BACKGROUND OF THE INVENTION
This invention relates to the preparation and use of a purified form of Isomer C of
rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid (CCI-779).
Rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid
(CCI-779) is an ester of rapamycin which has demonstrated significant inhibitory effects
on tumor growth in both in vitro and in vivo, models.
CCI-779 may delay the time to progression of tumors or time to tumor recurrence
which is more typical of cytostatic rather than cytotoxic agents. CCI-779 is considered to
have a mechanism of action that is similar to that of sirolimus. CCI-779 binds to and
forms a complex with the cytoplasmic protein FKBP, which inhibits an enzyme, mTOR
(mammalian target of rapamycin, also known as FKBP12-rapamycin associated protein
[FRAP]). Inhibition of mTOR's kinase activity inhibits a variety of signal transduction
pathways, including cytokine-stimulated cell proliferation, translation of mRNAs for
several key proteins that regulate the G1 phase of the cell cycle, and IL-2-induced
transcription, leading to inhibition of progression of the cell cycle from G1 to S. The
mechanism of action of CCI-779 that results in the Gl-S phase block is novel for an
aniicancer drug.
In vitro, CCI-779 has been shown to inhibit the growth of a number of
histologically diverse tumor cells. Central nervous system (CNS) cancer, leukemia (T-
cell), breast cancer, prostate cancer, and melanoma lines were among the most
sensitive to CCI-779. The compound arrested cells in the G1 phase of the cell cycle.
In vivo studies in nude mice have demonstrated that CCI-779'has activity against
human tumor xenografts of diverse histological types. Gliomas were particularly
sensitive to CCI-779 and the compound was active in an orthotopic glioma model in
nude mice. Growth factor (platelet-derived)-induced stimulation of a human
glioblastoma cell line in vitro was markedly suppressed by CCI-779. The growlh of
several human pancreatic tumors in nude mice as well as one of two breast cancer lines
studied in vivo also was inhibited by CCI-779.
The preparation and use of hydroxyesters of rapamycin, including CCI-779, are
disclosed in U.S. Patents 5,362,718 and 6,277,983, which are hereby incorporated by
reference.
SUMMARY OF THE INVENTION
We have now found that when prepared and isolated according the above
patents, CCI-779 exists as a mixture containing about 95 wt% of Isomer B and 30 wt%
isomer C (as measured by high performance liquid chromatograph (HPLC)). The
structures of each are provided below.

When formulated in solid form CCI-779 Isomer C is stable under storage
conditions for extended periods of time. CCI-779 Isomer C is also stable when in an
aqueous suspension.
Other aspects and advantages of the present invention will be readily apparent
from the followed detailed description of the invention.
DETAILED DESCRIPTION OF THE INVENTION
This invention provides Ihe preparation and use of purified CCI-779 Isomer C;
(his isomer is also referred to as the oxepane isomer of CCI-779. Accordingly, this
invention provides CCI-779 Isomer C substantially free of CCI-779 Isomer B.
As used herein, the term "CCI-779" when used without any reference to isomers
means rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid
containing about 95 wt% of CCI-779 Isomer B and 3 wt% CCI-779 Isomer C.
As used herein, the term "purified" means having a purity of greater than 50 wt%
of Isomer C. It is preferred that the purified CCI-779 Isomer C has a purity of greater
than 75 wt%, more preferred that the CCI-779 Isomer C has a purity of greater than 90
wt%, and most preferred that the CCI-779 Isomer C has a purity of greater than 95 wt%,
from Isomer B. Such purity can be readily measured by HPLC.
As used herein, the term "substantially free of" means CCI-779 Isomer C as a
compound, composition, or mixture which contains less than 10 wt% CCI-779 Isomer B,
and preferably less than 5 wt% CCI-779 Isomer B, more preferably less than 2 wt% CCI-
779-isomer B. The percent of Isomer C to Isomer B can readily be determined by
HPLC.
As used in accordance with this invention, the term "treatment" means alleviating
the symptoms in a mammal having a disease or disorder by providing said mammal with
an effective amount of CCI-779 Isomer C.
As used in accordance with this invention, the term "providing," with respect to
providing CCI-779 Isomer C means either directly administering CCI-779 Isomsr C, or
administering a prodrug, derivative, or analog which will form an effective amount of
CCI-779 Isomer C within the body.
The preparation of CCI-779 is described in U.S. Patent 5,362,718, which is
hereby incorporated by reference. A regiospecific synthesis of CCI-779 is described in
US Patent 6,277,983, which is hereby incorporated by reference. When prepared
according to the above patents, CCI-779 exists as an equilibrium mixture containing
about 95 wt% of Isomer B and 3 wt% isomer C (as measured by HPLC).
CCI-779 Isomer C exists in equilibrium with CCI-779 Isomer B, according to the
following scheme. Under conditions described herein, the equilibrium can be driven
from the greatly favored Isomer B state to the Isomer C state followed by isolation and
purification of CCI-779 Isomer C.
The conversion of CCI-779 Isomer B to CCI-779 Isomer C can be accomplished
in a mixture of aqueous buffer and organic solvent in the range of pH 4 to pH 10. More
particularly, the method of the invention involves dissolving CCI-779 in a solution
containing an organic solvent and an aqueous solvent, where the aqueous solvent has a
pH of from 4 to 10, 5 to 9, 6 to 9, or 7 to 9. The pH is preferred in the range of 8 and 9.
Most preferred pH is about 8.5.
Any suitable aqueous buffer including, without limitation, phosphate buffered
saline, triethylammonium acetate, and water with sodium citrate buffer, can be readily
selected by one of skill in the art. It is preferred that the organic solvent is a polar
aprotic solvent, i.e., a solvent that is polar but has no ability to be an H-bond donor.
Examples of suitable polar aprotic solvents include dimethylsulfoxide (DMSO; CH3-SO-
CH3), dimethylformamide, acetonitrile (H-CO-N(CH3)2), aldehydes( R-CHO), ketones ( R-
CO-R1),. In one example below, the aqueous buffer is triethylammonium acetate (TEAA)
and the polar aprotic solvent is acetonitrile. These are provided at a ratio of about 1:1,
by volume. However, other suitable ratios of this buffer and solvent combination will be
readily apparent to one of skill in the art. Similarly, other suitable aqueous buffers and
organic solvents useful in the method of the invention will be readily apparent to those of
skill in the art in view of this specification.
This conversion reaction can be performed at room temperature, i.e., about
22 °C to about 28 °C. Alternatively, the conversion may be performed at lower or higher
temperatures, as needed. Typically, conversion is allowed to proceed for about 30 to
45 minutes (or longer, as needed or desired) and is stopped by extraction of CCI-779
isomer C with a suitable organic solvent.
In one embodiment, the organic solvent used in the extraction is an aprotic
solvent, i.e., a solvent whose molecules have a zero molecular dipole and whose
hydrogen atoms are not bonded to an oxygen or nitrogen. Examples include the
hydrocarbons (e.g., alkanes, alkenes, and alkynes). In the examples below, the non-
polar aprotic solvent is methylene chloride. Other suitable aprotic solvents will be readily
apparent to one of skill in the art in view of the teachings herein.
Isolation of purified CCI-779 Isomer C can be accomplished using preparative
chromatography techniques, such as are well known to those of skill in the art. See,
generally, PREPARATIVE CHROMATOGRAPHY, by R.P.W. Scott, Chrom-Ed Book Series,
available on-line and Guiochon, G. et al, FUNDAMENTALS OF PREPARATIVE AND
NONLINEAR CHROMATOGRAPHY, 1st Ed. Academic Press (1994).
Antifungal activity for CCI-779 Isomer C was established by evaluating it against
several strains of fungi. Briefly the following procedure was used to evaluate such
activity. A 96 well microtiter plate was filled (50 µL/well) with RPMI 1640. The
compounds to be evaluated were placed in appropriate wells and serial diluted in
successive wells to provide several dilutions. The concentration ranged from 64 to 0.06
mg/mL An adjusted inoculum of fungi (50 µL) was added to each well and plates were
incubated at 35 °C for 24-48 h. The MIC is the lowest concentration of compound which
completely inhibited growth of organism in the wells. The following table shows the
results obtained in this standard pharmacological test procedure. Where the same fungi
name is listed more than once, it indicates that more than one strain was evaluated.

The results obtained in this standard pharmacological test procedure
demonstrate that the CCI-779 Isomer C is useful as an antifungal agent. CCI-779
Isomer C is also useful as an antineoplastic agent; in particular, it is useful against solid
tumors, including sarcomas and carcinomas; and more particularly against
astrocytomas, prostate cancer, breast cancer, colon cancer, small cell lung cancer, and
ovarian cancer; and adult T-cell leukemia/lymphoma. CCI-779 Isomer C is also useful
treatment or inhibition of transplantation rejection such as kidney, heart, liver, lung, bone
marrow, pancreas (islet cells), cornea, small bowel, and skin allografts, and heart valve
xenografts; in the treatment or inhibition of graft vs. host disease; in the treatment or
inhibition of autoimmune diseases such as lupus, rheumatoid arthritis, diabetes mellitus,
myasthenia gravis, and multiple sclerosis; and diseases of inflammation such as
psoriasis, dermatitis, eczema, seborrhea, inflammatory bowel disease, pulmonary
inflammation (including asthma, chronic obstructive pulmonary disease, emphysema,
acute respiratory distress syndrome, bronchitis, and the like) and ocular uveitis; adult T-
cell leukemia/lymphoma; fungal infections; hyperproliferative vascular diseases such as
restenosis; graft vascular atherosclerosis; and cardiovascular disease, cerebral vascular
disease, and peripheral vascular disease, such as coronary artery disease,
cereberovascular disease, arteriosclerosis, atherosclerosis, nonatheromatous
arteriosclerosis, or vascular wall damage from cellular events leading toward immune
mediated vascular damage, and inhibiting stroke or multiinfarct dementia.
When used for restenosis, it is preferred that CCI-779 Isomer C is used to treat
restenosis that occurs following an angioplasty procedure. When used for this treating
restenosis following an angioplasty, CCI-779 Isomer C can be administered prior to the
procedure, during the procedure, subsequent to the procedure, or any combination of
the above.
Dosage regimens are expected to vary according to the route of administration.
For example, dosages for oral administration are often up to five to tenfold greater than
for i.v. administration. For example, in one embodiment, an oral dosage of CCI-779 will
in the range of about 2 to about 100 mg/day, 5 mg/day to 75 mg/day, 10 mg/day to 50
mg/day, 15 mg/day to 35 mg/day, or about 20 mg/day to 25 mg/day for an adult.
However, this dosage can be adjusted upwardly or downwardly by one of skill in the art,
depending upon the indication being treated, the size of the patient, and other factors
which are known those of skill in the art.
Suitable oral formulations for CCI-779 isomer C can be prepared as described
for CCI-779, as described in WO 2004/026280 and US Patent Application No.
10/663,506, which is hereby incorporated by reference. Such an oral formulation
contains a granulation prepared using a wet granulation process. The granulation
contains CCI-779 isomer C, a water soluble polymer, a pH modifying agent, a surfactant,
and an antioxidant. In one embodiment, the formulation contains from 0.1 to 30 wt%,
from 0.5 to 25 wt%, from 1 to 20 wt%, from 5 to 15 wt%, or from 7 to 12 wt% (wt/wt)
CCI-779 isomer C, from 0.5 to 50 wt%, from 1 to 40 wt%, from 5 to 35 wt%, from 10 to
25 wt%, or from 15 to 20 wt% (wt/wt) water soluble polymer, from 0.5 to 10 wt%, 1 to 8
wt%, or 3 to 5 wt% (wt/wt) surfactant, and from 0.001 wt% to 1 wt%, 0.01 wt% to 1 wt%,
or 0.1 wt% to 0.5 wt% (wt/wt) antioxidant. However, other embodiments may contain
more, or less, of these components.
The oral formulation may also contain suitable chelating agents, fillers, binders,
surfactants, and the like to facilitate the granulation and tableting process. It is preferred
that the wet granulation be performed with a hydroalcoholic solvent system comprising
water and an alcohol, with ethanol being the preferred alcoholic component.
Typical water soluble polymers include, but are not limited to, polyvinylpyrrolidone
(PVP), hydroxypropylmethylcellulose (HPMC), polyethylene glycol (PEG), and
cyclodextrin or mixtures thereof. It is preferred that the water-soluble polymer is PVP,
and having a molecular weight of between 2.5 and 60 kilodaltons. Any given oral
formulation useful in the invention may contain multiple ingredients of each class of
component. For example, an oral formulation containing an antioxidant may contain one
or more antioxidants as the antioxidant component.
Acceptable pH modifying agents include, but are not limited to citric acid, sodium
citrate, dilute HCI, and other mild acids or bases capable of buffering a solution
containing CCI-779 isomer C to a pH in the range of about 4 to about 6. Acceptable
antioxidants include, but are not limited to, citric acid, d.l-a-tocopherol, BHA, BHT,
monothioglycerol, ascorbic acid, and propyl gallate. It is expected that the antioxidants
of the oral formulations used in this invention will be used in concentrations ranging from
0.001 wt% to 3wt%. Chelating agents, and other materials capable of binding metal
ions, such as ethylene diamine tetra acetic acid (EDTA) and its salts are capable of
enhancing the stability of CCI-779 isomer C. Surfactants may include polysorbate 80,
sodium lauryl sulfate, sodium dodecyl sulfate, salts of bile acids (taurocholate,
glycocholate, cholate, deoxycholate, etc.) that may be combined with lecithin.
Alternatively, ethoxylated vegetable oils, such as Cremophor EL, vitamin E tocopherol
propylene glycol succinate (Vitamin E TGPS), polyoxyethylene-polyoxypropylene block
copolymers, and poloxamers. Binders, fillers, and disintegrants such as sucrose,
lactose, microcrystalline cellulose, croscarmellose sodium, magnesium stearate, gum
acacia, cholesterol, tragacanth, stearic acid, gelatin, casein, lecithin (phosphatides),
carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethycellulose phthalate,
noncrystalline cellulose, cetostearyl alcohol, cetyl alcohol, cetyl esters wax, dextrates,
dextrin, lactose, dextrose, glyceryl monooleate, glyceryl monostearate, glyceryl
palmitostearate, polyoxyethylene alkyl ethers, polyethylene glycols, polyoxyethylene
castor oil derivatives, polyoxyethylene stearates, and polyvinyl alcohol, and the like may
also be incorporated into the oral formulation.
Oral formulations containing the active compounds of this invention may
comprise any conventionally used oral forms, including tablets, capsules, buccal forms,
troches, lozenges and oral liquids, suspensions or solutions. Capsules may contain
mixtures of the active compound(s) with inert fillers and/or diluents such as the
pharmaceutically acceptable starches (e.g. corn, potato or tapioca starch), sugars,
artificial sweetening agents, powdered celluloses, such as crystalline and
microcrystalline celluloses, flours, gelatins, gums, etc. Useful tablet formulations may be
made by conventional compression, wet granulation or dry granulation methods and
utilize pharmaceutically acceptable diluents, binding agents, lubricants, disintegrants,
surface modifying agents (including surfactants), suspending or stabilizing agents,
including, but not limited to, magnesium stearate, stearic acid, talc, sodium lauryl sulfate,
microcrystalline cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin,
alginic acid, acacia gum, xanthan gum, sodium citrate, complex silicates, calcium
carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate,
lactose, kaolin, mannitol, sodium chloride, talc, dry starches and powdered sugar.
Preferred surface modifying agents include nonionic and anionic surface modifying
agents. Representative examples of surface modifying agents include, but are not
limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol,
cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphates,
sodium dodecylsulfate, magnesium aluminum silicate, and triethanolamine. Oral
formulations herein may utilize standard delay or time release formulations to alter the
absorption of the active compound(s). The oral formulation may also consist of
administering the active ingredient in water or a fruit juice, containing appropriate
solubilizers or emulsifiers as needed.
In some cases it may be desirable to administer the compounds directly to the
airways in the form of an aerosol.
The compounds may also be administered parenteraily or intraperiioneally.
Solutions or suspensions of these active compounds as a free base or
pharmacologically acceptable salt can be prepared in water suitably mixed with a
surfactant such as hydroxy-propylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary
conditions of storage and use, these preparation contain a preservative to prevent the
growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous preparation of
sterile injectable solutions or dispersions. In all cases, the form must be sterile and must
be fluid to the extent that easy syringability exists. It must be stable under the conditions
of manufacture and storage and must be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol
and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
Particularly suitable injectable formulations for CCI-779 isomer C can prepared in
a manner similar to those described for CCI-779 in WO 2004/011000 and US Patent
Application No. 10/626,943, which is hereby incorporated by reference. In this
embodiment, the injectable formulation useful in the invention provides a CCI-779
isomer C cosolvent concentrate containing an parenterally acceptable solvent and an
antioxidant as described above and a parenteral formulation containing CCI-779 isomer
C, composed of CCI-779 isomer C, an parenterally acceptable cosolvent, an antioxidant,
a diluent solvent, and a surfactant. Any given formulation useful in this invention may
contain multiple ingredients of each class of component. For example, a parenterally
acceptable solvent can include a non-alcoholic solvent, an alcoholic solvent, or mixtures
thereof. Examples of suitable non-alcoholic solvents include, e.g., dimethylacetamide,
dimethylsulfoxide or acetonitrile, or mixtures thereof. "An alcoholic solvent," may contain
one or more alcohols as the alcoholic solvent component of the formulation. Examples
of solvents useful in the formulations invention include, without limitation, ethanol,
propylene glycol, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol
600, polyethylene glycol 1000, or mixtures thereof. These cosolvents are particularly
desirable because degradation via oxidation and lactone cleavage occurs to a lower
extent for these cosolvents. Further, ethanol and propylene glycol can be combined to
produce a less flammable product, but larger amounts of ethanol in the mixture generally
result in better chemical stability. A concentration of 30 to 100%v/v of ethanol in the
mixture is preferred.
In this embodiment, the stability of CCI-779 isomer C in parenterally acceptable
alcoholic cosolvents is enhanced by addition of an antioxidant to the formulation.
Acceptable antioxidants include, but are not limited to, citric acid, d.l-cx-tocopherol, BHA,
BHT, monothioglycerol, ascorbic acid, propyl gallate, and mixtures thereof. Generally,
the parenteral formulations useful in this embodiment of the invention will contain an
antioxidant component(s) in a concentration ranging from 0.001% to 1% w/v, or 0.01%
to 0.5% w/v, of the cosolvent concentrate, although lower or higher concentrations may
be desired. Of the antioxidants, d,I-o>tocopherol is particularly desirable and is used at a
concentration of 0.01 to 0.1 % w/v with a preferred concentration of 0.075% w/v of the
cosolvent concentrate.
In certain embodiments, the antioxidant component of the formulation of the
invention also exhibits chelating activity. Examples of such chelating agents include,
e.g., citric acid, acetic acid, and ascorbic acid (which may function as both a classic
antioxidant and a chelating agent in the present formulations). Other chelating agents
include such materials as are capable of binding metal ions in solution, such as ethylene
diamine tetra acetic acid (EDTA), its salts, or amino acids such as glycine are capable of
enhancing the stability of CCI-779 isomer C. In some embodiments, components with
chelating activity are included in the formulations of the invention as the sole "antioxidant
component". Typically, such metal-binding components, when acting as chelating agents
are used in the lower end of the range of concentrations for the antioxidant component
provided herein. In one example, citric acid enhanced the stability of CCI-779 isomer C
when used at a concentration of less than 0.01% w/v. Higher concentrations are less
stable solutions and thus, less desirable for products to be subject to long-term storage
in liquid form. Additionally, such chelating agents may be used in combination with other
antioxidants as part of the antioxidant component of the invention. For example, an
acceptable formulation may contain both citric acid and d.l-a-tocopherol. Optimal
concentrations for the selected antioxidant(s) can be readily determined by one of skill in
the art, based upon the information provided herein.
Advantageously, in certain embodiments of the parenteral formulations useful in
the invention, precipitation of CCI-779 isomer C upon dilution with aqueous infusion
solutions or blood is prevented through the use of a surfactant contained in the diluent
solution. The most important component of the diluent is a parenterally acceptable
surfactant. One particularly desirable surfactant is polysorbate 20 or polysorbate 80.
However, one of skill in the art may readily select other suitable surfactants from among
salts of bile acids (taurocholate, glycocholate, cholate, deoxycholate, etc.) which are
optionally combined with lecithin. Alternatively, ethoxylated vegetable oils, such as a
pegylated castor oil [e.g., such as PEG-35 castor oil which is sold, e.g., under the name
Cremophor EL, BASF], vitamin E tocopherol propylene glycol succinate (Vitamin E
TGPS), and polyoxyethylene-polyoxypropylene block copolymers can be used in the
diluent as a surfactant, as well as other members of the polysorbate family such as
polysorbate 20 or 60 Other components of the diluent may include water, ethanol,
polyethylene glycol 300, polyethylene 400, polyethylene 600, polyethylene 1000, or
blends containing one or more of these polyethylene glycols, propylene glycol and other
parenterally acceptable cosolvents or agents to adjust solution osmolarity such as
sodium chloride, lactose, mannitol or other parenterally acceptable sugars, polyols and
electrolytes. It is expected that the surfactant will comprise 2 to 100% w/v of the diluent
solution, 5 to 80% w/v, 10 to 75% w/v, 15 to 60 % w/v, and preferably, at least 5% w/v,
or at least 10% w/v, of the diluent solution.
A parenteral formulation useful in the invention can be prepared as a single
solution, or preferably can be prepared as a cosolvent concentrate containing CCI-779
isomer C, an alcoholic solvent, and an antioxidant, which is subsequently combined with
a diluent that contains a diluent solvent and suitable surfactant. Prior to use, the
cosolvent concentrate is mixed with a diluent comprising a diluent solvent, and a
surfactant. When CCI-779 isomer C is prepared as a cosolvent concentrate according
to this invention, the concentrate can contain concentrations of CCI-779 isomer C from
0.05 mg/mL, from 2.5 mg/mL, from 5 mg/mL, from 10 mg/mL or from 25 mg/mL up to
approximately 50 mg/ml. The concentrate can be mixed with the diluent up to
approximately 1 part concentrate to 1 part diluent, to give parenteral formulations having
concentrations of CCI-779 isomer C from 1 mg/mL, from 5 mg/mL, from 10 mg/mL, from
20 mg/mL, up to approximately 25 mg/ml. For example the concentration of CCI-779
isomer C in the parenteral formulation may be from about 2.5 to 10 mg/mL. This
invention also covers the use of formulations having lesser concentrations of CCI-779
isomer C in the cosolvent concentrate, and formulations in which one part of the
concentrate is mixed with greater than 1 part of the diluent, e.g., concentrate: diluent in a
ratio of about 1:1.5, 1:2, 1:3, 1:4 .1:5, or 1:9 v/v and so on, to CCI-779 isomer C
parenteral formulations having a CCI-779 isomer C concentration down to the lowest
levels of detection.
Typically the antioxidant may comprise from about 0.0005 to 0.5% w/v of the
formulation. The surfactant may for example comprise from about 0.5 % to about 10%
w/v of the formulation. The alcoholic solvent may for example comprise from about 10%
to about 90% w/v of the formulation.
The parenteral formulations useful in this invention can be used to produce a
dosage form that is suitable for administration by either direct injection or by addition to
sterile infusion fluids for intravenous infusion.
For the purposes of this disclosure, transdermal administrations are understood
to include all administrations across the surface of the body and the inner linings of
bodily passages including epithelial and mucosal tissues. Such administrations may be
carried out using the present compounds, or pharmaceutically acceptable salts thereof,
in lotions, creams, foams, patches, suspensions, solutions, and suppositories (rectal and
vaginal).
Transdermal administration may be accomplished through the use of a
transdermal patch containing the active compound and a carrier that is inert to the active
compound, is non toxic to the skin, and allows delivery of the agent for systemic
absorption into the blood stream via the skin. The carrier may take any number of forms
such as creams and ointments, pastes, gels, and occlusive devices. The creams and
ointments may be viscous liquid or semisolid emulsions of either the oil-in-water or
water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or
hydrophilic petroleum containing the active ingredient may also be suitable. A variety of
occlusive devices may be used to release the active ingredient into the blood stream
such as a semi-permeable membrane covering a reservoir containing the active
ingredient with or without a carrier, or a matrix containing the active ingredient. Other
occlusive devices are known in the literature.
Suppository formulations may be made from traditional materials, including
cocoa butter, with or without the addition of waxes to alter the suppository's melting
point, and glycerin. Water soluble suppository bases, such as polyethylene glycols of
various molecular weights, may also be used.
The purified CCI-779 isomer C of the invention may be formulated for any
suitable delivery route and vehicle and assembled in the form of a kit of parts. The
invention therefore includes a product containing (a) CCI-779 isomer C for use in
treating a mammal. The invention also includes a pharmaceutical pack containing a
course of treatment of a neoplasm for one individual mammal, wherein the pack
contains (a) units of CCI-779 isomer C in unit dosage form.
Thus, the CCI-779 Isomer C of the invention can be formulated as a
pharmaceutical composition and, optionally, assembled in the form of a kit, for use in
treatment of a mammal. Such a composition or kit can be used as an antineoplastic
agent, and in particular, in treatment of solid tumors, including sarcomas and
carcinomas; and more particularly against astrocytomas, prostate cancer, breast cancer,
colon cancer, small cell lung cancer, and ovarian cancer; and adult T-cell
leukemia/lymphoma. CCI-779 Isomer C-containing compositions and kits are also
useful treatment or inhibition of transplantation rejection such as kidney, heart, liver,
lung, bone marrow, pancreas (islet cells), cornea, small bowel, and skin allografts, and
heart valve xenografts; in the treatment or inhibition of graft vs. host disease; in (he
treatment or inhibition of autoimmune diseases such as lupus, rheumatoid arthritis,
diabetes mellitus, myasthenia gravis, and multiple sclerosis; and diseases of
inflammation such as psoriasis, dermatitis, eczema, seborrhea, inflammatory bowel
disease, pulmonary inflammation (including asthma, chronic obstructive pulmonary
disease, emphysema, acute respiratory distress syndrome, bronchitis, and the like) and
ocular uveitis; adult T-cell leukemia/lymphoma; fungal infections; hyperproliferative
vascular diseases such as restenosls; graft vascular atherosclerosis; and cardiovascular
disease, cerebral vascular disease, and peripheral vascular disease, such as coronary
artery disease, cereberovascular disease, arteriosclerosis, atherosclerosis,
nonatheromatous arteriosclerosis, or vascular wall damage from cellular events leading
toward immune mediated vascular damage, and inhibiting stroke or multiinfarct
dementia.
The following procedures describe one method for the preparation and
characterization of CCI-779 Isomer C. This example is illustrative only, and does not
limit the invention.
CCI-779 (300 mg 0.29 mmol) was dissolved in about 50 wt% 0.2 M TEAA buffer
pH 8.5 and 50 wt% acetonitrile. The solution was kept at room temperature for about 45
min. The conversion was stopped by CH2CI2 extraction. The organic layer was reduced
by rotary evaporation system to dryness. The isolation of isomer C was performed by
preparative NOVPAK™ HR C-18 column (300 x 19 mm) using the mobile phase
consisted of 38 wt% A, 62 wt% B. A is 90wt% 0.02 M TEAA buffer pH 3.5 10wt%
acetonitrile. B is 10wt% 0.02 M TEAA buffer pH 3.5 with 90wt% acetonitrile. The flow
rate was 20 mL/min. The fraction of isomer C (20 min) was collected and extracted with
CH2CI2 using a separatory funnel. The organic layer was combined and washed with 2 x
50 mL water then the organic layer was dried with anhydrous Na2SO4. The organic
solvent was reduced by rotary evaporation to about 1 mL. The product was transferred
into a vial and precipitated by adding n-hexane. A second purification by preparative
chromatography was usually necessary to obtain pure CCI-779 Isomer C. The white
powder was obtained by using N2 to blow away the solvent and the vial was put into
speed-vac to dry overnight. The purity of the isomer C for each purification step was
analyzed by analytical HPLC. (An Ultracarb™ ODS 150x4.6 mm, 5 µm in particle size
from Phenomenex was used.) The mobile phase consisted of 60wt% acetonitrile, 40wt%
water with 0.02 M sodium citrate buffer at pH 4.2. The column temperature was at 45°C
and flow rate was at 1.5 mL/min. The detection wavelength was set at 280 nm. The
injection volume was 10 µl). The retention time is 15 min. for CCI-779 isomer B and 1 7
min. for CCI-779 isomer C.) Based on AUC analysis of the HPLC, CCI-779 Isomer C
was produced in 98.4wt% purity, and contained 1.6wt% CCI-779 Isomer B. ESI mass
spectrometry indicates molecular ion [M+NH4]* m/e 1047.6 which is same as the
reference sample of CCI-779.
1H NMR data were acquired on both 400 and 600 MHz Varian Unity Plus™
spectrometers with a probe temperature of 25 °C. The sample was prepared in acetone-
d6 and compared to a solution of CCI-779 which was also prepared in acetone-d6.
Resonances that differentiate a seven membered ring hemiketal (CCI-779 Isomer C)
structure from the six membered ring hemiketal (CCI-779 Isomer B) structure include the
following:
(1) In the proton spectrum for CCI-779 Isomer C, C-12ccCH3 is observed at
1.17 ppm for both rotational isomers. This equivalence is deduced from the chemical
shift differences correlating the C-12aCH3 resonances in the COSY spectrum to two
distinct H-11 rotomer resonances at 1.22 and 1.25 ppm, and to H-12 resonances at 3.18
and 3.24 ppm which are further discussed below. This differs from the major, 80 wt%
rotomer resonance of C-12aCH3 observed at 0.92 ppm in the CCI-779 reference
spectrum.
(2) H-12 rotomer resonances are observed at 3.18 and 3.24 ppm in Isomer
C. These shifts are consistent with the structure of the H-12 methine proton to a ketone
carbonyl in CCI-779 Isomer C from the major rotomer resonance of CCI-779 observed at
2.21 ppm for H-12 methine proton to a hemiketal.
(3) Indirect detection shows three distinct pairs of rotomeric ketone carbonyl
resonances correlated to various proton resonances in the HMBC spectrum. All three
ketone carbonyls are downfield of 200 ppm in this spectrum. Using the HMBC data to
assign these ketone resonances to CCI-779 Isomer C, the C-27 carbonyl is at 210 ppm,
C-33 is at 213 ppm and C-14 is at 211 ppm.
Two deuterium exchangeable singlet rotomeric resonances for the C-15otOH are
observed in the proton spectrum at 5.9 and 6.1 ppm and correlate in the HMBC
spectrum to the C-14 ketone carbon resonances at 211 ppm, to the C-16 amide carbon
resonances at 169 ppm, and to the C-15 hemiketal carbon resonances at 99 ppm. This
triad of two and three bond OH couplings to non-protonated carbons is only consistent
with structure for CCI-779 Isomer C.
In the proton spectrum for the reference sample, CCI-779 a deuterium
exchangeable singlet resonance at 5.25 ppm correlates in the HMBC spectrum to the C-
15 ketone carbon at 198 ppm and to the C-14 hemiketal carbon at 100 ppm. No
correlation to the C-16 amide carbon at 168 ppm is observed. This data is consistent
with the CCI-779 structure.
All patents, patent applications, articles, and other documents referenced herein
are incorporated by reference. It will be clear to one of skill in the art that modifications
can be made to the specific embodiments described herein without departing from the
scope of the invention.
CLAIMS:
1. CCI-779 Isomer C having a purity of greater than 50wt%.
2. The CCI-779 Isomer C according to claim 1, wherein the purity of CCI-779
Isomer C is greater than 75wt%.
3. The CCI-779 Isomer C according to claim 1, wherein the purity of CCI-779
Isomer C is greater than 90wt%.
4. The CCI-779 Isomer C according to claim 1, wherein the purity of CCI-779
Isomer C is greater than 95wt%.
5. CCI-779 Isomer C substantially free of CCI-779 Isomer B.
6. The CCI-779 Isomer C according to claim 5, which contains less than
5wt% CCI-779 Isomer B.
7. The CCI-779 Isomer C according to claim 5, which contains less than
2wt% CCI-779 Isomer B.
8. CCI-779 Isomer C prepared from CCI-779 by
a) dissolving CCI-779 in a solution containing an organic solvent and
an aqueous solvent, said aqueous solvent having a pH in the range of 4 to 10;
b) extracting the CCI-779 Isomer C into an organic solvent.
9. The CCI-779 Isomer C prepared according to claim 8, wherein the organic
solvent in step a) is a polar aprotic solvent.
10. The CCI-779 Isomer C prepared according to claim 9, wherein the organic
solvent in step a) is acetonitrile.
11. The CCI-779 Isomer C prepared according to any one of claims 8 to 10,
wherein the pH is in the range of 7 to 9.
12. The CCI-779 Isomer C prepared according to claim 11, wherein the pH is
in the range of 7.5 to 8.5.
13. The CCI-779 Isomer C prepared according to any one of claims 8 to 12,
wherein the organic solvent of step b) is an aprotic solvent.
14. The CCI-779 Isomer C prepared according to claim 13, wherein the aprotic
solvent of step b) is methylene chloride.
15. A process for preparing purified CCI-779 Isomer C which comprises,
a) dissolving CCI-779 in a solution containing an organic solvent and
an aqueous solvent, said aqueous solvent having a pH in the range of 4 and 10;
b) extracting the CCI-779 Isomer C into an organic solvent.
16. The process according to claim 15, wherein the organic solvent in step a)
is a polar aprotic solvent.
17. The process according to claim 16, wherein the organic solvent in step a)
is acetonitrile.
18. The process according to any one of claims 15 to 17, wherein the pH is in
the range of 7 to 9.
19. The process according to claim 18, wherein the pH is in the range of 7.5 to
8.5.
20. The process according to any one of claims 15 to 19, wherein the organic
solvent of step b) is a non-polar aprotic solvent.
21. The process according to claim 20, wherein the organic solvent of step b)
is a non-polar aprotic solvent.
22. The process according to claim 21, wherein the organic solvent of step b)
is methylene chloride.
23. A pharmaceutical composition comprising purified CCI-779 isomer C
according to any one of claims 11o 7 and a pharmaceutically acceptable carrier.
24. A pharmaceutical pack containing a course of treatment for a mammal,
wherein the pack contains (a) units of purified CCI-779 isomer C according to any one of
claims 1 to 7 in unit dosage form.
CCL-779 ISOMER C
Purified CCI-779 isomer C is provided, as are pharmaceutical compositions and kits containing same