15 minutes and then stood at room temperature for seven days to allow more
precipitate to form.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20
minutes. The solution was decanted and the solid was washed with 10 mL cold DI H2O. This
solution was centrifuged at 2600 RPM for 20 minutes before the H2O was decanted. Two more
water washes occurred the same way. The sample was then washed with 10 mL 200 proof cold
EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted. Two more
EtOH washes were carried out the same way before the sample was placed on the lyophilizer for
four days.
9.2.6 R-type Zn complex of HIM2 at 5 g Scale
A HIM2 solution at approximately 10 g/L was prepared to a final pH ~3 with 10% HC1. Fifteen
hundred mircoliters of liquefied phenol was added to 450 mL of the above solution. The pH was
adjusted to 7.1 with concentrated ammonium hydroxide. Eighteen hundred uL of a 10% ZnC12
solution was added to the sample. The solution was stirred at room temperature for 15 minutes
and then stood at room temperature overnight to allow more precipitate to form.
The reaction performed above was split into three filtration trials. In trial one, the reaction
mixture was filtered through a fine fritted funnel and then washed with a 1% ZnC12 solution. The
material was dried overnight via vacuum filtration. The second trial was filtered over a medium
fritted filter which also contained filter paper. The substance was then washed with ethanol and
water and dried overnight via vacuum filtration. Finally, the third trial was filtered through a fine
fritted funnel, washed with a 1% ZnC12 solution and also washed with ethanol and water. This
material was also dried overnight under vacuum filtration.
9.2.7 R-type Zn Complex of HIM2 at Neutral pH
A HIM2 solution at approximately 10 g/L was prepared to a final pH ~3 with 10% HC1. One
hundred sixty five microliters of liquefied phenol was added to 50 mL of the above solution.
Then, two hundred microliters of a 10% ZnCl2 solution was added to the sample. The pH was
adjusted to 7.18 with concentrated ammonium hydroxide. The solution sat at room temperature
for two days to allow precipitate to form.
The reaction mixture was transferred to a centrifuge tube and centrifuged 'at 2800 RPM for 20
minutes. However, the material did not settle to the bottom of the tube initially and was
therefore, centrifuged for about 2 hours. The solution was decanted and the solid was washed
with 5 mL cold DI H2O. This solution was centrifuged at 2800 RPM for 60 minutes before the
HjO was decanted. The water wash was repeated two more times. The sample was then washed
with 5 mL 200 proof cold EtOH and centrifuged at 2800 RPM for 60 minutes before the EtOH
was decanted. Two more EtOH washes were carried out the same way. Material was cloudy
81
after the third EtOH wash and was placed in the refrigerator overnight to allow the reactions to
settle more. The solvent was decanted and the material was placed on the lyophilizer for 2 days.
and Analysis of Protamine Solids
9.3.1 Preparation of T-type Zn Complex of HIM2 with Protamine at Acidic pH
Protamine was added to a 10 g/L stock solution of HIM2 that had a final pH ~3 with 10% HC1.
Glacial acetic acid was added to a 10 mL aliquot (100 mg protein) of the above solution to a final
concentration of 0.25 M. Two hundred microliters of a 10% ZnCl2 solution was added to the
sample. The pH was adjusted with concentrated ammonium hydroxide to a pH ~5. The solution
was stirred at + 5 °C for 15 minutes and then stood at +5 °C for two days to allow solid formation
to occur.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20
minutes. The solution was decanted and the solid was washed with 10 mL cold DI H2O. This
solution was centrifuged at 2600 RPM for 20 minutes before the H2O was decanted. Two more
H2O washed occurred the same way. The sample was then washed with 10 mL 200 proof cold
EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted. Two more
EtOH washes were carried out the same way before the sample was placed on the lyophilizer for
two days.
9.3.2 Preparation of T-type Zn Complex of HIM2with Protamine at Neutral
A HIM2 solution at approximately 30 g/L was prepared to a final pH ~ 3 with 10% HC1. One
milliliter of glacial acetic acid was added to a 50 mL aliquot (1.5 g protein) of the above solution.
Six hundred microliters of a 10% ZnCl2 solution was added to the reaction followed by the
addition of 225 milligrams of protamine. The pH was adjusted to 6.95 with concentrated
ammonium hydroxide and the reaction stood for two days at +5 °C to allow solid formation to
occur.
The reaction mixture was transferred to a centrifuge rube and centrifuged at 2600 RPM for 20
minutes. The solution was decanted and the solid was washed with 10 mL cold Dl H2O. This
solution was centrifuged at 2600 RPM for 20 minutes before the H2O was decanted. Two more
H2? washed occurred the same way. The sample was then washed with 10 mL 200 proof cold
EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted. Two more
EtOH washes were carried out the same way before the sample was placed on the lyophilizer for
three days
9.3.3 Preparation of R-type Zn Complex of HIMlwith Protamine at Acidic pH
A HIM2 solution at approximately 10 g/L was prepared to a final pH ~ 3 with 10% HC1.
Liquified phenol (2.48mL) was added to a 150 mL aliquot (1.5 g protein) of the above solution.
The pH of the reaction was adjusted with concentrated ammonium hydroxide to a pH ~ 6.57.
Twelve microliters of a 10% ZnCl2 solution was added to the reaction followed by the addition of
225 milligrams of protamine. The reaction mixture stirred at room temperature for 15 minutes
before it stood for two days at room temperature to allow solid formation to occur.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2800 RPM for 15
minutes. The solution was decanted and the solid was washed with 50 mL cold DI H2?. This
solution was centrifuged at 2800 RPM for 15 minutes before the H2O was decanted. Two more
H2O washed occurred the same way. The sample was then washed with 10 mL 200 proof cold
EtOH and centrifuged at 2800 RPM for 15 minutes before the EtOH was decanted. Two more
EtOH washes were carried out the same way before the sample was placed on the lyophilizer for

9.3.4 Preparation of R-type Zn Complex of HIM2with Protamine at Neutral
PH
A HIM2 solution at approximately 10 g/L was prepared to a final pH ~ 3 with 10% HC1.
Liquefied phenol (495 mL) was added to 150 mL reaction. Then, 600 mililiters of a 10% ZnCl2
solution was added to reaction followed by the addition of 75 rng protamine. The pH was
adjusted with concentrated ammonium hydroxide to a pH of 7.01. The reaction stood for three
days at room temperature to allow solid formation.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2800 RPM for 15
minutes. The solution was decanted and the solid was washed with 50 mL cold DI H2O. This
solution was centrifuged at 2800 RPM for 15 minutes before the H2O was decanted. Two more
H2O washes occurred the same way. The sample was then washed with 50 mL 200 Proof cold
EtOH and centrifuged at 2800RPM for 15 minutes before the EtOH was decanted. Two more
EtOH washes were carried out the same way before the sample was placed on the lyophlizer for
two days.
9.4 Preparation and Analysis of Complexes of Insulin Compound Diconjugates
9.4.1 T-type Zn Complex at Al and B29 Insulin Compound Diconjugate
An insulin compound diconjugate having a modifying moiety -C(O)(CH2)5(OCH2CH2)70CH3
coupled at B29 and Al of human insulin (DICON-1) was added to solution at approximately 10
g/L and prepared to a final pH 3.15 with 10% HC1. Glacial acetic acid was added to a 3.75 mL
aliquot of the above solution to a final concentration of 0.25 M. Then 15 uL of a 10% ZnCl2
solution was added to the sample. The pH was adjusted to 4.90 with concentrated ammonium
hydroxide. The solution stirred for 15 minutes at +5°C and then stood for six days at +5°C to
allow solid formation (yielded a white solid).
9.4.2 R-type Zn Complex at Al and B29 Insulin Compound Diconjugate
DICON-1 was added to solution at approximately 10 g/L and prepared to a final pH 3.15 with
10% HC1. About 12 uL of liquefied phenol was added to a 3.75 mL aliquot of the above
solution. The pH was adjusted to 5.75 with concentrated ammonium hydroxide. Sixty uL of a
10% ZnCl2 solution was added to the sample. The solution was stirred at room temperature for
15 minutes and then stood at room temperature for six days to allow more precipitate to form
(yielded a white solid).
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20
minutes. The solution was decanted and the solid was washed with 5 mL cold DI H2O. This
solution was centrifuged at 2600 RPM for 20 minutes before the H2O was decanted and the solid
was washed with another 5 mL cold DI H20. Again, the sample was centrifuged at 2600 RPM
for 20 minutes before the H2O was decanted. The sample was washed with 5 mL 200 proof cold
EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted. The sample
was lyophilized for six days.
9.4.3 Diconjugate Bl, B29 (lOmg/mL)
DICON-1 was added to solution at approximately 10 g/L and 33 uL of liquified phenol was
added. The pH was adjusted to 5.34 with concentrated ammonium hydroxide. Then 160 uL of a
10% ZnCl2 solution was added to the sample. The solution stood at room temperature for two
weeks to allow solid formation to occur (yielded a white solid).
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2800 RPM for 15
minutes. The solution was decanted and the solid was washed three times with 5 mL cold DI
H2O. The solution was centrifuged for 15 minutes at 2800 RPM and decanted after each wash.
The sample was then washed three times with 5 mL 200 proof cold EtOH. Again the sample was
centrifuged at 2600 RPM for 15 minutes and decanted after each wash. The sample was
lyophilized for two days.
9.4.4 Diconjugate Bl, B29 (20mg/mL)
DICON-1 was added to solution at approximately 20 g/L and 66 microliters of liquified phenol
was added. The pH was adjusted to 7.65 with concentrated ammonium hydroxide. Then 320 uL
of a 10% ZnCl2 solution was added to the sample. The solution stood at room temperature for
two weeks to allow solid formation to occur (yielded a white solid).
85
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2800 RPM for 15
minutes. The solution was decanted and the solid was washed three times with 5 mL cold DI
H2O. The solution was centrifuged for 15 minutes at 2800 RPM and decanted after each wash.
The sample was then washed three times with 5 mL 200 proof cold EtOH. Again the sample was
centrifuged at 2600 RPM for 15 minutes and decanted after each wash. The sample was
lyophilized for two days.
9.5 Preparation and Analysis of T-type EV105 solids
9.5.1 T-type Zn Complex of IN105 Monoconjugate (10 g/L concentration)
A IN105 solution at approximately 10 g/L (lOOmg) was prepared having a final pH ~3 with 10%
HC1. 50 uL of a 10% w/w ZnCl2 solution was added to the sample. The pH was adjusted to 7.52
with concentrated ammonium hydroxide. The cloudy solution was stirred and then allowed to
stand for five days at room temperature to allow solid formation.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2900 RPM for 15
minutes. The solution was decanted and the solid was washed with 3x10 mL cold DI water. This
solution was centrifuged at 2900 RPM for 10 minutes before the water was decanted and the
solids were washed with another portion of cold DI water. The sample then was washed with
3x10 mL 200 proof cold EtOH and centrifuged at 2900 RPM for 10 minutes before the EtOH was
decanted. The sample was vacuum dried to provide white solid (90mg).
9.5.2 T-type Zn Complex of IN105 Monoconjugate (1 g Scale)
A IN1Q5 solution at approximately 10 g/L (Ig) was prepared to a final pH ~3 with 10% HC1.
Five hundred uL of a 10% ZnCl2 solution was added to the sample. The pH was adjusted to ~7.4
with concentrated ammonium hydroxide. The cloudy solution was stirred for 15 minutes and
then allowed to stand at room temperature for ~2 days before filtration.
The reaction mixture was filtered through sintered glass funnel (fine) under house vacuum. The
sintered glass funnel with filtered material was placed under vacuum in a glass dessicator over
night to result a white fine powder (900mg).
9.5.3 T-type Zn Complex of INI05 Monoconjugate at Neutral pH (5 g scale)
A INI05 solution at approximately 10 g/L (5g, lot#Nobex040706L) was prepared to a final pH ~3
with 10% HC1. Two mL of a 10% ZnCl2 solution was added to the sample. The pH was
adjusted to ~7.4 with concentrated ammonium hydroxide. The cloudy solution was then allowed
to stand at room temperature overnight to allow solid formation before filtration.
The reaction performed above was split into 4x50 mL centrifuge tubes and initially centrifuged at
3200 RPM for a total 2 hours. The material was then centrifuged at 9000 RPM for 20 minutes
and stored at 5°C over night. The supernatant was decanted and the solid was washed with 10 mL
cold DIH2O from each tube. The tubes were inverted and centrifuged at 3200 RPM for ~ 1 hour
before the H2O was decanted and the solids were washed with another 10 mL cold DI H2O.
Again, the sample was centrifuged at 3200 RPM for ~ 1 hour before the H2O was decanted. The
sample was washed with 2x10 mL 200 proof cold EtOH and centrifuged at 3200 RPM for 1 hour
before the EtOH was decanted. The sample was vacuum dried for two days to give 1.64g
(lot#Nobex040730L-A) of white powder.
9.6 Preparation and Analysis of R-type INI05 solids
9.6.1 R-type Zn Complex at IN105 Conjugate with Phenol at Neutral pH
AIN105 solution at approximately 10 g/L (500mg) was prepared to a final pH ~3 with 10% HC1.
Two hundred \iL of 10% ZnCl2 and 165 joL of liquefied phenol was added to the above solution.
The pH was adjusted to 7.37 with concentrated ammonium hydroxide. The cloudy solution sat at
room temperature for 2 days to allow solid formation before filtration.
The reaction mixture was filtered through sintered glass funnel (fine) under house vacuum. The
sintered glass funnel with filtered material was placed in under vacuum in a glass dessicator over
night to result in a white fine powder (440mg).
9.6.2 R-type Zn Complex of IN105 Conjugate at 5 g Scale
A IN105 solution at approximately 10 g/L (4.2g, lot#Nobex040706L) was prepared to a final pH
~3 with 10% HC1. 1.5 liquefied phenol and 1.8 mL of 10% ZnC12 solution was added to the
87
above solution. The pH was adjusted to ~7.4 with concentrated ammonium hydroxide. The very
cloudy solution stood at room temperature overnight to allow more precipitate to form.
The reaction performed above was split into 4x50 mL centrifuge tubes and initially centrifuged at
3200 RPM for 2 hours. The material was then centrifuged at 9000 RPM for 20 minutes and
stored at 5°C over night. The supernatant was decanted and the solid was washed with 10 mL
cold DI H2O from each tube. The tubes were inverted and centrifuged at 3200 RPM for ~ 1 hour
before the H2O was decanted and the solids were washed with another 10 mL cold DI H2O.
Again, the sample was centrifuged at 3200 RPM for ~ 1 hour before the H20 was decanted. The
sample was washed with 2x10 mL 200 proof cold EtOH and centrifuged at 3200 RPM for 1 hour
before the EtOH was decanted. The sample was vacuum dried for 2 days to give 2.34g of white
powder.
and Analysis of Protamine IN 105 solids
9.7.1 Preparation of R-type Zn Complex of BV105 Monoconjugate with
Protamine at Acidic pH
AIN105 solution at approximately 10 g/L is prepared to a final pH ~ 3 with 10% HC1. Liquified
phenol (248 uL) is added to a 15 mL aliquot (150 mg protein) of the above solution. The pH of
the reaction is adjusted with concentrated ammonium hydroxide to a pH ~ 6.50. One microliter
of a 10% ZnCl2 solution is added to the reaction followed by the addition of 22.5 milligrams of
protamine. The reaction mixture is stirred at room temperature for 15 minutes before it stood for
two days at room temperature to allow solid formation to occur.
The reaction mixture is transferred to a centrifuge tube and centrifuged at 2800 RPM for 15
minutes. The solution is decanted and the solid is washed with 5 mL cold DI H2O. This solution
is centrifuged at 2800 RPM for 15 minutes before the H2O is decanted. Two more H2O wash is
occurred the same way. The sample is then washed with 10 mL 200 proof cold EtOH and
centrifuged at 2800 RPM for 15 minutes before the EtOH is decanted. Two more EtOH washes
are carried out the same way before the sample is vacuum dried over two days.
9.7.2 Preparation of R-type Zn Complex of IN105 Conjugate With Protamine
at Neutral pH
AIN105 solution at approximately 10 g/L is prepared to a final pH ~ 3 with 10% HC1. Liquefied
phenol (49.5 uL) is added to 15 mL reaction. Then, 60 microliters of a 10% ZnCl2 solution is
added to reaction followed by the addition of 7.5 mg protamine. The pH is adjusted with
concentrated ammonium hydroxide to a pH of 7.00. The reaction is allowed to stand for three
days at room temperature to allow solid formation.
The reaction mixture is transferred to a centrifuge tube and centrifuged at 2800 RPM for 15
minutes. The solution is decanted and the solid was washed with 5.0 mL cold DI H2O. This
solution is centrifuged at 2800 RPM for 15 minutes before the H20 is decanted. Two more H2O
washes are occurred the same way. The sample is then washed with 50 mL 200 Proof cold EtOH
and centrifuged at 2800 RPM for 15 minutes before the EtOH is decanted. Two more EtOH
washes are carried out the same way before the sample is vacuum dried over two days.
9.7.3 Preparation of R-type Crystalline Zn Complex of INI 05
A crude 15mg/mL IN105 solution containing 25% organic was pH adjusted to 3.47 using 1M
HC1. Solid phenol was melted in a 40-60°C water bath and 0.218mL was added to reaction flask.
Then 0.4mL of 4% acidified aqueous ZnQ2 solution was added to reaction. The pH of the
solution was adjusted with 1M NaOH to a final pH of 6.6. While adjusting the pH, lOmL
aliquots were pulled at the following pH values: 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4 and 6.6.
The samples were allowed to sit without stirring for 24 hours. Needle-like crystals were observed
under a microsope.
9.7.4 Preparation of R-type Crystalline Zn Complex of IN105 Containing 30%
Organic
A fresh 15mg/mL solution of MPEG3 propionyl insulin compound was prepared in 250mM
ammonium acetate buffer and the pH was adjusted to 2.81 with 1M HC1. Liquefied phenol,
0.040mL, and 95% EtOH, 4.25mL, were added to the solution. Then, 0.400mL of a 4% acidified
ZnCl2 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.7
to 5.4 using 50% NH4OH and pulling ImL aliquots at each of the following desired pH: 4.0, 4.2,
4.4, 4.6, 4.8, 5.0, 5.2, 5.4. The samples were allowed to sit without stirring for 24 hours. The
89
microscope pictures taken after 24 hours showed needle-like crystals (see Figure 5) from the pH
range 4.0 to 5.2.
9.7.5 Preparation of R-type Crystalline Zn Complex of IN105 in lOOmM
ammonium Acetate Buffer (30,20 and 10% EtOH)
A fresh 15mg/mL solution of MPEGs propionyl insulin compound was prepared in lOOmM
ammonium acetate buffer and the pH was adjusted to 2.8 with 5M HC1. Liquefied phenol,
0.040mL, and 95% EtOH, 4.25mL, were added to the solution. Then, 0.400mL of a 4% acidified
ZnCl2 solution was added to the reaction mixture. The pH of the solution was adjusted from 2.9
to 5.6 using 5M NH4OH and pulling O.SmL aliquots at each of the following desired pH: 4.2,4.4,
4.6, 4.8, 5.0, 5.2, 5.4, 5.6. The samples were allowed to sit without stirring for 24 hours. The
microscope pictures taken after 24 hours showed needle-like crystals from the pH range 4.4 to
4.8.
A fresh 15mg/mL solution of MPEG3 propionyl insulin compound was prepared in lOOmM
ammonium acetate buffer and the pH was adjusted to 2.8 with 5M HC1. Liquefied phenol,
0.040mL, and 95% EtOH, 2.25mL, were added to the solution. Then, 0.400mL of a 4% acidified
ZnCU solution was added to the reaction mixture. The pH of the solution was adjusted from 2.9
to 5.6 using 5M NH4OH and pulling O.SmL aliquots at each of the following desired pH: 4.2, 4.4,
4.6, 4.8, 5.0, 5.2, 5.4, 5.6. The samples were allowed to sit without stirring for 24 hours. The
microscope pictures taken after 24 hours showed circle-like crystals from the pH range 4.8 to 5.4.
A fresh 15mg/mL solution of MPEG3 propionyl insulin compound was prepared in lOOmM
ammonium acetate buffer and the pH was adjusted to 2.8 with 5M HC1. Liquefied phenol,
0.040mL, and 95% EtOH, 1.15mL, were added to the solution. Then, 0.400mL of a 4% acidified
ZnCl2 solution was added to the reaction mixture. The pH of the solution was adjusted from 2.8
to 5.6 using 5M NH4OH and pulling O.SmL aliquots at each of the following desired pH: 4.2, 4.4,
4.6, 4.8, 5.0, 5.2, 5.4, 5.6. The samples were allowed to sit without stirring for 24 hours. The
microscope pictures taken after 24 hours showed needle-like crystals from the pH range 5.0 to
5.6.
9.7.6 Preparation of R-type Crystalline Zn Complex of IN105 in 20% Organic
with 0.1 and 0.2% Phenol
A fresh 15mg/mL solution of MPEG3 propionyl insulin compound was prepared in lOOmM
ammonium acetate buffer and the pH was adjusted to 3.0 with 5M HC1. Liquefied phenol,
90
O.OlOmL, and 95% EtOH, 2.5mL, were added to the solution. Then, 0.400mL of a 4% acidified
ZnClj solution was added to the reaction mixture. The pH of the solution was adjusted from 3.2
to 5.6 using 5M NH4OH and pulling O.SmL aliquots at each of the following desired pH: 4.2, 4.4,
4.6, 4.8, 5.0, 5.2, 5.4, 5.6. The samples were allowed to sit without stirring for 24 hours. The
microscope pictures taken after 24 hours showed circle-like crystals from the pH range 4.4 to 5.4.
A fresh 15mg/mL solution of MPEG3 propionyl insulin compound was prepared in lOOmM
ammonium acetate buffer and the pH was adjusted to 3.0 with 5M HC1. Liquefied phenol,
0.020mL, and 95% EtOH, 2.5mL, were added to the solution. Then, 0.400mL of a 4% acidified
ZnCl2 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.3
to 5.6 using 5M NH4OH and pulling O.SmL aliquots at each of the following desired pH: 4.2, 4.4,
4.6, 4.8, 5.0, 5.2, 5.4, 5.6. The samples were allowed to sit without stirring for 24 hours. The
microscope pictures taken after 24 hours showed circle-like crystals from the pH range 4.4 to 5.2.
9.7.7 Preparation of R-type Crystalline Zn Complex of IN105 at 8.0 gram
scale, pH 4.8 and Room Temperature
A fresh 15mg/mL solution of MPEG3 propionyl insulin compound was prepared in 250mM
ammonium acetate buffer and the pH was adjusted to 2.0 with 5M HC1. Liquefied phenol,
2.13mL, and 95% EtOH, 225mL, were added to the solution. Then, 21.3mL of a 4% acidified
ZnClz solution was added to the reaction mixture. The pH of the solution was adjusted to 4.8
using 5M NH4OH. The solution was allowed to sit without stirring for 24 hours before the
crystals were harvested. Needle-like crystals were observed at the T=0 microscope picture.
The crystals were harvested by splitting the reaction mixture into 6X250mL centrifuge tubes.
The tubes were spun at 10,OOORPM for 8 minutes at 10°C before the supernatant was decanted.
Then, to each tube, was added lOmL cold H2O before consolidating the 6 tubes into 2 tubes. The
centrifuge process was repeated once more with cold water and twice more with cold EtOH. The
crystals were then dried with a desktop lyophilizer for 2 days. The procedure produced 93%
yield (w/w) relative to the starting material.
9.7.8 Preparation of R-type Crystalline Zn Complex of IN105 at 1.5 gramscale, pH 4.8 and Room TemperatureA fresh solution of MPEG3 Propionyl Insulin compound (INI 05) was prepared by dissolving
1.52g of solid IN105 in 100 mL of 250 mM ammonium acetate pH 7.5. The solution was
adjusted to pH 2.8 using 5M HC1 / 5M NH4OH. Solid phenol was melted in a 40-60° C warm
91
water bath. 400uL of melted phenol and 42.5mL of 95% EtOH were added to the reaction flask.
Then 4mL of 4% acidified aqueous ZnCI2 was added to the reaction flask. The resulting solution
was then adjusted to pH 4.8 using 5M NH4OH. The reaction was then allowed to sit without
stirring for 48 hours before crystals were harvested. Needle-like crystal formation was observed
after 21 hours via microscope.
The crystals were harvested by splitting of the reaction slurry among 4 x 50mL centrifuge tubes.
The tubes were spun initially at 1000 RPM for 8 min. The supernatant was then decanted. The
crystals in each tube were washed with 1 x 5mL aliquot of ice-cold H2O then spun at 3000 RPM
for 8 min. The supernatant was then decanted. Repeated the washing/ spinning procedure with 1
x 5mL aliquot of ice-cold H2O then with 1 x 5 mL aliquot of ice-cold EtOH. The crystals were
then dried in a vacuum dessicator overnight. The procedure produced 73% yield (w/w) relative to
the starting material.
9.7.9 Preparation of R-type Crystalline Zn Complex of IN105 at 1.5 gram
scale, pH 4.4 and Room Temperature
A fresh solution of MPEG3 Propionyl Insulin compound (INI 05) was prepared by dissolving
1.50 g of solid INI 05 in 100 mL of 250 mM ammonium acetate pH 7.5. The solution was
adjusted to pH 2.6 using 5M HC1. Solid phenol was melted in a 40-60° C warm water bath.
400uL of melted phenol and 42.5mL of 95% EtOH were added to the reaction flask. Then 4mL
of 4% acidified aqueous ZnCl2 was added to the reaction flask. The resulting solution was then
adjusted to pH 4.4 using 5M NH4OH. The reaction was then allowed to sit without stirring for 22
hours before crystals were harvested. A mixture of needle-like crystal formation and precipitate
was observed after 2 hours via microscope. The reaction mixture appeared to be completely
crystalline after 21 hours via microscope.
The crystals were harvested by transferring the reaction slurry to a 1 x 250mL centrifuge tube.
The tube was spun initially at 10,000 RPM for 8 min. The supernatant was then decanted. The
crystals were washed with 1 x 20 mL aliquot of ice-cold H2O then spun at 10,000 RPM for 8 min.
The supernatant was then decanted. Repeated the washing/ spinning procedure with 1 x 20 mL
aliquot of ice-cold H2O then with 2 x 20 mL aliquots of ice-cold EtOH and a final 1 x 20 mL
aliquot of ice-cold H2O. The crystals were then dried in a vacuum dessicator overnight. The
procedure produced 67% yield (w/w) relative to the starting material.
92
9.7.10 Preparation of R-type Crystalline Zn Complex of IN105 at 8.0 Gram
Scale, pH 4.8 and Room Temperature
A fresh solution of MPEG3 Propionyl Insulin compound (INI05) was prepared by dissolving
7.98 g of solid IN105 in 533 mL of 250 mM ammonium acetate pH 7.5. The solution was
adjusted to pH 2.4 using 5M HC1. Solid phenol was melted in a 40-60° C warm water bath. 2.13
mL of melted phenol and 225 mL of 95% EtOH were added to the reaction flask. Then 21.3 mL
of 4% acidified aqueous ZnCl2 was added to the reaction flask. The resulting solution was then
adjusted to pH 4.8 using 5M NH4OH. The reaction was then allowed to sit without stirring for 21
hours before crystals were harvested. The reaction mixture appeared to be completely crystalline
after 2 hours via microscope.
The crystals were harvested by splitting the reaction slurry among 6 x 250mL centrifuge tubes.
The tubes were spun initially at 10,000 RPM for 8 min. The supernatant was then decanted. The
crystals in each tube were washed with 1 x 10 mL aliquots of ice-cold H2O then spun at 10,000
RPM for 8 min. The supernatant was then decanted Repeated the washing/ spinning procedure
with 1 x 10 mL aliquot of ice-cold H2O then with 2 x 10 mL aliquots of ice-cold EtOH and a final
1 x 10 mL aliquot of ice-cold H2O. The crystals were then dried in a vacuum dessicator for 2
days. The procedure produced 87% yield (w/w) relative to the starting material.
9.7.11 Preparation of R-type Crystalline Zn Complex of IN105 at 10.0 Gram
Scale, pH 4.8 and Room Temperature
A fresh solution of MPEG3 Propionyl Insulin compound (IN105) was prepared by dissolving
10.06 g of solid INI 05 in 670 mL of 250 mM ammonium acetate pH 7.5. The solution was
adjusted to pH 2.6 using 5M HC1. Solid phenol was melted in a 40-60° C warm water bath. 2.7
mL of melted phenol and 285 mL of 95% EtOH were added to the reaction flask. Then 27 mL of
4% acidified aqueous ZnCl2 was added to the reaction flask. The resulting solution was then
adjusted to pH 4.8 using 5M NH4OH. The reaction was then allowed to sit without stirring for 21
hours before crystals were harvested. The reaction mixture appeared to be completely crystalline
after 2.5 hours via microscope.
The crystals were harvested by splitting the reaction slurry among 6 x 250rnL centrifuge tubes.
The tubes were spun initially at 10°C , 10,000 RPM for 8 min. The supernatant was then
decanted The crystals in each tube were washed with 1x10 mL aliquots of ice-cold H2O, and
consolidated into 2 x 250mL centrifuge tubes then spun at 10°C , 10,000 RPM for 8 min. The
supernatant was then decanted Repeated the washing/ spinning procedure with 1x30 mL aliquot
93
of ice-cold H2O then with 2 x 30 mL aliquots of ice-cold EtOH and a final 1 x 30 mL aliquot of
ice-cold H2O. The crystals were then dried using a benchtop lyopholizer for 3 days. The
procedure produced 89% yield (w/w) relative to the starting material.
9.8 Preparation and analysis of cryatalline Zn compex of HIM2 using organic
solvent
9.8.1 Preparation of R-type Zn Complexes of HIM2
A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the
pH was adjusted to 2.95 with 5M HC1. Liquefied phenol, 40uL, and 95% EtOH, 3.5mL, were
added to the solution. Then, 600uL of a 4% acidified ZnClz solution was added to the reaction
mixture. The pH of the solution was adjusted from 3.14 to 6.0 using 5M NH4OH and pulling
SOOuL aliquots at each of the following desired pH: 4.2, 4.4 (See Figure 6A). 4.6, 4.8, 5.0, 5.2,
5.4 (See Figure 6B). 5.6, 5.8, 6.0. The samples were allowed to sit without stirring for 24 hours.
The microscope pictures taken after 24 hours showed needle-like crystals at pH 4.4. The pH
range from 4.6- 6.0 show large, crystalline like solids of various shapes and sizes.
A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the
pH was adjusted to 2.95 with 5M HC1. Liquefied phenol, 40uL, and 95% EtOH, 3.5mL, were
added to the solution. Then, 400uL of a 4% acidified ZnCl2 solution was added to the reaction
mixture. The pH of the solution was adjusted from 3.22 to 6.0 using 5M NH4OH and pulling
SOOuL aliquots at each of the following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0, 5.2 (See Figure 7A).
5.4, 5.6, 5.8, 6.0. The samples were allowed to sit without stirring for 24 hours. The microscope
pictures taken after 24 hours show crystalline-like solids from pH 4.2-6.0 of various shapes and
sizes.
A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the
pH was adjusted to 2.95 with 5M HC1. Liquefied phenol, 40uL, and 95% EtOH, 3.5mL, were
added to the solution. Then, 200uL of a 4% acidified ZnCl2 solution was added to the reaction
mixture. The pH of the solution was adjusted from 3.19 to 6.0 using 5M NH4OH and pulling
SOOuL aliquots at each of the following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0 (See Figure 7B). 5.2,
5.4, 5.6, 5.8, 6.0. The samples were allowed to sit without stirring for 24 hours. The microscope
pictures taken after 24 hours showed crystalline-like solids from pH 4.4-4.6 of various shapes and
sizes. The pH range of 4.8-5.2 show more uniform, needle-like crystals.
94
A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the
pH was adjusted to 2.95 with 5M HC1. Liquefied phenol, 40uL, and 95% EtOH, 2.6mL, were
added to the solution. Then, 600uL of a 4% acidified ZnCl2 solution was added to the reaction
mixture. The pH of the solution was adjusted from 3.04 to 6.0 using 5M NH4OH and pulling
SOOuL aliquots at each of the following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4 (See Figure
8A). 5.6,5.8, 6.0. The samples were allowed to sit without stirring for 24 hours. The microscope
pictures taken after 24 hours showed flat, snowflake-like crystals from pH 4.6-5.4.
A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the
pH was adjusted to 2.95 with 5M HC1. Liquefied phenol, 40uL, and 95% EtOH, 2.6mL, wereadded to the solution. Then, 400uL of a 4% acidified ZnCl2 solution was added to the reaction
mixture. The pH of the solution was adjusted from 3.05 to 6.0 using 5M NH«(OH and pulling
SOOuL aliquots at each of the following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0 (See Figure 8B), 5.2,
5.4,5.6, 5.8, 6.0. The samples were allowed to sit without stirring for 24 hours. The microscope
pictures taken after 24 hours showed needle-like crystals at pHS.O, crystal-like solids at pH5.2
and flat, snowflake-like crystals at pH5.4.
Rxn 6 A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer
and the pH was adjusted to 2.95 with 5M HC1. Liquefied phenol, 40uL, and 95% EtOH, 2.6mL,
were added to the solution. Then, 200uL of a 4% acidified ZnCl2 solution was added to the
reaction mixture. The pH of the solution was adjusted from 3.09 to 6.0 using 5M NH4OH and
pulling 500uL aliquots at each of the following desired pH: 4.2, 4.4, 4.6, 4.8 (See Figure 9A).
5.0, 5.2, 5.4, 5.6, 5.8, 6.0. The samples were allowed to sit without stirring for 24 hours. The
microscope pictures taken after 24 hours showed needle-like crystals and crystal like solid at
pH4.8-5.6.
A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the
pH was adjusted to 2.76 with 5M HC1. Liquefied phenol, 40uL, and 95% EtOH, 4.25mL, were
added to the solution. Then, 250uL of a 4% acidified ZnCl2 solution was added to the reaction
mixture. The pH of the solution was adjusted from 2.97 to 5.8 using 5M NH4OH and pulling
SOOuL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The
samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24
hours showed crystal-like precipitation from pH 4.6-5.8
A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the
pH was adjusted to 2.76 with 5M HC1. Liquefied phenol, 40uL, and 95% EtOH, 4.25mL, were
added to the solution. Then, 200uL of a 4% acidified ZnCl2 solution was added to the reaction
mixture. The pH of the solution was adjusted from 3.06 to 5.8 using 5M NH4OH and pulling
500uL aliquots at each of the following desired pH: 4.4,4.6, 4.8, 5.0, 5.2, 5.4 (See Figure 9B).
5.6, 5.8. The samples were allowed to sit without stirring for 24 hours. The microscope pictures
taken after 24 hours showed crystal-like precipitation from pH 4.6-5.6.
A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the
pH was adjusted to 2.76 with 5M HC1. Liquefied phenol, 40uL, and 95% EtOH, 4.25mL, were
added to the solution. Then, 150uL of a 4% acidified ZnCl2 solution was added to the reaction
mixture. The pH of the solution was adjusted from 3.09 to 5.8 using 5M NH4OH and pulling
SOOuL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The
samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24
hours showed crystals of various sizes and shapes frompH 5.0-5.2.
A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the
pH was adjusted to 2.76 with 5M HC1. Liquefied phenol, 40uL, and 95% EtOH, 4.25mL, were
added to the solution. Then, lOOuL of a 4% acidified ZnCl2 solution was added to the reaction
mixture. The pH of the solution was adjusted from 3.09 to 5.8 using 5M NH4OH and pulling
SOOuL aliquots at each of the following desired pH: 4.4,4.6, 4.8, 5.0 (See Figure 10A). 5.2, 5.4
(See Figure 10B). 5.6, 5.8. The samples were allowed to sit without stirring for 24 hours. The
microscope pictures taken after 24 hours showed needle-like crystals at pH 5.0 and various
shapes and sizes of crystalline material from pH 5.2-5.6.
A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the
pH was adjusted to 2.76 with 5M HC1. Liquefied phenol, 20uL, and 95% EtOH, 4.25mL, were
added to the solution. Then, 250uL of a 4% acidified ZnCl2 solution was added to the reaction
mixture. The pH of the solution was adjusted from 3.08 to 5.8 using 5M NH4OH and pulling
500uL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The
samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24
hours showed crystal-like precipitation from pH 4.8-5.8.
A fresh 15mg/mL solution of HDVI2 was prepared in 250mM ammonium acetate buffer and the
pH was adjusted to 2.76 with 5M HC1. Liquefied phenol, 20uL, and 95% EtOH, 4.25mL, were
96
added to the solution. Then, 200uL of a 4% acidified ZnCl2 solution was added to the reaction
mixture. The pH of the solution was adjusted from 3.05 to 5.8 using 5M NH4OH and pulling
500uL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The
samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24
hours showed very little crystal-like solids.
A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the
pH was adjusted to 2.76 with 5M HC1. Liquefied phenol, 20uL, and 95% EtOH, 4.25mL, were
added to the solution. Then, 200uL of a 4% acidified ZnCl2 solution was added to the reaction
mixture. The pH of the solution was adjusted from 3.05 to 5.8 using 5M NH4OH and pulling
500uL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The
samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24
hours showed very little crystal-like solids.
A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the
pH was adjusted to 2.76 with 5M HC1. Liquefied phenol, 20uL, and 95% EtOH, 4.25mL, were
added to the solution. Then, lOOuL of a 4% acidified ZnCl2 solution was added to the reaction
mixture. The pH of the solution was adjusted from 3.06 to 5.8 using 5M NH4OH and pulling
SOOuL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The
samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24
hours showed very little crystal-like solids.
9.9 Co-crystallization of HIM2 and IN105 with zinc
9.9.1 Preparation of R-type Co-crystallized Zn Complexes of HIM2 and ENflOS
9.9.2 50:50 (HIM2:IN105)
A fresh solution of HIM2 and INI 05 was prepared by dissolving 37.3mg HIM2 and 36.4mg
IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.84
using 5M HC1. Solid phenol was melted in a 40-60° C warm water bath. 16uL of melted phenol
and 1.75mL of 95% EtOH were added to the reaction flask. Then, 80uL of 4% acidified aqueous
ZnCl2 was added to the reaction flask. The pH of the solution was then adjusted from 3.19 to
5.60 using 5M NH4OH and pulling O.SOOmL aliquots at each of the following desired pH: 4.4,
4.6,4.8, 5.0, 5.2, 5.4 and 5.6. The samples were allowed to sit without stirring for 4 hours. The
97
microscope pictures taken after 4 hours show various sizes and shapes of crystals from the pH
range 4.4 to 5.6.
A fresh solution of HIM2 and IN105 was prepared by dissolving 37.1mg HIM2 and 35.9mg
IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 3.03
using 5M HC1. Solid phenol was melted in a 40-60° C warm water bath. 16uL of melted phenol
and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4% acidified aqueous
ZnCl2 was added to the reaction flask. The pH of the solution was then adjusted from 3.38 to
5.60 using 5M NH4OH and pulling O.SOOmL aliquots at each of the following desired pH: 4.4,
4.6, 4.8, 5.0 (See Figure 11 A). 5.2 (See Figure 11B). 5.4 and 5.6 (See Figure 12A). The
samples were allowed to sit without stirring for 4 hours. The microscope pictures taken after 4
hours show mostly short, needle-like crystals from the pH range 4.6 to 5.6.
9.9.3 70:30 (HIM2:IN105)
A fresh solution of HIM2 and INI 05 was prepared by dissolving 53.4mg HIM2 and 23,2 mg
IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.62
using 5M HC1. Solid phenol was melted in a 40-60° C warm water bath. 16uL of melted phenol
and 1.75mL of 95% EtOH were added to the reaction flask. Then, 80uL of 4% acidified aqueous
ZnCl2 was added to the reaction flask. The pH of the solution was then adjusted from 3.02 to
5.60 using 5M NH4OH and pulling O.SOOmL aliquots at each of the following desired pH: 4.4,
4.6, 4.8, 5.0, 5.2 (See Figure 12B). 5.4 and 5.6. The samples were allowed to sit without
stirring for 1 hour. The microscope pictures taken after 1 hour show various sizes and shapes of
crystal-like precipitation from the pH range 4.4 to 5.6.
A fresh solution of HIM2 and INI05 was prepared by dissolving 53.6mg HIM2 and 24.5mg
IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.89
using 5M HC1. Solid phenol was melted in a 40-60° C warm water bath. 16^L of melted phenol
and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4% acidified aqueous
ZnCl2 was added to the reaction flask. The pH of the solution was then adjusted from 3.28 to
5.60 using 5M NtLfOH and pulling O.SOOmL aliquots at each of the following desired pH: 4.4,
4.6, 4.8, 5.0, 5.2 (See Figure 13A). 5.4 and 5.6. The samples were allowed to sit without
stirring for 1 hour. The microscope pictures taken after 1 hour show mostly various sizes and
shapes of crystal-like precipitation from the pH range 4.6 to 4.8 and many, short, needle-like
crystals from the pH range 5.0 to 5.4.
9.9.4 30:70 (HIM2:IN105)
A fresh solution of HIM2 and IN 105 was prepared by dissolving 23.3mg HIM2 and 54.7mg
IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.84
using 5M HC1. Solid phenol was melted in. a 40-60° C warm water bath. 16u.L of melted phenol
and 1.75mL of 95% EtOH were added to the reaction flask. Then, 80uL of 4% acidified aqueous
ZnCl2 was added to the reaction flask. The pH of the solution was then adjusted from 3.27 to
5.60 using 5M NH4OH and pulling O.SOOmL aliquots at each of the following desired pH: 4.4,
4.6, 4.8, 5.0, 5.2, 5.4 and 5.6. The samples were allowed to sit without stirring for 1 hour. The
microscope pictures taken after 1 hour show mostly various sizes and shapes of crystal-like
precipitation from the pH range 4.4 to 5.0 and few, needle-like crystals from the pH range 5.2 to
5.6.
A fresh solution of H1M2 and IN105 was prepared by dissolving 24.8mg HIM2 and 54.9rng
INI05 in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 3.09
using 5M HC1. Solid phenol was melted in a 40-60° C warm water bath. 16u,L of melted phenol
and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4% acidified aqueous
ZnCl2 was added to the reaction flask. The pH of the solution was then adjusted from 3.47 to
5.60 using 5M NH4OH and pulling O.SOOmL aliquots at each of the following desired pH: 4.4,
4.6, 4.8, 5.0, 5.2, 5.4 and 5.6 (See Figure 13B). The samples were allowed to sit without
stirring for 1 hour. The microscope pictures taken after 1 hour show mostly various circular sizes
of crystal-like precipitation from the pH range 4.4 to 5.0 and crystals various shapes and sizes
from the pH range 5.2 to 5.6.
9.9.5 Preparation of R-type Co-crystallized Zn Complexes of HIM2 and IN105
9.9.6 50:50 (HIM2:IN105)
A fresh solution of HIM2 and IN105 was prepared by dissolving 37.4mg HIM2 and 35.9mg
INI 05 in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.60
using 5M HC1. Solid phenol was melted in a 40-60° C warm water bath. 16jiL of melted phenol
and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4% acidified aqueous
ZnCl2 was added to the reaction flask. The pH of the solution was then adjusted from 2.15 to
5.60 using 5M NHjOH and pulling O.SOOmL aliquots at each of the following desired pH: 4.4,
4.6,4.8, 5.0, 5.2, 5.4 and 5.6. The samples were allowed to sit without stirring for 24 hours. The
microscope pictures taken after 24 hours showed crystal solids of various shapes and sizes from
pH=4.6-5.6.
99
9.9.7 70:30 (HIM2:IN105)
A fresh solution of HIM2 and IN 105 was prepared by dissolving 57.0mg H1M2 and 24.5mg
INI05 in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.43
using 5M HC1. Solid phenol was melted in a 40-60° C warm water bath. 16uL of melted phenol
and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4% acidified aqueous
ZnCI2 was added to the reaction flask. The pH of the solution was then adjusted from 2.92 to
5.60 using 5M NH4OH and pulling O.SOOmL aliquots at each of the following desired pH: 4.4,4.6
(See Figure 14A). 4.8, 5.0, 5.2, 5.4 and 5.6. The samples were allowed to sit without stirring for
24 hours. The microscope pictures taken after 24 hours showed needle-like crystals from pH 5.0-
9.9.8 30:70 (HIM2:IN105)
A fresh solution of HIM2 and IN105 was prepared by dissolving 24.1mg HIM2 and 53.8mg
IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.35
using 5M HC1. Solid phenol was melted in a 40-60° C warm water bath. 16uL of melted phenol
and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4% acidified aqueous
ZnCl2 was added to the reaction flask. The pH of the solution was then adjusted from 2.60 to
5.60 using 5M NH,tOH and pulling O.SOOmL aliquots at each of the following desired pH: 4.4,
4.6, 4.8, 5.0 (See Figure 14B). 5.2, 5.4 and 5.6. The samples were allowed to sit without
stirring for 24 hours. The microscope pictures taken after 24 hours showed needle-like crystals
from pH 5.0-5.2.
9.9.9 Preparation of R-type Co-crystallized Zn Complexes of HIM2 and
Human Insulin
9.9.10 50:50 (HIM2:Insulin)
A fresh solution of HIM2 and Insulin was prepared by dissolving 39.2mg HIM2 and 36.7mg
Insulin in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.53
using 5M HC1. Solid phenol was melted in a 40-60° C warm water bath. 16uL of melted phenol
and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4% acidified aqueous
ZnCl2 was added to the reaction flask. The pH of the solution was then adjusted from 2.82 to
5.60 using 5M NH^OH and pulling O.SOOmL aliquots at each of the following desired pH: 4.4,
4.6,4.8, 5.0, 5.2 (See Figure ISA"). 5.4 and 5.6. The samples were allowed to sit without stirring
for 24 hours. The microscope pictures taken after 24 hours showed various shapes and sizes of
crystal-like solid from pH 5.2 and 5.4. Many tiny, needle-like crystals were observed at pH 5.6.
9.9.11 70:30 (HIM2:Insulin)
A fresh solution of HIM2 and Insulin was prepared by dissolving 56.5mg HIM2 and 20.2mg
Insulin in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 3.23
using 5M HC1. Solid phenol was melted in a 40-60° C warm water bath. 16uL of melted phenol
and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4% acidified aqueous
ZnClj was added to the reaction flask. The pH of the solution was then adjusted from 2.82 to
5.60 using 5M NH4OH and pulling O.SOOmL aliquots at each of the following desired pH: 4.4,
4.6,4.8, 5.0, 5.2, 5.4 and 5.6. The samples were allowed to sit without stirring for 24 hours. The
microscope pictures taken after 24 hours showed various shapes and sizes of crystal-like solid
from pH 5.2 and 5.6.
9.9.12 30:70 (HIM2:InsuIin)
A fresh solution of HIM2 and Insulin was prepared by dissolving 21.8mg HIM2 and 49.2mg
Insulin in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 3.23
using 5M HC1. Solid phenol was melted in a 40-60° C warm water bath. 16u,L of melted phenol
and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4% acidified aqueous
ZnCl2 was added to the reaction flask. The pH of the solution was then adjusted from 2.93 to
5.60 using 5M NH4OH and pulling O.SOOmL aliquots at each of the following desired pH: 4.4,
4.6,4.8, 5.0, 5.2, 5.4 (See Figure 15B) and 5.6. The samples were allowed to sit without stirring
for 24 hours. The microscope pictures taken after 24 hours showed flat, snowflake-Hke crystals
at pH 4.8. At pH 5.0, there was a mix of needle-like and snowflake-like crystals. From pH 5.2-
5.6 there were many tiny, needle-like crystals observed.
10 Aqueous Solubility of Zn Complexes
Two hundred microliters of 0.1M Phosphate Buffer Saline (PBS, filtered, pH = 7.4) was added to
a 1 mL conical reaction vial. To this vial, a small amount of sample was added slowly until
saturation is observed. Periodically, the solution was vortexed. Upon saturation, the vial was
placed in a small centrifuge tube and the sample was centrifuged at 2000RPM for 3min at RT.
After centrifugation, lOuL of the sample was removed from the supernatant and diluted in 490uL
buffer (0.1M PBS). This diluted sample was analyzed via HPLC to determine its' concentration.
11 In vitro Enzyme Resistance Examples for Zn IN105 Complexes
insulin compound conjugates (INI 05) were provided in 10 mM sodium phosphate buffer (a pH of
about 7.4) and their concentrations were determined by HPLC (the solutions are diluted with
buffer so that equimolar comparisons can be made between parent and conjugates -0.6 mg/mL).
Lyophilized chymotrypsin enzyme was resuspended in 1 mM HC1 to a concentration of -7.53
U/mL. A 1.53 mL aliquot of each sample was added to sample tubes and 0.850 mL into control
tubes. Samples were tested in duplicate along with four control tubes per sample. Aliquots were
incubated at 37°C in a thermomixer for 15 minutes. Then 17 uL of chymotrypsin enzyme was
added to each sample tube. Five uL of 1 mM HC1 was added to each control tube. Immediately
following the additions, 200 uL was removed from the sample and the control tubes and placed
into 50 uL of 1% TFA previously aliquoted out into centrifuge rubes. This sample serves as T=0.
The sampling procedure for Insulin compound (Zn free), INI 05 (Zn free) and Insulin compound
(regular insulin compound) was repeated at the following intervals: 0, 2, 5, 8, 12, 15, and 30
minutes. The control procedure was repeated at the following intervals: 0, 8, 15, 30 minutes. For
T-type and R-type samples, the procedure was repeated at the following intervals: 0, 5, 8, 12, 30,
40 and 60 minutes. The control procedure for the Zn complexes was repeated at the following
intervals: 0, 12, 40 and 60 minutes. Samples were stored at -20°C until analysis can occur via
HPLC. HPLC was performed to determine percent degradation relative to the respective T= 0
minute for each digest. The natural log of the percent remaining was plotted versus time and a
linear regression run for each digest. The half life was calculated using the equation: t ./,:
12 Formulation Examples
12.1 Liquid Formulation Examples
12.1.1 Buffer Solution for R6 type Zn-HIM2 Buffer Study
Components Amount in 1mL solution
Dibasic Sodium Phosphate 1.88mg
Insulin component 3.7 mg (100 units)
Glycerol 16.0 mg
Phenol (or m-Cresol) 3.0 mg
Zinc 0.037mg (1 % w/w of insulin)*
pH 7.4 to 7.8**
* Adjustment of the amount of Zinc to 0.037mg/ml per 3.7mg/ml insulin by adding Zinc chloride and to be
based the zinc content in the Zinc HIM2 solid.
** pH may be adjusted with HC1 10% and/or sodium hydroxide 10%
12.1.2 Preparation of Capric Acid/Laurie Acid Formulation Oral Liquid
Diluent
We transferee! approximately 60% of the required sterile water volume into a suitable container.
We added the appropriate amount (as indicated in the table below) of tromethamine, trolamine,
citric acid anhydrous, and sodium hydroxide pellets to the container and mixed well until
dissolved. We adjusted the temperature to 21 - 25° C (or room temperature) and measured the
pH of the liquid. We adjusted the pH to 7.7 - 7.9 as necessary using IN sodium hydroxide or IN
103
hydrochloric acid. We then adjusted the temperature to 45 - 50° C by warming on a hotplate and
maintained this temperature. We then added the capric acid to the warm solution and mixed until
the capric acid was dissolved. We adjusted the temperature to 21 - 25° C (or room temperature)
and measured the pH of the liquid. As needed, we adjusted the pH to 7.7 - 7.9 using IN sodium
hydroxide or IN hydrochloric acid. We then mixed the solution for 5 minutes. We added
appropriate amount of sterile water to equal 100% of the required volume and mixed well.
Component Percentage (%w/v)
Tromethamine 4.24
Trolamine 5.22
Citric Acid Anhydrous 6.72
Sodium Hydroxide Pellets 1.88
Capric Acid 0.50
Laurie Acid 0.50
Sodium Hydroxide, IN As Needed to Adjust pH 7.7-7.9
Hydrochloric Acid, IN As Needed to Adjust pH 7.7-7.9
Sterile Water Dilute to Required Volume
INI 05, HIM2 or ZnHM2 was weighed out in amounts necessary to achieve appropriate
concentration for dosing studies, e.g., 1 mg IN105 (of protein) was weighed out and combined
with 1 rnL of formulation to yield a 1 mg/mL IN105 in formulation.
12.1.3 Preparation of Oleic acid/capric Acid/Laurie Acid/Cholate Formulation
Oral Liquid Diluent
An oral liquid formulation of R-type Zn HIM2 was prepared having the components shown in the
following table:
Component Percentage (%w/v)
Tromethamine 4.24
Trolamine 5.22
Citric Acid Anhydrous 6.72
Sodium Hydroxide Pellets 1.88
Sodium Cholate 3.00
OleicAcid 1.00CapricAcid 0.50Laurie Acid 0.50Sucralose Solution, 25% 0.80Strawberry Flavor 0.40Sodium Hydroxide, 1N As Needed to Adjust pH7.7-7.9
Hydrochloric Acid, IN As Needed to AdjustpH 7.7- 7.9
Sterile Water Dilute to Required Volume
Oral liquid samples were prepared to contain 1 mg/mL protein equivalent of R-type Zn HIM2
(ZnHIM2-R). The ZnHIM2-R was removed from the Freezer (-20 °C), placed in a dessicator and
allowed to come to room temperature. A 1 mg/mL protein equivalent of ZnHIM2-R was
prepared in oral liquid diluent solution as follows. 6.4 mg of ZnHIM2-R was weighed. Then, 5.0
mL of oral liquid diluent was transferred into the container and gently swirled to mix. The
solution took approximately 45 minutes to dissolve. The resulting solution was a suspension
(cloudy appearance). Prior to dosing, the solution was gently swirled for 60 seconds to ensure the
solution was a homogeneous solution. For ZnHIM2-R, the protein content was 78.6%, 1 mg/mL
protein equivalent, quantity = 5 mL. Amount of ZnHIM2-R = (1 mg/mL) / (0.786)} * (5.0 mL) =
6.4 mg. ZnHIM2-R Concentration = (6.4 mg)/(5.0 mL) = 1.28 mg/mL (equivalent to 1 mg/mL
adjusted for protein content).
12.1.4 Preparation of Capric Acid Liquid Formulations
We transfered approximately 60% of the required sterile water volume into a suitable container.
We added the appropriate amount (as indicated in the table below) of tromethamine, trolamine,
citric acid anhydrous, and sodium hydroxide pellets to the container and mixed well until
dissolved. We adjusted the temperature to 21 - 25° C (or room temperature) and measured the
pH of the liquid. We adjusted the pH to 7.7 - 7.9 as necessary using IN sodium hydroxide or IN
hydrochloric acid. We then adjusted the temperature to 45 - 50° C by warming on a hotplate and
maintained this temperature. We then added the capric acid to the warm solution and mixed until
the capric acid was dissolved. We adjusted the temperature to 21 - 25° C (or room temperature)
and measured the pH of the liquid. As needed, we adjusted the pH to 7.7 — 7.9 using IN sodium
hydroxide or IN hydrochloric acid. We then mixed the solution for 5 minutes. We added
appropriate amount of sterile water to equal 100% of the required volume and mixed well.
Component % w/v
Tromethamine 4.24
Trolamine 5.22
Citric Acid Anhydrous 6.72
Sodium Hydroxide Pellets 1.88
Capric Acid 0.9, 1.5, 3.0 or 6.0
Sodium Hydroxide, IN As Needed to Adjust pH
Hydrochloric Acid, IN As Needed to Adjust pH
Sterile Water To Required Volume
INI 05 was weighed out in amounts necessary to achieve appropriate concentration for dosing
studies, e.g., 1 mg IN105 (of protein) was weighed out and combined with 1 mL of formulation to
yield a 1 mg/mL IN 105 in formulation.
12.1.5 Caprate and/or Laurate in Phosphate Buffer Liquid Formulations
Preparation of 100 mM Sodium Phosphate Buffer, pH 7.8 or 8.2. We transferred 1.17 grams of
Monosodium Phosphate Monohydrate to a 1-L flask. Approximately 500 mL of sterile water was
added and mixed well until dissolved. We then added 24.58 grams of Sodium Phosphate Dibasic
Heptahydrate and mixed well until dissolved. Diluted to volume with sterile water and mixed
well. Filtered through a 0.22 urn filter. We adjusted the pH to 7.8 or 8.2 with IN HC1 or IN
NaOH.
For IN105, we transferred 60% of the appropriate volume of the phosphate buffer pH 7.8 or 8.2
into a suitable container. We then added an amount of caprate calculated to produce 3% w/v of
the final solution and mixed well until dissolved. We then adjusted the pH to 7.8 or 8.2 with IN
HC1 or IN NaOH. We diluted to the appropriate volume (e.g., 100 mL) with phosphate buffer
pH 7.8 or 8.2.
Component % w/v
Sodium Caprate 3.0
100 mM Sodium Phosphate Buffer, pH 7.8 or 8.2 QS to 100%
106
For BN-054, we weighed 400 grams of 100 mM Sodium Phosphate Buffer, pH 7.8 in a suitable
container. We added 9.7 grams of Sodium Caprate and 11.1 grams Sodium Laurate and mixedwell until dissolved. We added appropriate amount of 100 mM Sodium Phosphate Buffer, pH
7.8, to equal a net weight of 500 grams.
Component % w/w
Sodium Caprate 1.94
Sodium Laurate 2.22
100 mM Sodium Phosphate Buffer, pH 7.8 QS to 100%
12.1.6 Liquid Formulation with Arginine or Trolamine
Preparation of 100 mM Sodium Phosphate Buffer, pH 7.8. We transferred 1.17 grams of
Monosodium Phosphate Monohydrate to a 1-L flask. Approximately 500 mL of sterile water was
added and mixed well until dissolved. We then added 24.58 grams of Sodium Phosphate Dibasic
Heptahydrate and mixed well until dissolved. Diluted to volume with sterile water and mixed
well. Filtered through a 0.22 urn filter. We adjusted the pH to 7.8 with IN HC1 or IN NaOH.
We transferred 60% of the appropriate volume of the phosphate buffer pH 7.8 into a suitable
container. We added the appropriate amount (as indicated in the table below) of arginine or
trolamine to the container and mixed well until dissolved. We then added an amount of caprate
calculated to produce 3% w/v of the final solution and mixed well until dissolved. We then
adjusted the pH to 7.8 with IN HC1 or IN NaOH. We diluted to the appropriate volume (e.g.,
100 mL) with phosphate buffer pH 7.8.
Component % w/v
Sodium Caprate 3.0
Arginine or Trolamine 0.4 or 1.2
100 mM Sodium Phosphate Buffer, pH 7.8 QS to 100%
INI 05 was weighed out in amounts necessary to achieve appropriate concentration for dosing
studies, e.g., 1 mg IN 105 (of protein) was weighed out and combined with 1 mL of formulation to
yield a 1 mg/mL INI 05 in formulation.
12.1.7 Liquid Formulation with Caprylic Acid
We transfered approximately 60% of the required sterile water volume into a suitable container.
We added the appropriate amount (as indicated in the table below) of tromethamine, trolamine,
citric acid anhydrous, and sodium hydroxide pellets to the container and mixed well until
107
dissolved. We adjusted the temperature to 21 - 25° C (or room temperature) and measured the
pH of the liquid. We adjusted the pH to 7.7 - 7.9 as necessary using IN sodium hydroxide or IN
hydrochloric acid. We then adjusted the temperature to 45 - 50° C by warming on a hotplate and
maintained this temperature. We then added the caprylic acid to the warm solution and mixed
until the caprylic acid was dissolved. We adjusted the temperature to 21 - 25° C (or room
temperature) and measured the pH of the liquid. As needed, we adjusted the pH to 7.7 - 7.9
using IN sodium hydroxide or IN hydrochloric acid. We then mixed the solution for 5 minutes.
We added appropriate amount of sterile water to equal 100% of the required volume and mixed
well.
Component
INI 05 was weighed out in amounts necessary to achieve appropriate concentration for dosingstudies, e.g., 1 mg IN105 (of protein) was weighed out and combined with 1 mL of formulation to
yield a 1 mg/mL INI 05 in formulation.
12.1.8 Liquid Formulation with Linoleic Acid
Preparation of 100 mM Sodium Phosphate Buffer, pH 7.8. We transferred 1.17 grams of
Monosodium Phosphate Monohydrate to a 1-L flask. Approximately 500 mL of sterile water was
added and mixed well until dissolved. We then added 24.58 grams of Sodium Phosphate Dibasic
Heptahydrate and mixed well until dissolved. Diluted to volume with sterile water and mixed
well. Filtered through a 0.22 urn filter. We adjusted the pH to 7.8 with IN HC1 or IN NaOH.
We transferred 60% of the appropriate volume of the phosphate buffer pH 7.8 into a suitable
container. We then added an amount of linoleic acid sodium salt calculated to produce 3% w/v of
the final solution and mixed well until dissolved. We then adjusted the pH to 7.8 with IN HC1 or
IN NaOH. We diluted to the appropriate volume (e.g., 100 mL) with phosphate buffer pH 7.8.
108
Component % w/v
Linoleic Acid Sodium Salt 3.0
100 mM Sodium Phosphate Buffer, pH 7.8 QS to 100%
12.1.9 Preparation of Capric Acid and Laurie Acid Liquid Formulations
We transferee! approximately 60% of the required sterile water volume into a suitable container.
We added the appropriate amount (as indicated in the table below) of tromethamine, trolamine,
citric acid anhydrous, and sodium hydroxide pellets to the container and mixed well until
dissolved. We adjusted the temperature to 21 - 25° C (or room temperature) and measured the
pH of the liquid. We adjusted the pH to 7.7 - 7.9 as necessary using IN sodium hydroxide or IN
hydrochloric acid. We then adjusted the temperature to 45 - 50° C by warming on a hotplate and
maintained this temperature. We then added the capric acid and/or lauric acid to the warm
solution .and mixed until the capric acid and/or lauric acid were dissolved. We adjusted the
temperature to 21 - 25° C (or room temperature) and measured the pH of the liquid. As needed,
we adjusted the pH to 7.7 - 7.9 using IN sodium hydroxide or IN hydrochloric acid. We then
mixed the solution for 5 minutes. We added appropriate amount of sterile water to equal 100% of
the required volume and mixed well.
.
12.2 Solid Dosage Formulation Examples
12.2.1 Preparation and dissolution profile of caprate/laurate solid dosage
formulation using Nobex-IN105-[854]-
Transfer approximately 58 mg of sodium caprate, 57 mg of sodium laurate, 286 mg mannitol, 30
mg of sodium starch glycolate, and 6 mg (protein) of Nobex-IN105 onto a piece of weigh paper
and blend thoroughly. Transfer the blend to the press and compress at approximately 350 psi to
form a tablet.
Solid Dosage Form (Tablets) Formulation Nobex-IN105-r8541 58 mg Caprate and 57 mg
Laurate per Tablet
Component mg per Tablet
The dissolution testing was carried out using a USP apparatus 2 dissolution unit. The medium
was water, paddle speed 50 rpm, and the medium volume was 500 mL. The dissolution samples
were analyzed by HPLC using a gradient system. The mobile phases were water with 0.1% TFA
(mobile phase A) and acetonitrile with 0.1% TFA (mobile phase B). The gradient utilized was: 0
minutes 100% mobile phase A, 11 minutes 65% mobile phase A, 15 minutes 20% mobile phase
A, 16 minutes 20% mobile phase A, 17 minutes 100% mobile phase A. The wavelength was 214
nm and column was a C18 (150 * 2 mm). The following tables and graphs summarize the
dissolution data obtained for the dissolution testing of Nobex-Zn-IN105 Tablets Formulation
[854] containing 6 mg Zn-IN105 (protein), 286 mg Mannitol, 58 mg Sodium Caprate, 57 mg
Sodium Laurate, and 30 mg sodium starch glycolate (Bxplotab):
Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets [8541, % IN 105
12.2.2 Solid Dosage Form (Tablet) Formulation Preparation 143 mg Caprate
and 140 mg Laurate per Tablet
Preparation of Formulation Nobex-IN105-[8561
Transfer approximately 143 mg of sodium caprate, 140 mg of sodium laurate, 150 mg mannitol,
30 mg of sodium starch glycolate, and 6 mg (protein) of Nobex-IN105 onto a piece of weigh
paper and blend thoroughly. Transfer the blend to the press and compress at approximately 350
psi to form a tablet.
Solid Dosage Form (Tablets) Formulation Nobex-lNlOS-18561 143 me Caprate and 140 mg
Laurate per Tablet
Component mg per Tablet
Sodium Caprate 143
Sodium Laurate 140
Mannitol 150
Explotab (sodium starch glycolate) 30
Nobex-IN105 (protein) 6
The dissolution testing was carried out using a USP apparatus 2 dissolution unit. The medium
was water, paddle speed 50 ipm, and the medium volume was 500 mL. The dissolution samples
were analyzed by HPLC using a gradient system. The mobile phases were water with 0.1% TFA
(mobile phase A) and acetonitrile with 0.1% TFA (mobile phase B). The gradient utilized was: 0
minutes 100% mobile phase A, 11 minutes 65% mobile phase A, 15 minutes 20% mobile phase
A, 16 minutes 20% mobile phase A, 17 minutes 100% mobile phase A. The wavelength was 214
nm and column was a CIS (150 * 2 mm). The following tables and graphs summarize the
dissolution data obtained for the dissolution testing of Nobex-Zn-IN105 Tablets containing 6 mg
Zn-IN105 (protein), 150 mg Mannitol, 143 mg Sodium Caprate, 140 mg Sodium Laurate, and 30
mg sodium starch glycolate (Explotab):
Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets [8561. % IN105
Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets [8561, % Laurate
12.2.3 Solid Dosage Form (Tablet) Formulation Preparation 143 mg Caprate
per Tablet
Preparation of Formulation Nobex-IN105-I8591
Transfer approximately 143 mg of sodium caprate, 150 mg mannitol, 30 mg of sodium starch
glycolate, and 6 mg (protein) of Nobex-IN105 onto a piece of weigh paper and blend thoroughly.
Transfer the blend to the press and compress at approximately 350 psi to form a tablet.
Solid Dosage Form (Tablets^ Formulation Nobex-IN105-r8591143 mg Caprate per Tablet
Component mg per Tablet
The dissolution testing was carried out using a USP apparatus 2 dissolution unit. The medium
was water, paddle speed 50 rpm, and the medium volume was 500 mL. The dissolution samples
were analyzed by HPLC using a gradient system. The mobile phases were water with 0.1% TFA
(mobile phase A) and acetonitrile with 0.1% TFA (mobile phase B). The gradient utilized was: 0
minutes 100% mobile phase A, 11 minutes 65% mobile phase A, 15 minutes 20% mobile phase
A, 16 minutes 20% mobile phase A, 17 minutes 100% mobile phase A. The wavelength was 214
nm and column was a CIS (150 x 2 mm). The following tables and graphs summarize the
dissolution data obtained for the dissolution testing of Nobex-Zn-IN105 Tablets containing 6 mg
Zn-IN105 (protein), 150 mg Mannitol, 143 mg Sodium Caprate, and 30 mg sodium starch
glycolate (Explotab):
Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets 18591. % IN1Q5
12.2.4 Solid Dosage Form (Tablet) Formulation Preparation 286 mg Caprate
per Tablet
Preparation of Formulation Nobex-IN105-[8601
Transfer approximately 286 mg of sodium caprate, 150 mg manm'tol, 30 mg of sodium starch
glycolate, and 6 mg (protein) of Nobex-IN105 onto a piece of weigh paper and blend thoroughly.
Transfer the blend to the press and compress at approximately 350 psi to form a tablet.
Solid Dosage Form (Tablets) Formulation Nobex-IN105-r8601 286mg Caprate per Tablet
Component mg per Tablet
The dissolution testing was carried out using a USP apparatus 2 dissolution unit. The medium
was water, paddle speed 50 rpm, and the medium volume was 500 mL. The dissolution samples
were analyzed by HPLC using a gradient system. The mobile phases were water with 0.1% TFA
(mobile phase A) and acetonitrile with 0.1% TFA (mobile phase B). The gradient utilized was: 0
minutes 100% mobile phase A, 11 minutes 65% mobile phase A, 15 minutes 20% mobile phase
A, 16 minutes 20% mobile phase A, 17 minutes 100% mobile phase A. The wavelength was 214
nm and column was a CIS (150 x 2 mm). The following tables and graphs summarize the
dissolution data obtained for the dissolution testing of Nobex-Zn-IN105 Tablets containing 6 mg
Zn-IN105 (protein), 150 mg Mannitol, 286 mg Sodium Caprate, and 30 mg sodium starch
glycolate (Explotab):
Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets [8601. % IN105
12.2.5 Solid Dosage Form (Tablet) Formulation Preparation 100 mg Caprate
per Tablet
Preparation of Formulation Nobex-IN105-[8611
Transfer approximately 100 mg of sodium caprate, 150 mg mannitol, 25 mg of sodium starch
glycolate, and 6 mg (protein) of Nobex-IN105 onto a piece of weigh paper and blend thoroughly.
Transfer the blend to the press and compress at approximately 350 psi to form a tablet.
115
Solid Dosage Form (Tablets) Formulation Nobex-IN105-F86n 100 me Caprate per Tablet
Component mg per Tablet
The dissolution testing was carried out using a USP apparatus 2 dissolution unit. The medium
was water, paddle speed 50 rpm, and the medium volume was 500 mL. The dissolution samples
were analyzed by HPLC using a gradient system. The mobile phases were water with 0.1% TFA
(mobile phase A) and acetonitrile with 0.1% TFA (mobile phase B). The gradient utilized was: 0
minutes 100% mobile phase A, 11 minutes 65% mobile phase A, 15 minutes 20% mobile phase
A, 16 minutes 20% mobile phase A, 17 minutes 100% mobile phase A. The wavelength was 214
nm and column was a CIS (150 x 2 mm). The following tables and graphs summarize the
dissolution data obtained for the dissolution testing of Nobex-Zn-IN105 Tablets containing 6 mg
Zn-IN105 (protein), 150 mg Mannitol, 100 mg Sodium Caprate, and 25 mg sodium starch
glycolate (Explotab):
12.2.6 Solid Dosage Form (Tablet) Formulation Preparation 150 mg Caprate
per Tablet
Preparation of Formulation Nobex-IN105-[8621
Transfer approximately 150 mg of sodium caprate, 150 mg mannitol, 25 mg of croscarmellose
sodium, and 6 mg (protein) of Nobex-lN105 onto a piece of weigh paper and blend thoroughly.
Transfer the blend to the press and compress at approximately 350 psi to form a tablet.
Solid Dosage Form (Tablets) Formulation Nobex-IN105-[8621 150 mg Caprate per Tablet
Component mg per Tablet
The dissolution testing was carried out using a USP apparatus 2 dissolution unit. The medium
was water, paddle speed 50 rpm, and the medium volume was 500 mL. The dissolution samples
were analyzed by HPLC using a gradient system. The mobile phases were water with 0.1% TFA
(mobile phase A) and acetoniteile with 0.1% TFA (mobile phase B). The gradient utilized was: 0
minutes 100% mobile phase A, 11 minutes 65% mobile phase A, 15 minutes 20% mobile phase
A, 16 minutes 20% mobile phase A, 17 minutes 100% mobile phase A. The wavelength was 214
nm and column was a CIS (150 * 2 mm). The following tables and graphs summarize the
dissolution data obtained for the dissolution testing of Nobex-Zn-IN105 Tablets containing 6 mg
Zn-IN105 (protein), 150 mg Mannitol, 150 mg Sodium Caprate, and 25 mg Croscarmellose
Sodium (Explotab):
13 In vitro Enzyme Resistance Examples
insulin compound conjugates (HIM2) were provided in 10 mM sodium phosphate buffer (a pH of
about 7.4) and their concentrations are determined by HPLC (the solutions are diluted with buffer
so that equimolar comparisons can be made between parent and conjugates ~0.6 mg/mL).
Lyophilized chymotrypsin enzyme was resuspended in 1 mM HC1 to a concentration of 7.53
U/mL. A 1.53 mL aliquot of each sample was added to sample tubes and 0.850 mL into control
tubes. Samples were tested in duplicate along with four control tubes per sample. Aliquots were
incubated at 37°C in a thermomixer for 15 minutes. Then 17 uL of chymotrypsin enzyme was
added to each sample tube. Five \\L of 1 mM HC1 was added to each control tube. Immediately
following the additions, 200 uL was removed from the sample and the control tubes and placed
into 50 uL of 1% TFA previously aliquoted out into centrifuge tubes. This sample serves as T=0.
The sampling procedure for Insulin (Zn free), HIM2 (Zn free) and Insulin (regular insulin
compound) was repeated at the following intervals: 0, 2, 5, 8, 12, 15, and 30 minutes. The
control procedure was repeated at the following intervals: 0, 8, 15, 30 minutes. For T-type and
R-type samples, the procedure was repeated at the following intervals: 0, 5, 8, 12, 30, 40 and 60
minutes. The control procedure for the Zn complexes was repeated at the following intervals: 0,
12, 40 and 60 minutes. Samples were stored at -20°C until analysis can occur via HPLC. HPLC
was performed to determine percent degradation relative to the respective T= 0 minute for each
digest. The natural log of the pecent remaining was plotted versus time and a linear regression
run for each digest. The half life was calculated using the equation: t./,: = -0.693/slope.
14.1 Extended Mouse Blood Glucose Assay (MBGA)
Six paired-dose groups of 5 male CF-1 mice (Charles River Laboratories; 25-30 g) received
subcutaneous injections of either the insulin compound conjugate (test article) or recombinant
human insulin. The test article was reconstituted with phosphate buffer (0.01M, a pH of about
7.4) containing 0.1% w/w bovine serum albumin and dosed at 100, 66.6, 43.3, 30, 20, and 13.3
|ig/kg. Insulin was reconstituted with phosphate buffer (0.01 M, a pH of about 7.4) containing
0.1% w/w bovine serum albumin and dosed at 50, 33.3, 21.7, 15, 10, and 6.7 ng/kg. After
receiving a subcutaneous dose in the pocket formed by the thigh and groin, animals were returned
to their cages for 30 minutes at room temperature and then were quickly anesthetized and
terminally bled. Blood samples were collected in heparin tubes for glucose assay. If glucose
assay was delayed, the tubes were stored in ice water and re-warmed to room temperature before
assay.
Plasma glucose was measured with a glucometer (e.g., One Touch® Basic; Lifescan), which was
calibrated at the beginning of each day of use according to the manufacturer's instructions. The
potency of the insulin compound conjugate was then calculated relative to the standard curve that
was generated for the recombinant human insulin response. Calculations were based upon the
assumption that recombinant human insulin has a potency of 27.4 IU/mg.
Results are shown in Figures 16-20. Figure 16 shows MBGA biopotency profiles for HIM2.
Figure 17 shows MBGA biopotency profiles for Zn-HIM2 insulin compound product R-type.
Figure 18 shows MBGA biopotency profiles for Zn-HIM2 insulin compound product T-type.
Figure 19 shows MBGA biopotency profiles for Zn-HIM2 insulin compound product with
protamine. Figure 20 shows glucose lowering effect of R-type protamine complex at 30 and 90
minutes post dose. These results show that the biopotency of HIM2 is not significantly reduced
by complexation with Zn"1"1". The R-type protamine complex (see Figure 17 [71) shows greater
glucose reduction at 30 minutes than 90 minutes.
Further, Figures 21-24 show MBGA biopotency profiles for IN-186, IN-192, IN-190, IN-191,
IN-189, IN-178, IN-193, IN-194, IN-185, IN-196 and IN-197 having structures as follows:
14.2.1 Initial HIM2 Studies
Dogs (n=3 or 6) were prepared surgically (isoflurane anesthesia) by placing a catheter in a
femoral artery. The animals were allowed to recover for 16-17 days after which they were fasted
overnight and studied in the conscious state. After a 60 min equilibration period, there was a 20
min control period after which the drug was given by mouth. Zn^-HIM2 R-type Insulin
compound was tested in a buffer solution, prepared as shown in Example Error! Reference
source not found.. In addition, R-type and NPH-type complexes were tested in oral liquid
formulation that contains caprate acid and laurate acid, prepared as shown in Example Error!
Reference source not found..
All 3 test samples were tested at only one dose level (the dose level were identified based on the
previous experimental results). The plasma glucose level was then be clamped at a euglycemic
value by infusion of D-20 through a leg vein for 4 h. Blood samples (4 ml) were taken at -20, 0,
5, 10, 20, 30, 45, 90, 120, 180 and 240 min for measurement of glucose, insulin compound and
C-peptide. Arterial blood samples were obtained as required to clamp the plasma glucose level.
A total of 72 ml of blood was taken in each experiment.
The following measurements were performed: glucose infusion rate, insulin compound
concentration, C-peptide concentration, and plasma C-peptide levels (to allow an estimation of
endogenous insulin compound release). The glucose infusion rate required to maintain
euglycemia provides an index of insulin compound action.
Following the experiment the free end of the catheter was buried subcutaneously and the dogs
were allowed to recover for two weeks prior to another study in which a different test article was
used. Animals were randomized to dose and used a total of 3 times. Total number of dogs was 6.
Figures 25 and 26 show the results.
14.2.2 Initial IN105 Studies
The study was conducted on six (6) overnight fasted conscious mongrel dogs which had been fed
a diet of 34% protein, 46% carbohydrate, 14.5% fat and 5.5% fiber based on dry weight. Each
animal had a silastic catheter inserted into the femoral artery as described elsewhere (1)
approximately three weeks prior to the experiment. On the day of experiment the catheter was
removed from its subcutaneous pocket under local anesthesia. Test article: Nobex-IN105 (Lot.#
KJ-173-095 & KJ-173-116) was provided in the oral fatty acid fonnulation (Nobex-IN-[753]-
040422) at a concentration of 1.0 mg/ml. Each dog received 0.25 mg/kg oral dose of Nobex-
IN105 (1.0 mg/ml@0.25 ml/kg dosing volume). Nobex-IN105 was given at t=0 and glucose (D-
20) was infused through a cephalic vein in order to maintain euglycemia. Arterial blood samples
were drawn for the measurement of insulin and glucose as previously described (1). After the
experiment was completed, the arterial catheter was buried subcutaneously as it was during the
initial surgery.
During the experiment, one dog vomited immediately after the dosing and only a portion of the
dose administered. Therefore, the results from this experiment were reported with and without the
data obtained from this dog.
Arterial plasma insulin levels rose in all six dogs, including outlier (Oral-2i), following the oral
administration of Nobex-IN105. Mean arterial insulin rose from 6.0 ±1.4 uU/ml (6.3 ±1.7
uU/ml, n=5) to a peak of 109.4 ± 31.4 uU/ml (127.8 ± 30.1 uU/ml, n=5) at 10 min postadministration
and then fell so that by 150 min all dogs returned to baseline insulin levels
(Figures 27 and 28).
Euglycemia was maintained by glucose infusion. The glucose infusion rate required to maintain
euglycemia as greatest in the animals with larger rise in arterial insulin (Figures 27 and 28).
Mean Area-Under-The-Glucose Infusion Rate Curve (AUCO-240) was 578.5 ± 144.5 mg/kg/inin
(669.4 ± 137.7 mg/kg/min, n=5).
14.2.3 Solid Formulations
Formulation screening studies using the glucose-clamp model were conducted on overnight
fasted conscious mongrel dogs which had been fed a diet of 34% protein, 46% carbohydrate,
14.5% fat and 5.5% fiber, based on dry weight. Each animal had a silastic catheter inserted into
the femoral artery as described elsewhere (reference 1) approximately three weeks prior to the
experiment. On the day of experiment the catheter was removed from its subcutaneous pocket
under local anesthesia. The test article contained l.Omg/mL Zn INI05 in different liquid
formulations or 5-6mg of IN105 per capsule or tablet. Each dog in every experiment received an
oral liquid dose of approximately 0.25 rug/kg or a capsule or tablet containing 5 or 6mg of IN 105
twice in succession, the first at t = 0 and the second at t = 120 minutes. Glucose (D-20) was
infused through a cephalic vein in order to maintain euglycemia. hi some cases study time was
extended after dosing where the effect lasted beyond 120 minutes. Arterial blood samples were
drawn for the measurement of insulin, glucose and C-peptide as previously described (reference
1). After the experiment was completed, the arterial catheter was replaced into the subcutaneous
tissue.
Formulations, both solution and solid dosage forms, ware prepared with different levels of fatty
acids, buffers, diluents and disintegrants. To minimize variables, the liquid and the solid
formulations contained consistent levels of IN 105 and each excipient (Capric, Laurie, Caprylic,
Myristic, Linoleic), thus varying only the relative amounts of fatty acid content, buffer, diluents
(mannitol or micro crystalline cellulose), and/or disintegrant (Explotab). The glucose infusion
rate and IN105 absorption (plasma insulin immuno-reactivity) data were evaluated and compared
with each dosed formulation.
Initially, experiments were carried out to simplify and refine the optimized liquid formulation
(reference 2) to a liquid formulation that would be more readily converted to a solid dosage form.
This was carried out by substituting the free fatty acid with the corresponding sodium salt (e.g.
capric acid replaced by sodium caprate) as well as removing the buffer components (citric acid,
trolamine, tromethamine, sodium hydroxide) that were deemed to be no longer required.
Additionally, the effect of other fatty acids such as linoleic, caprylic and myristric acids and the
amino acid, arginine, were examined for their effects on the absorption of IN105.
After an initial set of prototype screens using one to two dogs per experiments few prototype
formulations were selected and tested in additional dogs to better determine the variability and
consistency between formulations and individual animals.
A dissolution method was developed and dissolution studies were carried out on a variety of
candidate formulations to evaluate dissolution profiles of IN105 and the fatty acids contents.
RESULTS
In the initial experiments, dogs dosed with INI 05 in 3% w/v capric acid sodium salt in a
phosphate buffer without additional excipients, showed (Figures 29-30) a similar response
compared to the optimized liquid formulation containing 3% w/v capric acid in Trolamine/Citric
acid/Tromethamine/Sodium Hydroxide buffer. This demonstrated that the sodium salt of the fatty
acid form behaves comparably to the acid form and that the additional buffer components did not
contribute to the formulation.
In separate studies evaluating alternative fatty acids substituting 3% caprate with either 3%
caprylic acid or 3% linoleic acid, neither of the alternatives exhibited significant effects. The use
of caprylic acid resulted in the need for low to moderate levels of glucose while the use of linoleic
acid did not result in requiring any glucose infusion suggesting lack of effect. Both formulations
showed relatively low levels of arterial insulin. A liquid formulation containing arginine showed
relatively no benefit on GIR or IN105 levels.
In the primary studies, solid formulations were evaluated as both powder blend filled into hard
gelatin capsules and tablets compressed by hand using a Carver Press. The capsules, powder
blend of 6mg of IN105 (insulin equivalent, 0.25mg/Kg) with 57mg of caprate / 57 mg laurate
showed no significant effect in the first dosing up to 120mins, and in the second dosing a
significant effect was observed with the GIR concurrent with IN 105 absorption where the levels
were well above the base line form 0 to 120 mins. This data suggests a variable but potential
delayed response in INI 05 in the capsule dosage form. Dissolution was only slightly delayed
with the capsule relative to the tablets although there may be little to no in-vitro/in-vivo
correlation.
In the initial prototype tablet screening studies, tablets containing 6mg of INI 05 and 150mg
Mannitol, 30mg sodium starch glycolate with 143mg Caparte with or with out 143 mg laurate
showed significantly higher GIR and IN105 absorption than the same tablets with 54mg Caprate
or/ and 54mg Laurate (Figures 31, 32, and 33\ This suggests a reasonable dose response of
ligher GIR and IN 105 levels relative to increasing levels of caprate and laurate. The tables
showed an early and consistent GIR response time to the IN 105 filled capsules. Additionally, the
IN105 tablets showed an arterial plasma rise in insulin in all dogs dosed.
In a series of final studies, three prototype tablet formulations (Formuluation [856] contained 143
mg caprate and 140 mg laurate, [860] contained 286 mg caprate, and [862]] contained 150 mg
caprate] were selected for evaluation in 5 dogs (3 different dogs for each formulation) to assess
the consistency of performance (Figures 34-37). Arterial plasma insulin levels rose in all 3 dogs
and at all 18 doses (3 dogs X 3 tablets X 2 doses each ) with corresponding GIR response
following the oral administration of 6mg (Insulin equivalent) of IN105 formulated in the tablets
containing either 150mg or 280 mg caprate or 143mg/140mg caparte/laurate (Figures 31-32 and
38-42). The c-peptide levels (ng/ml), with 150mg and 280mg caprate tablets, the average (n=2),
showed a decrease from an initial level of 0.30 ± 0.05 to 0.22 ± 0.02 during the first dosing and
0.1 ± 0.05 to 0.02 ± 0.0 in the second dosing, and with 140mg/140mg caprate/laurate tablets,
showed 0.21 ± 0.05 to 0.05 ± 0.02 during the fist dosing and 0.18 ± 0.05 to 0.18 ±0.01. This is
indicative of suppression of C-peptide secretion from the pancreas as result of the exogenous
IN 105 insulin.
All three prototype tablet formulations of IN105 showed consistent levels of IN105 absorption
and resultant glucose infusion rate among doses and within and between dogs, including on
different days
During the final studies, in which sets of 6 dogs were utilized, one dog (dog #3) experienced less
response with all liquid and solid dosages. To more accurately represent the results, the data is
presented with and without results from dog #3. Data from dogs that did not receive a complete
dose (bad gavage, vomiting, etc) or had endogenous insulin are omitted.
Dissolution study: Representative samples of tablets and capsules were subject to dissolution
testing (described above).
Discussion
These studies demonstrate that the prototype INI 05 tablet containing caprate or caprate and
laurate sodium salts with mannitol and the disintegrant sodium starch glycolate and containing
6mg IN105 (approximately 0.25 mg/kg) delivered orally resulted in significant and consistent
elevation of arterial plasma insulin that required glucose infusion to preserve euglycemia.
These prototype tablets resulted in IN105 levels and GIR rates at least as good and likely "to be
better than the liquid formulations containing comparable levels of caprate or laurate. The
prototype tablets forms maintain the absorption profile of the oral liquid formulation. The relative
oral bio-efficacy of the selected prototype tablet formulations (e.g., 280mg and 150mg caprate
containing tablets, n=6, AUC for GIR= 496±117 and 500±275) appears to be better than liquid
formulations (e.g., 3% w/v capric acid liquid formulation, n=5, AUC for GIR =182±92 and 198
±119).
The data suggests that the tablets containing sodium caprate as the only fatty acid along with
mannitol and the disintegrant, sodium starch glycolate would be useful in the further development
of solid dosage forms for use in clinical studies. Data also suggest that Insulin levels following
the oral administration of IN 105 in the selected prototype caprate tablets forms (sodium caprate at
either 150mg or 286mg) peaked steadily with a typical Tmax at around 20 min post-dose and a
Cmax of about 59.0 ± 20.1 and 62.9. ± 25.4 μUnits/ml, in both doses. The plasma insulin levels
remained elevated close to the Cmax level for 10-15 minutes and above basal levels throughout
120 min following each dose. The GIR required maintaining euglycemia using these tablets
reached Tmax at or around 30 to 40 min in both doses and GIR Cmax reached an average of 8.4 ±
1.99 and 7.41 ± 2.18 mg/kg/min. The tablet dosage forms required higher GIR Cmax (7.4-8.4 vs.
4.5-5.4) and required glucose infusion for a longer duration (100-120 mins vs. 60-90min) to
maintain euglycemia then the optimized liquid formulation.
In comparison with arterial plasma insulin levels of historical SQ and inhaled insulin, it appears
that these prototype tablets provide maximum insulin levels similar to SQ and inhaled delivery
and resembles an insulin profile comparable to that of inhaled insulin (Figures 34-37).
The prototype tablet experiments suggests the selected prototype tablets are suitable as solid
dosage formulations for further evaluation of IN 105 with future development to focus on
producing a clinical formulation that can be produced using a tablet press.
This specification is divided into sections with subject for ease of reference only. Sections and
subject headings are not intended to limit the scope of the invention. The embodiments
described herein are for the purpose of illustrating the many aspects and attributes of the
invention and are not intended to limit the scope of the invention.

We claim
1. A complex comprising:
an insulin conjugate comprising a native insulin or insulin polypeptide analog thereof
having an A chain and B chain of amino acids conjugated to a modifying moiety, wherein
the insulin polypeptide analog has the structure of a human native insulin with one or
more amino acid additions, deletions and/or substitutions and exhibits similar activity
relative to the native insulin, wherein the modifying moiety comprises from 2 to 10
polyethylene glycol subunits (OCH2CH2) forming a PEG component coupled to a
lipophilic component, where the lipophilic component is an alkyl, wherein the modifying
moiety is coupled to a lysine within 5 amino acide residues of the C-terminus of the B
chain thereby providing a monoconjugate;
and a cation selected from the group consisting of Zn++, Mn++, Ca++, Fe++, Ni++,
Cu++, Co++ and Mg++, wherein the insulin conjugate is complexed to the cation.
2. The complex as claimed in claim 1 wherein the modifying moiety is coupled to B29 of
the native insulin or insulin polypeptide analog thereof.
3. The complex as claimed in claim 1 where the insulin conjugate has a relative lipophilicity
of 1 or less than 1 relative to the corresponding parent insulin.
4. The complex as claimed in claim 1 where the complex is a solid.
5. The complex as claimed in claim 1 where:
a) the solubility of the calion-insulin conjugate complex at a pH of 7.4 is less than the
solubility of the insulin conjugate in solution at a pH of 7.4, and
b) the cation-insulin conjugate remains soluble at greater than 1 g/L in aqueous solution
across a pH range from 5.8 to about 8.5.
6. The complex as claimed in claim 1 where:
a) the complex is an R-type Zn complex comprising protamine; and
b) at a pH of 7.4, the aqueous solubility of the complex is from 10 to 110 g/L.
7. The complex as claimed in claim 1 where:
a) the complex is a T-type Zn complex comprising protamine; and
b) at a pH of 7.4, the aqueous solubility of the complex is from 10 to 150 g/L. f,
8. The complex as claimed in claim 1 where the insulin compound conjugate produces
crystals in aqueous solution at a pH which is equal to the pi plus or minus 2.5.
9. The complex as claimed in claim 1 wherei the modifying moiety is coupled at a free
amino group in the insulin to form a carbamate bond.
10. The complex as claimed in claim 1 where:
a) the insulin comprises a free hydroxyl group, and
b) the modifying moiety is coupled to the free hydroxyl group to form a carboxyllic,
ether, or carbonate bond.
11. The complex as claimed in claim 1 where the cation component corhprises Zn++.
12. The complex as claimed in claim 1 where molar ratio of insulin compound conjugate to
cation component is between 1:1 and 1:100.
13. The complex as claimed in claim 1 where the modifying moiety has a structure selected
from the group consisting of:
O
' where n is 1 to 4, and m is 2 to5;
o
O
HO ^-^^ ro ^^ ^^
\ / 3
5
o
HO ^^^ yo ^^, ^
9
o
HO^ ^ - ^ ^O ^ ^ ^
o
HO-^ ^--^ ^-^ Y^ ^O-^j
9 :
O
HO . \0 ^-73 ^ ^ ^
• ' 9 !
O
HO \0 ^/3
O
9
O
^ • L ^ ^ ^ ^ - - - ^ ^0(mPEG)7
HO ^ - ^ ^ ^ - ^ ^ ^ ^
9 •
O
!
^ \ ^ . ' ' \ ^ ^ ^ - ^ /.^~\^ ^ ' - ^ / ^ ' \ ^ _^0(mPEG)7
f
o o
HO ^0(PEG)3 ^ ^ ^ \ ^ ^ .
O 0
HO 0(PPG)3 ^^^^^ ^ ^ ^ ^ ^ ^ .
0 0
HO 0(PEG)3 ^ ^ ^ ^ ^ ^ ^ ^ . . - ' ^ ^ x ^ ^ \ ^ \v
O
HO- 0(PEG)4 ^ ^ - ^ ^^^-^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ' ' ^ ^ ' " - ' ^ ^ ^ ^ ^ ^ .
O
^ > f \ / ^ ^ ^ \ ^ ^ ^ N O
H
O MPEGO4 ^ * \ ^0MPEG4
14. A composition comprising at least one as claimed in the complexes of claims 1 to 13 and '
a pharmaceutically acceptable carrier.
15. The composition as claimed in claim 14, further comprising a second component
comprising an insulin conjugate or an unconjugated insulin.
16. The composition as claimed in claim 14, wherein the complex is a fortn selected from the
group consisting of a crystalline solid , an amorphous solid and co-crystal comprising ^t
least two insulin compound conjugates, wherein the insulin conjugates are the same or
different.
17. The composition as claimed in claim 14 wherein the composition is provided as a
lyophilized powder, a solid mixture or .a hybrid complex.
f
I
h •
18. The composition as claimed in claim 14 Comprising more than one complex where the
insulin conjugates of the complexes comprise different insuliri or different insulin
conjugates. >
19. The composition as claimed in claim 18 wherein the different insulin conjugates have
different properties selected from the group consisting of different solubilities; different
circulation half life, and different modifying moieties.
20. The composition as claimed in claim 19 where in one of the complexes has a rapid-acting
profile; and the other of the complexes has a medium-to-long acting profile.
21. The composition as claimed in claim 19 wherein one of the complexes has profile
suitable for basal insulin compound control; and the other of the complexes has a profile
suitable for post-prandial glucose control.
22. The composition as claimed in claim 15 where the composition is a. co-crystal of at least
two components selected from the group consisting of HIM2, insulin compound and
IN105, wherein HIM2 and INIOS have the following structures:
O
o I —I
II HIM2
Insulin ' ^ ^ ^ O ^ ° " - ^ / J \ 0 ^ ' ^^
23. The composition as claimed in claim 15 where the composition is a co-crystal having a
PK/PD profile suitable for post-prandial glucose control or overnight basal insulin
compound control.
24. The composition as claimed in claim 14 further comprising a complexing agent, where
the complexing agent is selected from the group consisting of protaminjes, surfen, globin
proteins, spermine, spermidine albumin, amino acids, carboxyllic acids, polycationic
polymer compounds, cationic polypeptides, polylysine, anionic polypeptides, nucleotides,
and antisense.
25. The composition as claimed in claim 14 devoid of protamine.
26. The composition as claimed in claim 16 wherein the complex is in the form of a
crystalline solid, a rod shaped crystal, a crystal having irregular morphology, a mixture of
amorphous and crystalline solids, or a powder.
f
27. The composition as claimed in claim 14, comprising a stabilizing agent selected from the
group consisting of phenol, m-cresol and paraben.
Dated this 15 day of February, 2007. /"^ T C ^ / A ^
[klESH KUMAR]
OFREMFRY&SAGAR
ATTORNEYIOF THE APPLICANTS
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