PROCESS FOR THE PREPARATION OF PURIFIED
CRYSTALLINE CCI-779
BACKGROUND OF THE INVENTION
The present invention provides purified crystalline CCI-779 and processes
for preparing same.
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
has been demonstrated to be effective in multiple applications, including as an
anticancer agent for treating central nervous system cancer, leukemia, breast
cancer, prostate cancer, melanoma, gliomas, and glioblastoma.
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 Gl to S. The mechanism of action
of CCI-779 that results in the G1-S phase block is novel for an anticancer 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 Gl 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 growth 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.
CCI-779 has been purified by several recrystallization processes, which
produce a form of CCI-779 that contains unacceptable amounts of impurities.
Other routes to more purified CCI-779 include chromatography purifications,
which provide a purer form of CCI-779. These chromatography purifications,
however, cannot be scaled up to produce any appreciable amounts of purified CCI-
779, nor would doing so be financially beneficially.
What is needed in the art are alternate process for preparing purified forms
of CCI-779, especially processes performed on a larger scale.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 provides the differential scanning calorimetry thermogram of one
representative sample of purified crystalline CCI-779 prepared according to the
present invention.
Figure 2 provides the X-ray diffraction pattern of one representative sample
of purified crystalline CCI-779 prepared according to the present invention.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides purified crystalline CCI-779.
In a further aspect, the present invention provides purified crystalline CCI-
779 which process does not include chromatography.
In another aspect, the present invention provides purified crystalline CCI-
779 which purification thereof does not include chromatography.
In yet a further aspect, the present invention provides a process for
preparing purified crystalline CCI-779.
In still another aspect, the present invention provides a process for
purifying CCI-779.

In a further aspect, the present invention provides a method for monitoring
crystallization and/or purification of CCI-779.
Other aspects and advantages of the present invention are described further
in the following detailed description of the preferred embodiments thereof.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a process for preparing purified crystalline
CCI-779 with good crystallinity. This process also can be performed successfully
on a large scale, without adversely affecting the purity of the CCI-779 product and
in good yields.
The good crystallinity of the purified CCI-779 contributes to the improved
stability of the purified CCI-779 and longer shelf life, and contains less oxidative
impurities. In one embodiment, the purified crystalline CCI-779 of the present
invention degrades less than about 3%, less than about 2%, or less than about 1%
over a period of about 1 month. Desirably, the purified crystalline CCI-779 retains
greater than 97% strength over a period of about 3 months, and more desirably
about 6 months at about 5 °C as measured by high performance liquid
chromatography (HPLC), among other techniques. The strength is similarly
maintained at elevated temperatures up to about 70 °C, desirably up to about 51
°C, more desirably temperatures up to about 25 °C and greater relative humidities
up to about 60%, and most desirably temperatures at about 5 °C, or combinations
thereof. In one embodiment, the purified crystallized CCI-779 prepared according
to the present invention maintains its stability at a temperature of about 25 °C and
relative humidity of about 60% for up to about 3 months, and more desirably up to
about 6 months.
The purified crystalline CCI-779 prepared according to the present
invention typically contains less than 0.6% wt/wt of oxidative impurities.
Desirably, the purified CCI-779 also contains less than 0.1% wt/wt of
phenylboronic acid and less than about 0.3% wt/wt of acetone. The term
"oxidative impurity" as used herein refers to impurities that are produced due to

degradation of residual phenylboronic acid in the solid sample of purified
crystalline CCI-779.
The purified crystalline CCI-779 typically has a differential scanning
calorimetry (DSC) thermogram having an endotherm peak greater than about 165
°C. Desirably, the endotherm peak is at about 165 °C, 166 °C, 167 °C, 168 °C,
169 °C, or 170 °C. Similarly, the X-ray diffraction (XRD) peak pattern of the
purified crystalline CCI-779 typically contains peaks at 2θ with an area intensity of
greater than about 25% ± a 20 of 0.2. For example, the XRD pattern of the
purified crystalline CCI-779 typically contains peaks at 2θ of about 7.7, 9.0,11.4,
12.6, 13.3,15.0, 15.4, 16.2,66.5, 34.8,43.7, 31.4, and 58.
In one embodiment, the present invention provides purified crystalline
CCI-779 having a DSC thermogram having an endotherm peak greater than about
165 °C; and an XRD peak pattern having peaks at 2θ of about 7.7, 9.0,11.4,12.6,
13.3, 15.0, 15.4, 16.2, 66.5, 34.8, 43.7, 31.4, and 58.
In another embodiment, the present invention provides purified crystalline
CCI-779 which comprises less than about 0.6 % wt/wt of phenylboronic acid.
A. Process for Preparing Purified Crystalline CCI-779
Also provided by the present invention is a process for preparing purified
crystalline CCI-779. The process of the invention advantageously provides a route
to purified crystalline CCI-779 without the use of chromatography for the
purification. The present process also provides purified crystalline CCI-779 on a
large scale.
The first step in the process of the invention includes dissolving unpurified
CCI-779 in a first solvent. Desirably, the CCI-779 is highly soluble in the first
solvent. Typically, the first solvent is a ketone. In one embodiment, the first
solvent is acetone, methyl ethyl ketone, diethyl ketone, or methyl isobutyl ketone.
In a further embodiment, the first solvent is acetone. In another embodiment, the
first solvent is methyl ethyl ketone., among others. However, one of skill in the art
would readily be able to select a suitable first solvent for use in the present

invention by using the teachings of the present invention. Once dissolved in the
first solvent, the solution is typically filtered to remove any solid particles.
The term "unpurified CCI-779" or "crude CCI-779" as used herein refers to
a less pure and/or crystalline form of CCI-779. There are a variety of methods for
preparing unpurified or crude CCI-779 and include US Patent Nos. 5,362,718 and
6,277,983, which are hereby incorporated by reference. Alternatively, CCI-779
can be purchased commercially (e.g., Wyeth). The unpurified or crude CCI-779
can be non-micronized or micronized as described in US Patent Application
Publication No. US-2005-0152983-A1, which is hereby incorporated by reference.
The first solvent is then removed from the filtered solution, typically using
reduced pressures, optionally in the presence of heat One of skill in the art would
readily be able to use adjust the conditions for removing the first solvent or would
be able to use other methods for removing the first solvent.
Once the first solvent is removed, CCI-779 is obtained, typically as a foam,
solid, or combination thereof. Typically, trace amounts of the first solvent remains
in the residual CCI-779 foam, solid, or combination thereof. However, the CCI-
779 obtained as the foam, solid, or combination thereof can lack any first solvent
therein. In one embodiment, the CCI-779 is amorphous. In another embodiment,
the CCI-779 is partially crystalline.
The CCI-779 obtained after removal of the first solvent is then dissolved in
a second solvent. Desirably, the CCI-779 is less soluble in the second solvent than
in the first solvent. The CCI-779 is dissolved in a minimal amount of the second
solvent. The term "minimal amount" refers to the smallest volume of second
solvent that permits dissolution of the majority of the CCI-779. Alternatively, the
CCI-779 is first dissolved in greater amounts of the second solvent and then the
volume of the solution reduced by using techniques known to those of skill in the
art including reduced pressures. One of skill in the art would also be able to
readily recognize when the volume of the solution has been reduced to acceptable
amounts.
Suitably, the solubility of the CCI-779 in the second solvent is determined
by visual inspection. Typically, the solution containing the CCI-779 and second

solvent is visually inspected to ensure that most of the CCI-779 dissolved. One of
skill in the art would readily be able to utilize techniques to effect further
dissolution of the CCI-779 in the second solvent. Alternatively, the solubility of
the solid CCI-779 is determined by an analytical device such as a turbidity probe
or a focused beam reflectance measurement (FBRM® probe). In one embodiment,
the second solvent is an ether. In another embodiment, the second solvent has a
polarity similar to the polarity of diethyl ether. In a further embodiment, the
second solvent is diethyl ether. The solution is also optionally filtered after
dissolution of the CCI-779 in the second solvent.
Once dissolved, the content of the first solvent, typically acetone, in the
solution of the second solvent/CCI-779 is measured. Desirably, the content of the
first solvent is less than about 4 vol%, and more desirably the content of the
acetone is less than about 3 vol%. Typically, the content of the first solvent in the
second solvent is measured using HPLC. If the content of the first solvent in the
solution exceeds 4 vol%, the second solvent is removed using reduced pressures,
optionally in the presence of heat, to re-form solid CCI-779. The solid CCI-779 is
treated with a second aliquot of the second solvent and the content of the first
solvent again measured. These steps are repeated until the content of the first
solvent in a solution of the CCI-779/second solvent is less than 4 vol%. Typically,
the second solvent is removed at least once before the acceptable first solvent
content is reached.
In addition to measuring the acetone content, the present invention also
provides measuring the nucleation point of purified CCI-779 prior to
crystallisation of the same in the second solvent. The term "nucleation" as used
herein refers to the spontaneous formation of crystalline CCI-779 from a
supersaturated solution of CCI-779 in the second solvent, such as ether. The
"nucleation point", i.e., the point at which nucleation begins, is typically measured
by focused beam reflectance measurement (FBRM®), of which there are a variety
of FBRM® instruments available in the art and useful in the present invention and
include the Lasentec® D 600L or S400 FBRM® systems. The FBRM®
instrument is useful to ensure high crystallinity and purity of the CCI-779

crystalline product. Typically, the nucleation point is achieved when the chord
count of the particles of CCI-779 formed in the solution is greater than 1500
chords/second for 1 to 5 m size particles of the purified crystalline CCI-779.
The term "chord" as used herein refers to a straight-line path across the
cross-section of a particle and is an averaged dimension of the length of the
particle. One of skill in the art may also describe a chord simplistically as a
measure of the width of the particle. Desirably, the term "chord" as used herein
refers to a CCI-779 crystalline particle. The term "chord count" as used herein
refers to the number of particles of a certain chord length or size fraction.
The nucleation point can be adjusted by varying the nucleation hold time
during measurement with the FBRM® instrument until the desired chord count is
achieved. If a scavenging agent is utilized in the present invention, the "nucleation
hold time" refers to the period of time that the CCI-779 in the second solvent is
maintained before the scavenging agent is added. If a scavenging agent is omitted
from the process of the invention, the "nucleation hold time" refers to the period of
time that the CCI-779 in the second solvent is maintained before the anti-solvent is
added. The nucleation point is desirably determined by monitoring the FBRM®
signal as described above.
Once the desired content of the first solvent in the second solvent and
nucleation point has been achieved, a scavenging agent is optionally added to
remove residual phenylboronic acid. The inventors have found that residual
phenylboronic acid can degrade in solid samples of CCI-779, thereby resulting in
the formation of oxidative impurities, which can lead to the breakdown of CCI-
779, particularly during storage. Desirably, the scavenging agent is pentanediol
and is added to the solution containing the second solvent and the CCI-779.
Crystallization of purified CCI-779 can begin at various stages during
purification and can vary from one run to another. Typically, crystallization begins
after addition of the second solvent. Desirably, crystallization begins (i) during
measurement of the first solvent in the second solvent, (ii) during measurement of
the nucleation point, or a combination thereof.

After addition of the optional scavenging agent, an anti-solvent is added to
obtain solid purified crystalline CCI-779 or purified crystalline CCI-779 that had
not yet crystallized in the solution. The term "anti-solvent" as used herein refers to
a solvent that minimally or does not dissolve the purified crystalline CCI-779. If a
scavenging agent is utilized in the process, the anti-solvent is added to the solution
containing the second solvent, CCI-779, and scavenging agent. If a scavenging
agent is not utilized in the process, the anti-solvent is added to the solution
containing the second solvent and CCI-779. In one embodiment, the anti-solvent
is a hydrocarbon and is desirably a straight chain or branched saturated
hydrocarbon. Typically, the hydrocarbon has about 6 to about 10 carbon atoms. In
one embodiment, the hydrocarbon is heptane, 2-methyl pentane, n-hexane, and
combinations thereof, among others. In another embodiment, the hydrocarbon is
heptane. In another embodiment, the anti-solvent is heptane and further contains
other hydrocarbons.
The inventors found that the crystallinity and yield of the purified
crystalline CCI-779 is affected by the acetone content, induction time, and anti-
solvent addition. Specifically, highly crystalline purified CCI-779 was obtained in
high yields with low levels of acetone, a long induction time, and a non-linear
addition of the anti-solvent.
As used herein, the phrase "induction time" refers to the period of time
between addition of the first solvent and the start of the addition of the anti-solvent.
In one embodiment, the induction time is about 0.5 to about 5 hours. In yet
another embodiment, the induction time is about 1 to 3 hours. However, the
induction time is quite variable and depends on the amount of time required to
obtain an acceptable content of the first solvent in the second solvent and amount
of time required to adjust the nucleation time during measurement with the
FBRM® instrument.
The term "non-linear" as used herein refers to the rate of the anti-solvent
addition, whereby the rate varies with time. Desirably, the anti-solvent is initially
slowly added to the second solvent and thereafter the rate of addition is increased
over time. The inventors have found that if the initial addition of the anti-solvent

is too fast, small-needle-like particles of CCI-779 with poor flow are formed as an
oil, a fine, poorly crystalline material, or a fine, non-crystalline material, thereby
indicating primary nucleation. A non-linear addition of the anti-solvent also
results in the formation of crystalline purified CCI-779 with consistent morphology
and size. In one embodiment, crystalline purified CCI-779 is prepared as rod-like
crystalline particles. In another embodiment, the crystalline purified CCI-779
contains particles about 10 to about 100 m. in length.
The term "primary nucleation" as used herein refers to precipitation of
CCI-779, whereby crystal particles of CCI-779 are not formed. This term differs
from the crystallization of CCI-779, whereby crystalline CCI-779 is formed.
Desirably, the flow rate of the anti-solvent is normalized and expressed as a
1:1 ratio of the rate of anti-solvent addition (L/hr) to the amount of crude CCI-779
(kg). However, one of skill in the art would also readily be able to utilize a slower
flow rate. Typically, the anti-solvent is initially added at a rate of about 2
L/hour/kg crude CCI-779. Thereafter, the anti-solvent addition rate is increased,
desirably to about 3 L/hour/kg crude CCI-779. This increased anti-solvent
addition rate can be maintained or thereafter increased to a faster addition, such as
about 11 L/hour/kg crude CCI-779. In one embodiment, the anti-solvent is added
over a period of about 120 to about 240 minutes. In another embodiment, the anti-
solvent is added over a period of about 180 minutes. In a further embodiment, the
anti-solvent is initially added at a rate of 1 L/hour/kg crude CCI-779, thereby
increased to a rate of 3 L/hour/kg crude CCI-779 in the second hour, and finally
increased to a rate of 11 L/hour/kg crude CCI-779 in the last 30 minutes.
The purified crystalline CCI-779 is collected using techniques known to
those of skill in the art and include, without limitation, filtration, decanting,
centrifugation, among others. Once collected, the purified crystalline CCI-779 is
optionally washed, once or multiple times, with the anti-solvent, second solvent, or
combination thereof. In one embodiment, the purified crystalline CCI-779 is
washed with an ether and an anti-solvent. In another embodiment, the purified
crystalline CCI-779 is washed with a diethyl ether and heptane solution. Once
collected and optionally washed, the purified crystalline CCI-779 is dried using

techniques known to those of skill in the art, and optionally micronized using the
micronization techniques as described above for unpurified CCI-779- Typically,
the purified crystallized CCI-779 prepared according to the present invention is
micronized to prepare particles of less than about 30 m.
The purified crystalline CCI-779 can also be further purified by repeating
the steps of the process of the invention.
In one example, the present invention provides a process for purifying CCI-
779 including (i) dissolving CCI-779 in a ketone; (ii) filtering the product of step
(i); (iii) removing the ketone from the product of step (ii); (iv) dissolving the
product of step (iii) in an ether; (v) measuring the content of the ketone in the
product of step (iv), wherein if the ketone content is greater than 4 vol%, the ether
is removed and steps (iv) and (v) are repeated; (vi) measuring the nucleation point
of the product of step (iv) by focused beam reflectance measurement and adjusting
the nucleation hold time until the chord count is greater than 1500 chords/second
for 1 to 5 m particles of the CCI-779; (vii) optionally adding pentanediol to the
product of step (vi); (viii) adding an anti-solvent to the product of step (vii); and
(ix) collecting purified crystalline CCI-779, wherein steps (i) to (ix) and/or steps
(iv) to (ix) are optionally repeated with said purified CCI-779.
In another example, the present invention provides a process for purifying
CCI-779 including (i) dissolving unpurified CCI-779 in acetone; (ii) filtering the
solution of step (i); (iii) removing the acetone from the product of step (ii); (iv)
dissolving the product of step (iii) in diethylether; (v) measuring the content of the
acetone in the product of step (iv), wherein if the acetone content is greater than 4
vol%, the diethylether is removed and steps (iv) and (v) are repeated; (vi)
measuring the nucleation point in the product of step (iv) by focused beam
reflectance measurement and adjusting the nucleation hold time until the chord
count is greater than 1500 chords/second for 1 to 5 m particles of the CCI-779;
(vii) optionally adding pentanediol to the diethylether; (viii) adding heptane to the
product of step (vii) over a period of 120 to 240 minutes; (ix) collecting purified
CCI-779; and (x) drying purified CCI-779 under reduced pressures at a
temperature of about 25 to about 50 °C, wherein step (vii) is performed about 0.5

to about 5 hours after step (iv) and wherein steps (i) to (x) and/or (iv) to (ix) are
optionally repeated with said purified CCI-779.
In a further example, the present invention provides a method for
monitoring crystallization of CCI-779, including (i) dissolving unpurified CCI-779
in a first solvent; (ii) removing the first solvent from the product of (i); (iii)
dissolving the product of step (ii) in a second solvent; (iv) measuring the content of
the first solvent in the product of (iii), wherein if the content of the first solvent is
greater than a predetermined solvent content, the second solvent is removed and
steps (ii) and (ii) are repeated; (v) measuring the nucleation point of CCI-779 in the
second solvent by focused beam reflectance measurement and adjusting the
nucleation hold time until the chord count is the same or greater than a
predetermined nucleation chord count.
B. Compositions Containing Purified Crystalline CCI-779
The present invention also provides compositions, preferably
pharmaceutical compositions, containing purified crystalline CCI-779 alone or in
combination with unpurified CCI-779. The compositions typically contain a
pharmaceutically acceptable carrier, but can also contain other suitable
components. Typically, the additional components are inert and do not interfere
with the function of the required components of the compositions. The
compositions of the present invention can thereby further include other adjuvants,
syrups, elixirs, diluents, binders, lubricants, surfactants, granulating agents,
disintegrating agents, emollients, metal chelators, pH adjusters, surfactants, fillers,
disintegrants, and combinations thereof, among others.
Adjuvants can include, without limitation, flavoring agents, coloring
agents, preservatives, and supplemental antioxidants, which can include vitamin E,
ascorbic acid, butylated hydroxytoluene (BHT) and butylated hydroxyanisole
(BHA).
Binders can include, without limitation, povidone, cellulose,
methylcellulose, hydroxyrnethylcellulose, carboxymethylcellulose calcium,
carboxymethylcellulose sodium, hydroxypropylcellulose,

hydroxypropylmethylcellulose phthalate, noncrystalline cellulose,
polypropylpyrrolidone, polyvinylpyrrolidone (povidone, PVP), gelatin, gum arabic
and acacia, polyethylene glycols, starch, sugars such as sucrose, kaolin, dextrose,
and lactose, cholesterol, tragacanth, stearic acid, gelatin, casein, lecithin
(phosphatides), cetostearyl alcohol, cetyl alcohol, cetyl esters wax, dextrates,
dextrin, glyceryl monooleate, glyceryl monostearate, glyceryl palmitostearate,
polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives,
polyoxyethylene stearates, polyvinyl alcohol, and gelatin, among others. In one
embodiment, the binder is povidone.
Lubricants can include light anhydrous silicic acid, talc, stearic acid,
sodium lauryl sulfate, magnesium stearate and sodium stearyl furamate, among
others. In one embodiment, the lubricant is magnesium stearate.
Granulating agents can include, without limitation, silicon dioxide, starch,
calcium carbonate, pectin, crospovidone, and polyplasdone, among others.
Disintegrating agents or disintegrants can include starch,
carboxymethylcellulose, substituted hydroxypropylcellulose, sodium bicarbonate,
calcium phosphate, calcium citrate, sodium starch glycolate, pregelatinized starch
or crospovidone, among others.
Emollients can include, without limitation, stearyl alcohol, mink oil, cetyl
alcohol, oleyl alcohol, isopropyl laurate, polyethylene glycol, olive oil, petroleum
jelly, palmitic acid, oleic acid, and myristyl myristate.
Surfactants can include polysorbates, sorbitan esters, poloxamer, or sodium
lauryl sulfate. In one embodiment, the surfactant is sodium lauryl sulfate.
Metal chelators can include physiologically acceptable chelating agents
including edetic acid, malic acid, or fumaric acid. In one embodiment, the metal
chelator is edetic acid.
pH adjusters can also be utilized to adjust the pH of a solution containing
CCI-779 to about 4 to about 6. In one embodiment, the pH of a solution
containing CCI-779 is adjusted to a pH of about 4.6. pH adjustors can include
physiologically acceptable agents including citric acid, ascorbic acid, fumaric acid,
or malic acid, and salts thereof. In one embodiment, the pH adjuster is citric acid.

Additional fillers that can be used in the composition of the present
invention include mannitol, calcium phosphate, pregelatinized starch, or sucrose.
C. Methods of Using Purified Crystalline CCI-779
The invention further provides methods of delivering purified CCI-779 to a
patient, where the method includes administering purified CCI-779 according to
the invention. .
The dosage requirements of purified crystalline CCI-779 may vary based
on the severity of the symptoms presented and the particular subject being treated.
Treatment can be initiated with small dosages less than the optimum dose of
purified crystalline CCI-779. Thereafter the dosage is increased until the optimum
effect under the circumstances is reached. Precise dosages will be determined by
the administering physician based on experience with the individual subject
treated. In general, purified crystalline CCI-779 is most desirably administered at
a concentration that will generally afford effective results without causing any
unacceptable harmful or deleterious side effects. For example, an effective amount
of purified crystalline CCI-779 is generally, e.g., about 0.1 to about 50 mg, about
10 mg to about 30 mg, or about 0.5 to about 2 mg.
Purified crystalline CCI-779 is therefore useful as an antineoplastic agent,
and in particular, in treatment of sarcomas and carcinomas, astrocytomas, prostate
cancer, breast cancer, colon cancer, small cell lung cancer, ovarian cancer, central
nervous system cancer, melanoma, gliomas, glioblastoma, and adult T-cell
leukemia/lymphoma. Purified crystalline CCI-779 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; graft vs. host disease; autoimmune diseases such as lupus,
rheumatoid arthritis, diabetes mellitus, myasthenia gravis, and multiple sclerosis;
for treating 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; fungal infections;

hyperproliferative vascular diseases such as restenosis, graft vascular
atherosclerosis, cardiovascular disease, cerebral vascular disease, 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,
stroke or multiinfarct dementia.
Purified crystalline CCI-779 can be formulated in any form suitable for the
desired route of delivery using apharmaceutically effective amount of purified
crystalline CCI-779. For example, purified crystalline CCI-779 can be delivered by
a route such as oral, dermal, transdermal, intrabronchial, intranasal, intravenous,
intramuscular, subcutaneous, parenteral, intraperitoneal, intranasal, vaginal, rectal,
sublingual, intracranial, epidural, intratracheal, or by sustained release. Preferably,
delivery is oral.
For example, purified crystalline CCI-779 may be formulated for
administration orally in such forms as tablets, capsules, microcapsules, dispersible
powders, granules, or suspensions containing, for example, from about 0.05 to 5%
of suspending agent, syrups containing, for example, from about 10 to 50% of
sugar, and elixirs containing, for example, from about 20 to 50% ethanol, and the
like. The preferred pharmaceutical compositions from the standpoint of ease of
preparation and administration are solid compositions, particularly tablets and
hard-filled or liquid-filled capsules.
Purified crystalline CCI-779 may also be administered parenterally or
intraperitoneally. Solutions or suspensions of purified crystalline CCI-779 as a
free base or pharmacologically acceptable salt can be prepared in water suitably
mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be
prepared in glycerol, liquid, polyethylene glycols and mixtures thereof in oils.
Under ordinary conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms. Typically, such sterile
injectable solutions or suspensions contain from about 0.05 to 5% suspending
agent in an isotonic medium. Such pharmaceutical preparations may contain, for

example, from about 25 to about 90% of the active ingredient in combination with
the carrier, more usually between about 5% and 60% by weight.
In another embodiment, purified crystalline CCI-779 is delivered
intravenously, intramuscularly, subcutaneously, parenterally and intraperitoneally
in the form of sterile injectable solutions, suspensions, dispersions, and powders
which are fluid to the extent that easy syringe ability exits. Such injectable
compositions are sterile, stable under conditions of manufacture and storage, and
free of the contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for example,
water, ethanol (e.g., glycerol, propylene glycol and liquid polyethylene glycol),
oils, and mixtures thereof. Preferably the liquid carrier is water. In one
embodiment, the oil is vegetable oil. Optionally, the liquid carrier contains a
suspending agent. In another embodiment, the liquid carrier is an isotonic medium
and contains 0.05 to about 5% suspending agent.
In a further embodiment, purified crystalline CCI-779 is delivered rectally
in the form of a conventional suppository.
In another embodiment, purified crystalline CCI-779 is delivered vaginally
in the form of a conventional suppository, cream, gel, ring, or coated intrauterine-
device (IUD).
In yet another embodiment, purified crystalline CCI-779 is delivered
intranasally or intrabronchially in the form of an aerosol.
In a further embodiment, purified crystalline CCI-779 is delivered
transdermally or by sustained release through the use of a transdermal patch
containing purified crystalline CCI-779 and an optional carrier that is inert to
purified crystalline CCI-779, is nontoxic to the skin, and allows for delivery of
purified crystalline CCI-779 for systemic absorption into the blood stream. Such a
carrier can be a cream, ointment, paste, gel, or occlusive device. The creams and
ointments can be viscous liquid or semisolid emulsions. Pastes include absorptive
powders dispersed in petroleum or hydrophilic petroleum. Further, a variety of
occlusive devices can be utilized to release purified crystalline CCI-779 into the

blood stream and include semi-permeable membranes covering a reservoir contain
the active reagents, or a matrix containing the reactive reagents.
The use of sustained delivery devices can be desirable, in order to avoid the
necessity for the patient to take medications on a daily basis. The term "sustained
delivery" is used herein to refer to delaying the release of an active agent, i.e.,
purified crystalline CCI-779, until after placement in a delivery environment,
followed by a sustained release of the agent at a later time. A number of sustained
delivery devices are known in the art and include hydrogels (US Patent Nos.
5,266,325; 4,959,217; 5,292,515), osmotic pumps (US Patent Nos. 4,295,987 and
5,273,752 and European Patent No. 314,206, among others); hydrophobic
membrane materials, such as ethylenemethacrylate (EMA) and
ethylenevinylacetate (EVA); bioresorbable polymer systems (International Patent
Publication No. WO 98/44964 and US Patent Nos. 5,756,127 and 5,854,388); and
other bioresorbable implant devices composed of, for example, polyesters,
polyanhydrides, or lactic acid/glycolic acid copolymers (US Patent No. 5,817,343).
For use in such sustained delivery devices, purified crystalline CCI-779 can be
formulated as described herein. See, US Patent Nos. 3,845,770; 3,916,899;
3,536,809; 3,598,123; and 4,008,719.
Purified crystalline CCI-779 typically formed into a suitable dosing unit for
delivery to a patient. Suitable dosing units include oral dosing units, such as a
directly compressible tablets, capsules, powders, suspensions, microcapsules,
dispersible powders, granules, suspensions, syrups, elixirs, and aerosols. These
dosing units are readily prepared using the methods described herein and those
known to those of skill in the art.
In one embodiment, when administered orally, the capsules utilized in the
present invention include hydroxypropyl methylcellulose, hypromellose capsule, or
a hard shell gelatin capsule. The tablets or caplets of the present invention that
contain purified crystalline CCI-779 are optionally film-coated. Suitable film-
coatings are known to those of skill in the art. For example, the film-coating can
be selected from among polymers such as hydroxypropylmethylcellulose, ethyl
cellulose, polyvinyl alcohol, and combinations thereof.

A pharmaceutically effective amount of purified crystalline CCI-779 can
vary depending on the other components of the composition being delivered, mode
of delivery, severity of the condition being treated, the patient's agent and weight,
and any other active ingredients used in the composition. The dosing regimen can
also be adjusted to provide the optimal therapeutic response. Several divided doses
can be delivered daily, e.g., in divided doses 2 to 4 times a day, or a single dose
can be delivered. The dose can however be proportionally reduced or increased as
indicated by the exigencies of the therapeutic situation. In one embodiment, the
delivery is on a daily, weekly, or monthly basis. In another embodiment, the
delivery is on a daily delivery. However, daily dosages can be lowered or raised
based on the periodic delivery.
When utilized for treating neoplastic disease, carcinomas, and
adenocarcinomas, purified crystalline CCI-779 can be administered in conjunction
with one or more chemotherapeutic agents which can readily be selected by one of
skill in the art.
D. Kits Containing Purified CCI-779
The present invention also provides kits or packages containing purified
crystalline CCI-779. Kits of the present invention can include purified crystalline
CCI-779 or in combination with less pure forms and a carrier suitable for
administration to a mammalian subject as discussed above.
Preferably, the daily dosage of purified crystalline CCI-779 remains fixed
in each particular phase in which it is delivered. The kit can further contain
instructions for administering purified crystalline CCI-779.
The following examples are provided to illustrate the invention and do not
limit the scope thereof. One skilled in the art will appreciate that although specific
reagents and conditions are outlined in the following examples, modifications can
be made which are meant to be encompassed by the spirit and scope of the
invention.

EXAMPLES
Example 1 - General Process for Preparing Purified Crystalline CCl-779
This example provides a process for preparing purified, crystalline CCI-779
from crude CCI-779.
Crude CCI-779 (200 g, 19.4 mol) was dissolved in acetone (1.61 L) in a 3L
jacketed reactor (Chemglass) equipped with an external heater/chiller, vacuum
distillation apparatus, 1 baffle housing the reactor temperature probe, overhead
stirrer with a 4 pitched-blade turbine impeller. The reactor was also fitted with
Lasentec® D600L focused beam reflectance measurement system (FBRM®
system) using a 18" hastealloy probe (0.7" OD) fitted with a sapphire window
(from Mettler Toledo Inc). The solution was then clarified through filtration using
a 0.45 m polypropylene filter element. Acetone was removed from the resulting
solution under vacuum to obtain a foam. Diethyl ether (1.27 L) was added and the
distillation continued until the level of acetone in the resulting solution was less
than 4 vol%. The solution was stirred until a slurry was obtained.
The nucleation process was monitored by using the FBRM® instrument
and the hold time was adjusted to achieve stable nucleation until the chord count
was greater than about 1500 chords/second for particles about 1 to about 5 m.
The slurry was stirred for additional time until the chord count for 1 to 5 m
particles indicated the end of the spontaneous nucleation event The slurry was
treated with 2-methyl-2,4-pentanediol (0.13 L) at 20-25 °C to scavenge the residual
phenylboronic acid- Heptane (3.14 L) was added to the slurry using a
programmable piston pump (Stepdos) which was used for controlled anti-solvent
addition at a segmented non-linear addition rate. Upon dilution with heptane by
controlled addition over 3 hours, the slurry was filtered to give large white crystals
of purified crystalline CCI-779 (mp onset = 169 °C) in 84% yield, with total
impurities of 1.25 %, where 0.54% of the impurities were oxidative impurities.

Example 2 - PREPARATION OF PURIFIED CRYSTALLINE CCI-779
USING SLOWER FILTRATION
Purified crystalline CCI-779 was prepared using the description provided in
Example 1 except that the CCI-779 solution in acetone was filtered using a
medium grit fritted glass funnel (75 mm ID). The filtration was slow and
necessitated the use of 2 funnels in parallel. The total time required for the
filtration was 2.1 hours.
The acetone was then removed as described in Example 1. The increased
viscosity of the amorphous CCI-779 resulted in the agitator slowing down. The
amorphous CCI-779 was held overnight under vacuum with the agitator switched
off.
Following the addition of diethyl ether as described in Example 1, in which
dissolution took about 11 minutes, nucleation of the CCI-779 was monitored. A
sample of the solution was then withdrawn for acetone content analysis and was
determined to be about 3.2 vol% acetone. The induction time for the appearance
of nuclei was 54 minutes (see Figure 1).
Following nucleation, the batch was aged for 34 minutes as indicated by the
FBRM® instrument to obtain stabilization of the chord count. The pentanediol
and heptane were added as described in Example 1. The isolated purified
crystalline CCI-779 was washed and dried at 40 °C under vacuum for 48 hours. A
yield of 176.3 g of purified crystalline CCI-779 was obtained (87.6 %). See Table
1 for the HPLC data for the purified CCI-779 obtained immediately after
crystallization and samples tested after 2 weeks and 1 month at varying conditions.


Example 3 - PREPARATION OF PURIFIED CRYSTALLINE CCI-779 WITH
FASTER FILTRATION
This example was performed using the same protocol as described in
Example 2. However, filtration of the crude CCI-779 solution in acetone was
completed in 30 minutes by using a medium grit fritted funnel. Following
distillation of the acetone, the batch was held as an amorphous foam/oil for 2 hours
under reduced pressures. Diethyl ether was then added to the foam/oil and the
diethyl ether solution was evaluated for acetone content (8.5 vol%). The
diethylether (containing acetone) was then removed by distillation to obtain an
amorphous foam. This foam was maintained under reduced pressures for 30
minutes and diethyl ether again added. The diethylether was again monitored for
acetone contact and determined to be less than 3.6 vol% acetone. The
crystallization was the continued as in Example 2. The induction time for
nucleation, i.e., the time required to obtain a chord count for particles of about 1 to
about 5 m particles of greater than about 1500 chords/s was 26 minutes.
Following the nucleation, the slurry was stirred for additional time to ensure
stabilization of the nucleation. Following isolation and drying as outlined above, a
final yield of 175.1 g of purified crystalline CCI-779 was obtained (87.3 %). See
Table 2, for the HPLC data for the purified crystalline CCI-779 obtained
immediately after crystallization and samples tested after 2 weeks and 1 month, at
varying conditions.


Example 4 - PREPARATION OF PURIFIED CRYSTALLINE CCI-779 WITH
CONTROLLED HEPTANE ADDITION
This example provides a semi-batch crystallization process to improve
crystallinity. The process is carried out in a reactor with accurate temperature
control, overhead stirring and ability to carry out controlled solvent feed. For
optimal control, the reactor is fitted with a Lasentec® FBRM® probe measuring
chord lengths in the 1-500 m range using "fines" electronics.
Acetone (5.7 L) is added to crude CCI-779 (1 kg). The acetone solution is
stirred at high revolutions per minute at 22 °C (jacket) until a clear solution is
obtained as monitored by the Lasentec® system particle count. The clear solution
is filtered through a 0.45 m in-line filter. The filter is washed with acetone (2 L)
and the acetone wash added to the filtered solution. The acetone of the filtered
solution is removed under reduced pressures by distillation at a temperature of
about 5-20 °C until a foam is obtained. The foam is maintained under reduced
pressures for about 2 hours.
Diethyl ether (3.4 L) is added to the dried foam and the solution stirred at
22 °C. After 10 minutes, the solid is collected by filtration and the diethylether
solution is monitored by gas chromatography (GC) to monitor the amount of
acetone remaining in the diethylether. The diethylether wash is repeated if the
acetone content is greater than about 3 vol% of the diethylether.
The diethyl ether washed sample is again combined with diethyl ether, is
mixed for 30 minutes, and the particle count is measured using the FBRM®
instrument After verification that the particle count is greater than 1500
chords/second for 1-5 m range, the slurry is mixed for an additional 30 minutes.
A visual inspection is also performed to confirm that a slurry is present.
A pentanediol solution (0.7 L pentanediol in 0.4 L diethyl ether) is then
added to the diethyl ether slurry over a period of at least 15 minutes. The solution
is mixed for 60 minutes at a temperature of about 22 °C or until the Lasentec©
system particle count is stable. Heptane (10.7 L) is added to the solution while
maintaining a temperature of about 22 °C over aperiod of about 150 minutes using
the following profile:

1. Add the first 2 Lin first 60 minutes;
2. Add 3 L in second 60 minutes; and
3. Add 5.7 L in next 30 minutes.
The slurry is then mixed for 4 hours at a temperature of about 22 °C, the
slurry filter, and the solid washed 3 times with a solvent mixture of ether/heptanes
(0.9/4.8 L). The solid is dried at a temperature of about 40 °C under reduced
pressures.
All publications cited in this specification are incorporated herein by
reference herein. While the invention has been described with reference to a
particularly preferred embodiment, it will be appreciated that modifications can be
made without departing from the spirit of the invention. Such modifications are
intended to fall within the scope of the appended claims.

CLAIMS:
1. A purified crystalline form of CCI-779 which retains greater than
97% strength over a period of at least about 3 months,
wherein said purified form of CCI-779 is not purified by
chromatography.
2. The crystalline form according to claim 1, which comprises less
than 0.1% wt/wt of phenylboronic acid.
3. The crystalline form according to claim 1 or claim 2, which
comprises less than 0.6% wt/wt of oxidative impurities.
4. The crystalline form according to any one of claims 1 to 3, which
comprises less than about 0.3% wt/wt of acetone.
5. The crystalline form according to any one of claims 1 to 4, having a
differential scanning calorimetry thermogram having an endotherm peak of greater
than about 165 °C.
6. The crystalline form according to claim 1, having an X-ray
diffraction peak pattern having peaks at 2 of 7.7,9.0, 11.4,12.6,13.3,15.0,15.4,
16.2, 66.5, 34.8,43.7,31.4, and 58.
7. A purified crystalline form of CCI-779 according to claim 1,
having:
(i) a differential scanning calorimetry thermogram having an
endotherm peak greater than about 165 °C;
(ii) an X-ray diffraction peak pattern having peaks at 20 of 7.7,
9.0,11.4,12.6,13.3,15.0,15.4,16.2, 66.5, 34.8,43.7,31.4, and 58.

8. A process for purifying CCI-779 comprising:
(i) dissolving CCI-779 in a ketone;
(ii) filtering the product of step (i);
(iii) removing said ketone from the product of step (ii);
(iv) dissolving the product of step (iii) in an ether;
(v) measuring the content of said ketone in the product of step (iv);
wherein if the ketone content is greater than 4 vol%, said ether is
removed and steps (iv) and (v) are repeated;
(vi) measuring the nucleation point of the product of step (iv) by
focused beam reflectance measurement and adjusting the nucleation hold time until
the chord count is greater than 1500 chords/second for 1 to 5 m particles of said
CCI-779;
(vii) optionally adding pentanediol to the product of step (vi);
(viii) adding an anti-solvent to the product of step (vii); and
(ix) collecting purified CCI-779;
wherein steps (i) to (ix) or step (iv) to (ix) are optionally repeated with said
purified CCI-779.
9. The process according to claim 8, wherein the product of step (iii) is
amorphous.
10. The process according to claim 8 or claim 9, wherein said ketone is
acetone.
11. The process according to any one of claims 8 to 10, wherein said
ether is similar in polarity to diethyl ether.
12. The process according to claim 11, wherein said ether is diethyl
ether.

13. The process according to any one of claims 8 to 12, wherein said
anti-solvent is heptane.
14. The process according to claim 13, wherein said anti-solvent further
comprises other hydrocarbons.
15. The process according to any one of claims 8 to 14, wherein step
(vii) is performed about 0.5 to about 5 hours after step (iv).
16. The process according to claim 15, wherein step (vii) is performed
about 1 to 3 hours after step (iv).
17. The process according to any one of claims 8 to 16, wherein
crystallization of purified CCI-779 begins in step (iv), step (v), (vi), or between
steps (iv) and (vi).
18. The process according to any one of claims 8 to 17, wherein said
anti-solvent is added non-linearly.
19. The process according to claim 18, wherein the initial rate of
addition of said anti-solvent is slow.
20. The process according to claim 19, wherein the rate of addition of
said anti-solvent is increased over time.
21. The process according to any one of claims 8 to 20, wherein said
anti-solvent is added over a period of about 120 to about 240 minutes.
22. The process according to claim 21, wherein said anti-solvent is
added over a period of about 180 minutes.

23. The process according to any one of claims 8 to 22, wherein step
(vii) is optionally performed about 0.5 to about 5 hours after step (iv).
24. The process according to claim 8 to 23, wherein said purified CCI-
779 is washed with an ether and an anti-solvent.
25. The process according to any one of claims 8 to 23, wherein said
purified CCI-779 is dried.
26. The process according to any one of claims 8 to 24, further
comprising the step of drying the purified CCI-779 under reduced pressures at a
temperature of about 25 to about 50 °C, and wherein the steps are optionally
repeated with said purified CCI-779.
27. A product prepared by the process of any of claims 8 to 26.
28. A method for monitoring crystallization of CCI-779, comprising:
(i) dissolving unpurified CCI-779 in a first solvent;
(ii) removing the first solvent from the product of (i);
(iii) dissolving the product of step (ii) in a second solvent;
(iv) measuring the content of said first solvent in the product of
wherein if the content of the first solvent is greater than a
predetermined solvent content, said second solvent is removed and steps (ii) and
(ii) are repeated;
(v) rneasuring the nucleation point of CCI-779 in said second
solvent by focused beam reflectance measurement and adjusting the nucleation
hold time until the chord count is the same or greater than a predetermined
nucleation chord count.

29. A kit comprising (i) purified crystalline CCI-779 of any one of
claims 1 to 7; and (ii) a carrier suitable for administration to a mammalian subject.
30. A pharmaceutical composition comprising purified crystalline CCI-
779 of any one of claims 1 to 7 and one of more of:
(i) a metal chelator;
(ii) a pH adjuster;
(iii) a surfactant;
(iv) at least one filler;
(v) a binder;
(vi) a disintegrant; and
(vii) a lubricant.
31. A method for treating central nervous system cancer, leukemia,
breast cancer, prostate cancer, melanoma, gliomas, and glioblastoma comprising
administering the purified crystalline form of CCI-779 of any of claims 1 to 7 to a
mammalian subject in need thereof.
32. Use of a purified CCI-779 according to any one of claims 1 to 7 in
preparation of a medicament.
33. Use of a purified CCI-779 according to claim 32, wherein the
medicament is useful for treatment of a condition selected from central nervous
system cancer, leukemia, breast cancer, prostate cancer, melanoma, gliomas, and
glioblastoma.

The present invention provides purified crystalline CCI-779 and processes for preparing the same.