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
SUBSTITUTE SHEET (RULE 26)
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)
20 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,
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 acid
modifying 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.
7 Detailed Description of the Invention
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 insulin
precursors 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 or
more, 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
18
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:
O
-f*-CH—(CH2)m-X(C2H40)n-Y
^ (Formula II),
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:
O
Me
, and
23
(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.
24
The modifying moiety may, for example, have a formula:
\PAGn-X
(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
25
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, 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
26
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:
27
'" O
(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:
O
HO"
HO'
HO
28
o
HO \0'
O
HO
HO"
, and
The following modifying moieties can be particularly preferred for use in a basal insulin
compound replacement regimen.

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.
39
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
40
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.
41
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
cation to biologically active agent needed can be determined by one of ordinary skill in the art by
44
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.
46
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.
48
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
50
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
52
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.

We claim
1. A solid or liquid pharmaceutical composition formulated for oral administration by
ingestion, comprising:
from about 40 to about 60 % w/w fatty acid component, where the fatty acid component
comprises saturated or unsaturated C 4.12 fatty acids and/or salts of such fatty acids;
a therapeutic agent, wherein the therapeutic agent is a PEGylated protein or peptide wherein the
protein or peptide is an insulin or an insulin analog thereof and where the insulin or insulin
analog is coupled at Al, Bl and/or B29 to a linear or branched PEG moiety having from 2 to 10
PEG subunits to form an insulin conjugate.
2. The pharmaceutical composition of claim 1 comprising a diluent selected from the
group consisting of binders, disintegrants, fillers, diluents, lubricants, glidants, flow enhancers,
compression aids, colors, sweeteners, preservatives, suspensing agents, dispersing agents, film
formers, coatings, flavors, printing inks, celluloses, sugars, mannitols, lactoses, dissolution
enhancers, and crosscarmaloses.
3. The pharmaceutical composition of claim 1 or 2 prepared in a form selected from the
group consisting of tablets, minitabs, powders, hard gelatin capsules, or soft gelatin capsules.
4. The pharmaceutical composition of any one of claims 1 to 3, wherein the fatty acid
component is capric acid and/or lauric acid and/or salts of capric acid and/or lauric acid.
5. The pharmaceutical composition of any one of claims 1 to 4 comprising a buffer salt
selected from the group consisting of phosphate buffer, sodium phosphate, tris buffer, citric acid
buffer, and ethanolamine buffer.
6. The pharmaceutical composition of any one of claims 1 to 5 comprising a buffer salt
selected to achieve a buffering capacity at the site of absorbtion to maintain a local pH of from
4.8 to 9.5.
7. The pharmaceutical composition of any one of claims 1 to 6 where the insulin
conjugate consists of human insulin coupled at Al, Bl and/or B29 to a modifying moiety having
a structure:
8. The pharmaceutical composition of claim 1 prepared in a form selected from the
group consisting of semisolids, suspensions, microemulsions, and emulsions.
9. The pharmaceutical composition of claim 7, wherein the fatty acid component is
capric acid and/or lauric acid and/or salts of capric acid and/or lauric acid.
10. The pharmaceutical composition of claim 7 comprising a buffer salt selected from
the group consisting of phosphate buffer, sodium phosphate, tris buffer, citric acid buffer,
ethanolamine buffer, and triethylamine buffer.
11. The pharmaceutical composition of claim 7 or 8 wherein the fatty acid component is
sodium caprate.
12. The pharmaceutical composition of claim 11, further comprising mannitol.
13. The pharmaceutical composition of claim 11 to 12 wherein the modifying moiety is
conjugated to B29 of the insulin or insulin analog.