U.S. patent application number 11/881033 was filed with the patent office on 2007-11-22 for methods of preparing pimecrolimus.
Invention is credited to Viktor Gyollai, Csaba Szabo.
Application Number | 20070270451 11/881033 |
Document ID | / |
Family ID | 36097009 |
Filed Date | 2007-11-22 |
United States Patent
Application |
20070270451 |
Kind Code |
A1 |
Gyollai; Viktor ; et
al. |
November 22, 2007 |
Methods of preparing pimecrolimus
Abstract
The present invention provides a process for the preparation of
pimecrolimus from ascomycin in which ascomycin is reacted with a
conversion reagent that converts ascomycin to its activated
derivative at C-32. The activated ascomycin is then reacted with
chloride ion. The process of the invention requires fewer process
steps than prior art processes, and does not require the protection
of the ascomycin C-24 hydroxyl group or the purification of the
activated ascomycin derivative.
Inventors: |
Gyollai; Viktor; (Debrecen,
HU) ; Szabo; Csaba; (Debrecen, HU) |
Correspondence
Address: |
KENYON & KENYON LLP
ONE BROADWAY
NEW YORK
NY
10004
US
|
Family ID: |
36097009 |
Appl. No.: |
11/881033 |
Filed: |
July 24, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11293353 |
Dec 1, 2005 |
7279571 |
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11881033 |
Jul 24, 2007 |
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60632372 |
Dec 1, 2004 |
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60633926 |
Dec 6, 2004 |
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60641697 |
Jan 5, 2005 |
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60641868 |
Jan 5, 2005 |
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60641869 |
Jan 5, 2005 |
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60662440 |
Mar 16, 2005 |
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60705681 |
Aug 3, 2005 |
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60709160 |
Aug 17, 2005 |
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Current U.S.
Class: |
514/291 ;
540/456 |
Current CPC
Class: |
C07D 498/18 20130101;
A61P 37/08 20180101; C07H 19/01 20130101; A61P 29/00 20180101; A61P
37/00 20180101; A61K 9/1688 20130101; A61P 37/06 20180101; A61P
31/00 20180101; A61P 17/00 20180101; C07D 491/14 20130101; A61P
37/02 20180101; A61K 9/145 20130101; A61P 31/04 20180101; A61K
31/4745 20130101 |
Class at
Publication: |
514/291 ;
540/456 |
International
Class: |
A61K 31/453 20060101
A61K031/453; A61P 17/00 20060101 A61P017/00; C07D 405/14 20060101
C07D405/14 |
Claims
1. Pimecrolimus having a purity of at least about 95% area by
HPLC.
2. The pimecrolimus of claim 1, having a purity of at least about
98% area by HPLC.
3. A pharmaceutical formulation comprising a therapeutically
effective amount of the pimecrolimus of claim 1.
4. A method for treating a patient suffering from atopic
dermatitis, comprising the step of administering to the patient the
pharmaceutical formulation of claim 3.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/293,353, which claims benefit of U.S. Provisional Patent
Applications Nos. 60/632,372, filed Dec. 1, 2004, 60/633,926, filed
Dec. 6, 2004, 60/641,697, filed Jan. 5, 2005, 60/641,868, filed
Jan. 5, 2005, 60/641,869, filed Jan. 5, 2005, 60/662,440, filed
Mar. 16, 2005, 60/705,681, filed Aug. 3, 2005, and 60/709,160,
filed Aug. 17, 2005, the contents of which are incorporated herein
in their entirety by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to methods of preparing the
anti-inflammatory compound pimecrolimus and to pure
pimecrolimus.
BACKGROUND OF THE INVENTION
[0003] Pimecrolimus is an anti-inflammatory compound derived from
the macrolactam natural product ascomycin, produced by certain
strains of Streptomyces. Pimecrolimus is sold in the United States
under the brand name ELIDEL.RTM., and is approved for the treatment
of atopic dermatitis. The systematic name of pimecrolimus is
(1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-12-[(1E)-2-{(1R,3R,4S)-4--
chloro-3-methoxycyclohexyl}-1-methylvinyl]-17-ethyl-1,14-dihydroxy-23,25-d-
imethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.0.sup.4-
,9]octacos-18-ene-2,3,10,16-tetraone. Pimecrolimus is the
32-epichloro derivative of ascomycin. Its empirical formula is
C.sub.43H.sub.68ClNO.sub.11 and its molecular weight is 810.47.
[0004] European Patent EP 427 680 B1 discloses a method of
synthesizing pimecrolimus in the form of a colorless foamy resin.
The disclosed method comprises the following four reaction steps:
[0005] (a) protection of the two hydroxyl groups at C-24 and C-32
with t-butyldimethylsilyl ethers; [0006] (b) deprotection of the
silyl-protected hydroxyl group at C-32, while the hydroxyl group at
C-24 remains protected; [0007] (c) substitution of chlorine for the
free hydroxyl group at C-32 with an inversion of configuration; and
[0008] (d) deprotection of the silyl-protected hydroxyl group at
C-24.
[0009] EP 427 680 does not disclose the yield of each step in the
synthesis, but does disclose that each step is followed by the
chromatographic purification of the product of that step.
Accordingly, it would be expected that the overall yield of the
process disclosed in EP 427 680 is low, given the number of
reaction steps and chromatographic purifications required.
[0010] Therefore, a process for the preparation of pimecrolimus
having a reduced number of steps, particularly chromatographic
steps, would be advantageous.
[0011] The present invention provides such processes.
SUMMARY OF THE INVENTION
[0012] The present invention provides methods for the synthesis of
pimecrolimus, comprising dissolving ascomycin in an organic
solvent, combining the solution of ascomycin with a base to obtain
a reaction mixture, combining the reaction mixture with a
conversion reagent to obtain an activated ascomycin derivative,
combining the activated derivative of ascomycin with a chloride ion
source until obtaining pimecrolimus, and recovering the
pimecrolimus.
[0013] Preferably, the pimecrolimus obtained by the above process
is purified by column chromatography.
[0014] Preferably, the purified pimecrolimus has a purity of at
least about 95% area by HPLC, more preferably of at least about 98%
area by HPLC.
[0015] The present invention also provides pimecrolimus having a
purity of at least about 95% area by HPLC, more preferably of at
least about 98% area by HPLC.
[0016] In another embodiment the present invention provides a
pharmaceutical formulation comprising a therapeutically effective
amount of the above pure pimecrolimus.
[0017] In another embodiment the present invention provides a
method for treating a patient suffering from atopic dermatitis,
comprising the step of administering to the patient the
pharmaceutical formulation described above.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1: A chromogram of the pimecrolimus.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention provides methods for the synthesis of
pimecrolimus, comprising dissolving ascomycin in an organic
solvent, combining the solution of ascomycin with a base to obtain
a reaction mixture, combining the reaction mixture with a
conversion reagent to obtain an activated ascomycin derivative,
combining the activated derivative of ascomycin with a chloride ion
source until obtaining pimecrolimus, and recovering the crude
pimecrolimus. Optionally, the hydroxyl group at position C-24 of
ascomycin is not protected during the process. Contrary to the
procedure described in EP 427 680, the selective protection of the
hydroxyl group of ascomycin at C-24 is not necessary, since the
hydroxyl group at C-32 is activated regioselectively (hydroxyl
group at C-24 remains intact). The conversion reagent converts
ascomycin to an activated ascomycin derivative at C-32.
##STR1##
[0020] Preferably, the activated ascomycin derivative is a
sulfonate ester, more preferably, a tosylate or mesylate, and, most
preferably, a triflate.
[0021] Preferably, the organic solvent is selected from the group
consisting of: dichloromethane, chloroform, diethylether,
diisopropylether, methyl-t-butylether, toluene, ethyl acetate,
i-butylacetate, acetone, methylethylketone, acetonitrile,
N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide,
and mixtures thereof. More preferably, the organic solvent is
toluene or acetonitrile. Preferably, the ascomycin is dissolved in
the organic solvent at a temperature of more than about 25.degree.
C. The resulting ascomycin solution is then stirred under an inert
atmosphere, e.g., nitrogen, at a low temperature of less than about
25.degree. C., preferably, less than about 0.degree. C., and, more
preferably, less than about -20.degree. C. The low temperature is
necessary for selective reaction and for obtaining low level of
side products. The stirring is preferably continued for a time
sufficient to dissolve substantially all the ascomycin.
[0022] Preferably, the base is in an amount of about 1 to about 4
equivalents. The base can be added dropwise, in parts, or all at
once. The base may be an organic or inorganic base. Preferably, the
base is selected from the group consisting of triethylamine,
diisopropyl-ethylamine (EDIPA), N-methyl-morpholine,
N,N-dimethylaniline, pyridine, and substituted pyridine
derivatives, such as 2,6-lutidine, s-collidine, and
4-dimethylaminopyridine. More preferably, the base is selected from
the group consisting of: diisopropyl-ethylamine (EDIPA),
s-collidine and 2,6-lutidine. The base may also be added to the
ascomycin solution together with the activating conversion reagent
in the following step, as described below. The added base may be in
solution, where the solvent is preferably selected from the group
consisting of: dichloromethane, chloroform, diethylether,
diisopropylether, methyl-t-butylether, toluene, ethyl acetate,
i-butylacetate, acetone, methylethylketone, acetonitrile,
N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide,
n-hexane, n-heptane, cyclohexane and mixtures thereof. More
preferably, the solvent is toluene. Preferably, the base is added
under neat conditions. The base is preferably added to prevent an
increase in the acidity of the reaction mixture, as, during the
reaction, the activating agent forms an acid that, in the absence
of the base, would acidify the reaction mixture. For example, when
the activating agent is trifluoromethanesulfonic anhydride,
trifluoromethanesulfonic acid is formed during the reaction, and,
when the activating agent is an acyl chloride, such as
trifluoromethanesulfonyl chloride, HCl is formed during the
reaction. Without the addition of the base, the formation of an
acid in the reaction mixture increases the acidity over the course
of the reaction, slowing the rate of the reaction, and decomposing
the macrocycle. Therefore, the base is preferably added to the
reaction mixture to neutralize the acid produced in the
process.
[0023] Following or with the addition of the base the reaction
mixture is combined with the activating conversion reagent,
dropwise, in parts, or all at once. Preferably, the progress of the
reaction is monitored, e.g., with thin layer chromatography (TLC),
until the reaction is complete or nearly complete.
[0024] Preferably, the conversion reagent is selected from the
group consisting of: fluorosulfonic anhydride, fluorosulfonyl
chloride, trifluoromethanesulfonic anhydride,
trifluoromethanesulfonyl chloride, methanesulfonic anhydride,
methanesulfonyl chloride, phenylmethanesulfonic anhydride,
phenylmethanesulfonyl chloride, p-toluenesulfonic anhydride,
p-toluenesulfonyl chloride, benzenesulfonic anhydride, and
benzenesulfonyl chloride. More preferably, the conversion reagent
is selected from the group consisting of: trifluoromethanesulfonic
anhydride and trifluoromethanesulfonyl chloride. The added
conversion reagent may be in solution, where the solvent is
selected from the same group of solvents that is used to form the
solution with the base, as described above. Preferably, the solvent
is selected from the group consisting of: toluene, n-hexane,
n-heptane and cyclohexane.
[0025] The time required for the complete or near complete
conversion of the ascomycin to the corresponding activated
ascomycin, such as a ascomycin 32-triflate, can vary somewhat,
depending upon the reaction conditions, such as the temperature,
and the solvent, base, and activating agent utilized. Those skilled
in the art will understand how to monitor the reaction, e.g., by
TLC, at appropriate time intervals, depending on the conditions
chosen. As a non-limiting example, when trifluoromethanesulfonic
anhydride as the activating agent is used, the reaction is almost
instantaneous, even at -40.degree. C., provided that the reaction
mixture contains a sufficient amount of base, i.e., about 3 to
about 4 equivalents. In contrast, for example, with the use of
p-toluenesulfonyl chloride as the activating conversion agent, the
reaction requires 1 to 3 hours at 0.degree. C. with 3 to 4
equivalents of base, and the reaction may require at least a day
with 1 equivalent of base.
[0026] After the addition of the activating conversion reagent, the
reaction mixture is then combined with a solution of a chloride-ion
source. Preferably, the solution of chloride-ion source is added to
the reaction mixture. Useful chloride-ion sources include, but are
not limited to lithium chloride, sodium chloride, potassium
chloride, magnesium chloride, calcium chloride, aluminum chloride,
iron(II)chloride, iron(III)chloride, ammonium chloride,
hydrochlorides of organic bases, quaternary ammonium chlorides,
quaternary phosphonium chlorides, tetrabutylammonium chloride,
benzyl-triethylammonium chloride and similar quaternary ammonium
chlorides, and hydrochlorides of the bases discussed above.
Preferably, chloride-ion source is benzyltriethylammonium chloride.
Useful solvents for the chloride-ion source include those discussed
above for the base and activating agent. Alternatively, the
chloride-ion source can be added to the reaction mixture neat.
[0027] After the addition of the chloride-ion source the reaction
mixture is stirred at a temperature above about 0.degree. C., but
below the reflux temperature of the solvent or mixture of solvents
used in the reaction mixture. Preferably, the temperature is at
least about 25.degree. C. Again, the progress of the reaction is
monitored to determine completion, such as with TLC.
[0028] The time required for the disappearance of the intermediate
activated ascomycin derivative, such as 32-triflate, will vary
somewhat depending on the precise conditions used, and depends
mainly on the reaction temperature and the solubility of the
selected chloride-ion source. Typically, the time is between about
1 hour and 1 day at room temperature. At a lower temperature and/or
with a chloride-ion source having a lower solubility, the reaction
is significantly slower. Longer reactions are less favored, as the
possibility of the formation of undesirable side products is
increased.
[0029] The pimecrolimus recovering step comprises: adding water
with a water immiscible organic solvent to obtain a two phase
system; separating the two phase system; extracting the organic
phase with an aqueous KHSO.sub.4 solution, NaHCO.sub.3 solution and
brine, concentrating the organic phase; and drying.
[0030] Alternatively, the recovering step comprises: adding water
to the reaction mixture to obtain a two phase system; separating
the two phase system; and concentrating the organic phase.
[0031] The crude product is obtained as an amorphous solid by
employing high vacuum during the last part of the concentration
process.
[0032] Preferably, the crude product is purified by column
chromatography prior to the drying step. The solution containing
the crude product is concentrated and subjected to chromatography
column. After the chromatography, the pure fractions are combined
and concentrated to obtain an amorphous solid.
[0033] Preferably, the pimecrolimus obtained after the purification
step has a purity of at least about 95% area by HPLC, more
preferably of at least about 98% area by HPLC.
[0034] In one embodiment, the present invention comprises
pimecrolimus having a purity of at least about 95% area by HPLC,
more preferably of at least about 98% area by HPLC.
[0035] Another embodiment of the present invention is a
pharmaceutical formulation comprising a therapeutically effective
amount of the above purified Pimecrolimus, and an amount of
pharmaceutically acceptable excipient.
[0036] "Therapeutically effective amount" means the amount of the
purified pimecrolimus, when administered to a patient for treating
a disease or other undesirable medical condition, is sufficient to
have a beneficial effect with respect to that disease or condition.
The "therapeutically effective amount" will vary depending on the
purity, the disease or condition and its severity, and the age,
weight, etc., of the patient to be treated. Determining the
therapeutically effective amount of a given pure pimecrolimus is
within the ordinary skill of the art, and requires no more than
routine experimentation.
[0037] Another embodiment of the present invention is a method for
treating a patient suffering from Atopic dermatitis, comprising the
step of administering to the patient the pharmaceutical formulation
comprising a therapeutically effective amount of purified
Pimecrolimus produced by the present invention. A further
embodiment of the present invention is a method of providing
immunosuppression to a patient in need thereof comprising the step
of administering to the patient the pharmaceutical formulation
comprising a therapeutically effective amount of purified
Pimecrolimus produced by the present invention.
[0038] Pharmaceutical formulations of the present invention contain
the purified Pimecrolimus produced by the processes of the present
invention. In addition to the active ingredient(s), the
pharmaceutical formulations of the present invention may contain
one or more excipients. Excipients are added to the formulation for
a variety of purposes.
[0039] Diluents may be added to the formulations of the present
invention. Diluents increase the bulk of a solid pharmaceutical
composition, and may make a pharmaceutical dosage form containing
the composition easier for the patient and caregiver to handle.
Diluents for solid compositions include, for example,
microcrystalline cellulose (e.g., AVICEL.RTM.), microfine
cellulose, lactose, starch, pregelatinized starch, calcium
carbonate, calcium sulfate, sugar, dextrates, dextrin, dextrose,
dibasic calcium phosphate dihydrate, tribasic calcium phosphate,
kaolin, magnesium carbonate, magnesium oxide, maltodextrin,
mannitol, polymethacrylates (e.g., EUDRAGIT.RTM.), potassium
chloride, powdered cellulose, sodium chloride, sorbitol, and
talc.
[0040] Solid pharmaceutical compositions that are compacted into a
dosage form, such as a tablet, may include excipients whose
functions include helping to bind the active ingredient and other
excipients together after compression. Binders for solid
pharmaceutical compositions include acacia, alginic acid, carbomer
(e.g., carbopol), carboxymethylcellulose sodium, dextrin, ethyl
cellulose, gelatin, guar gum, hydrogenated vegetable oil,
hydroxyethyl cellulose, hydroxypropyl cellulose (e.g.,
KLUCEL.RTM.), hydroxypropyl methyl cellulose (e.g., METHOCEL.RTM.),
liquid glucose, magnesium aluminum silicate, maltodextrin,
methylcellulose, polymethacrylates, povidone (e.g., KOLLIDON.RTM.,
PLASDONE.RTM.), pregelatinized starch, sodium alginate, and
starch.
[0041] The dissolution rate of a compacted solid pharmaceutical
composition in the patient's stomach may be increased by the
addition of a disintegrant to the composition. Disintegrants
include alginic acid, carboxymethylcellulose calcium,
carboxymethylcellulose sodium (e.g., AC-DI-SOL.RTM.,
PRIMELLOSE.RTM.), colloidal silicon dioxide, croscarmellose sodium,
crospovidone (e.g., KOLLIDON.RTM., POLYPLASDONE.RTM.), guar gum,
magnesium aluminum silicate, methyl cellulose, microcrystalline
cellulose, polacrilin potassium, powdered cellulose, pregelatinized
starch, sodium alginate, sodium starch glycolate (e.g.,
EXPLOTAB.RTM.), and starch.
[0042] Glidants can be added to improve the flowability of a
non-compacted solid composition, and to improve the accuracy of
dosing. Excipients that may function as glidants include colloidal
silicon dioxide, magnesium trisilicate, powdered cellulose, starch,
talc, and tribasic calcium phosphate.
[0043] When a dosage form such as a tablet is made by the
compaction of a powdered composition, the composition is subjected
to pressure from a punch and dye. Some excipients and active
ingredients have a tendency to adhere to the surfaces of the punch
and dye, which can cause the product to have pitting and other
surface irregularities. A lubricant can be added to the composition
to reduce adhesion, and ease the release of the product from the
dye. Lubricants include magnesium stearate, calcium stearate,
glyceryl monostearate, glyceryl palmitostearate, hydrogenated
castor oil, hydrogenated vegetable oil, mineral oil, polyethylene
glycol, sodium benzoate, sodium lauryl sulfate, sodium stearyl
fumarate, stearic acid, talc, and zinc stearate.
[0044] Flavoring agents and flavor enhancers make the dosage form
more palatable to the patient. Common flavoring agents and flavor
enhancers for pharmaceutical products that may be included in the
composition of the present invention include maltol, vanillin,
ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol,
and tartaric acid.
[0045] Solid and liquid compositions may also be dyed using any
pharmaceutically acceptable colorant to improve their appearance,
and/or facilitate patient identification of the product and unit
dosage level.
[0046] In liquid pharmaceutical compositions prepared using
purified Pimecrolimus produced by the processes of the present
invention, Pimecrolimus and any other solid excipients are
dissolved or suspended in a liquid carrier such as water, vegetable
oil, alcohol, polyethylene glycol, propylene glycol or
glycerin.
[0047] Liquid pharmaceutical compositions may contain emulsifying
agents to disperse uniformly throughout the composition an active
ingredient or other excipient that is not soluble in the liquid
carrier. Emulsifying agents that may be useful in liquid
compositions of the present invention include, for example,
gelatin, egg yolk, casein, cholesterol, acacia, tragacanth,
chondrus, pectin, methyl cellulose, carbomer, cetostearyl alcohol,
and cetyl alcohol.
[0048] Liquid pharmaceutical compositions may also contain a
viscosity enhancing agent to improve the mouth-feel of the product
and/or coat the lining of the gastrointestinal tract. Such agents
include acacia, alginic acid bentonite, carbomer,
carboxymethylcellulose calcium or sodium, cetostearyl alcohol,
methyl cellulose, ethylcellulose, gelatin guar gum, hydroxyethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,
maltodextrin, polyvinyl alcohol, povidone, propylene carbonate,
propylene glycol alginate, sodium alginate, sodium starch
glycolate, starch tragacanth, and xanthan gum.
[0049] Sweetening agents such as sorbitol, saccharin, sodium
saccharin, sucrose, aspartame, fructose, mannitol, and invert sugar
may be added to improve the taste.
[0050] Preservatives and chelating agents such as alcohol, sodium
benzoate, butylated hydroxyl toluene, butylated hydroxyanisole, and
ethylenediamine tetraacetic acid may be added at levels safe for
ingestion to improve storage stability.
[0051] A liquid composition may also contain a buffer such as
guconic acid, lactic acid, citric acid or acetic acid, sodium
guconate, sodium lactate, sodium citrate, or sodium acetate.
Selection of excipients and the amounts used may be readily
determined by the formulation scientist based upon experience and
consideration of standard procedures and reference works in the
field.
[0052] The solid compositions of the present invention include
powders, granulates, aggregates and compacted compositions. The
dosages include dosages suitable for oral, buccal, rectal,
parenteral (including subcutaneous, intramuscular, and
intravenous), inhalant, and ophthalmic administration. Although the
most suitable administration in any given case will depend on the
nature and severity of the condition being treated, the most
preferred route of the present invention is oral. The dosages may
be conveniently presented in unit dosage form, and prepared by any
of the methods well-known in the pharmaceutical arts.
[0053] Dosage forms include solid dosage forms like tablets,
powders, capsules, suppositories, sachets, troches, and lozenges,
as well as liquid syrups, suspensions, and elixirs.
[0054] The oral dosage form of the present invention is preferably
in the form of an oral capsule having a dosage of about 10 mg to
about 160 mg, more preferably from about 20 mg to about 80 mg, and
most preferably capsules of 20, 40, 60, and 80 mg. Daily dosages
may include 1, 2, or more capsules per day.
[0055] The dosage form of the present invention may be a capsule
containing the composition, preferably a powdered or granulated
solid composition of the invention, within either a hard or soft
shell. The shell may be made from gelatin, and, optionally, contain
a plasticizer such as glycerin and sorbitol, and an opacifying
agent or colorant.
[0056] A composition for tableting or capsule filling may be
prepared by wet granulation. In wet granulation, some or all of the
active ingredients and excipients in powder form are blended, and
then further mixed in the presence of a liquid, typically water,
that causes the powders to clump into granules. The granulate is
screened and/or milled, dried, and then screened and/or milled to
the desired particle size. The granulate may then be tableted, or
other excipients may be added prior to tableting, such as a glidant
and/or a lubricant.
[0057] A tableting composition may be prepared conventionally by
dry blending. For example, the blended composition of the actives
and excipients may be compacted into a slug or a sheet, and then
comminuted into compacted granules. The compacted granules may
subsequently be compressed into a tablet.
[0058] As an alternative to dry granulation, a blended composition
may be compressed directly into a compacted dosage form using
direct compression techniques. Direct compression produces a more
uniform tablet without granules. Excipients that are particularly
well suited for direct compression tableting include
microcrystalline cellulose, spray dried lactose, dicalcium
phosphate dihydrate, and colloidal silica. The proper use of these
and other excipients in direct compression tableting is known to
those in the art with experience and skill in particular
formulation challenges of direct compression tableting.
[0059] A capsule filling of the present invention may comprise any
of the aforementioned blends and granulates that were described
with reference to tableting, however, they are not subjected to a
final tableting step.
[0060] The active ingredient and excipients may be formulated into
compositions and dosage forms according to methods known in the
art.
[0061] Having described the invention with reference to certain
preferred embodiments, other embodiments will become apparent to
one skilled in the art from consideration of the specification. The
invention is further defined by reference to the following examples
describing in detail the preparation of the composition and methods
of use of the invention. It will be apparent to those skilled in
the art that many modifications, both to materials and methods, may
be practiced without departing from the scope of the invention.
Instruments
[0062] Chromatography Method for Measuring Purity: TABLE-US-00001
Eluent: n-Hexane:acetone:acetonitrile 20:2:1 (by volume) Flow:
20-40 mL/min Detection: TLC (UV, 254 nm) Sample conc: 400-500 g/L
Sample vol: 60-70 mL Column temp: 25.degree. C. Detection limit:
not determined
EXAMPLES
[0063] The following non-limiting examples are merely illustrative
of the preferred embodiments of the present invention, and are not
to be construed as limiting the invention, the scope of which is
defined by the appended claims.
Example 1
[0064] A 3.0 g sample of crude ascomycin was purified by passing it
through a short silica column. The 3.0 g of syrup obtained was
dissolved in 25 ml of anhydrous dichloromethane. The solution was
cooled to -15.degree. C. with a slow stream of dried nitrogen gas
bubbling through the solution.
[0065] A 5 percent by weight solution of trifluoromethanesulfonic
anhydride in anhydrous dichloromethane was added to the above
solution in 6 ml parts, together with 0.3 g parts of 2,6-lutidine.
After the addition of a total of 24 ml trifluoromethanesulfonic
anhydride solution, 28 g of a 10 percent by weight lithium chloride
solution was added to the reaction mixture, and the reaction
mixture was allowed to warm to room temperature, i.e., about
21.degree. C. The reaction mixture was then stirred for 4 days.
[0066] The reaction mixture was diluted with a mixture of 200 ml
ethyl acetate and 25 ml of water, and poured into a separating
funnel, and shaken. After extraction, 25 ml of a 10 percent by
weight aqueous solution of KHSO.sub.4 was added. Following
additional shaking, the aqueous layer was removed, and the organic
phase was washed three times with 25 ml of a 10 percent by weight
aqueous KHSO.sub.4 solution, twice with 25 ml of a saturated
aqueous NaHCO.sub.3 solution, and twice with 25 ml of brine. The
organic phase was dried over anhydrous MgSO.sub.4. After
filtration, it was concentrated in vacuum, and, then, finally,
under high vacuum to completely remove the solvent. The yield was
2.94 g of crude amorphous pimecrolimus.
[0067] The product was purified by flash chromatography using
n-hexane/acetone (2:1, V/V) as an eluent. The yield was 2.54 g of
amorphous pimecrolimus.
Example 2
[0068] A 31.4 gram sample of ascomycin, assayed at 92.7 percent,
was dissolved in 200 ml of toluene. The solution was concentrated
at 40.degree. C. to dryness (syrup). Anhydrous toluene was added to
provide a 230 g solution, and 275 ml of anhydrous acetonitrile was
then added. The solution was cooled to a jacket temperature of
-15.degree. C. with a slow stream of dried nitrogen over the
surface of the solution. Simultaneously, 315 ml of anhydrous
toluene was cooled in a smaller reactor, having a jacket
temperature of -15.degree. C., also with a slow stream of dried
nitrogen over the surface of the liquid. When the temperature of
the toluene reached about -12.5.degree. C., 13.85 g of
trifluoromethanesulfonic anhydride (triflic anhydride) was added
dropwise. At about the same time, 11.33 g of ethyldiisopropylamine
(EDIPA) was added dropwise to the ascomycin solution. After a few
minutes, the triflic anhydride solution was transferred to the
ascomycin solution. When the addition was complete, the temperature
of the jacket was set to 26.degree. C. When the temperature of the
reaction mass reached about 0.degree. C., 300 g of a 12.5 percent
by weight solution of benzyl-triethylammonium chloride
("BnEt.sub.3NCl") in anhydrous acetonitrile was added. Water in an
amount of 200 ml was added 45 minutes after the temperature of the
reaction mass reached about 24.degree. to about 25.degree. C.
Following a period of vigorous stirring, the aqueous phase was
removed, and an additional 200 ml of water was added, and, again,
removed after mixing. The organic phase was concentrated at
40.degree. C. until almost all of the acetonitrile was removed. The
solution was diluted with the same volume of toluene with stirring.
The precipitating solids were filtered, and washed with toluene.
The filtrate was concentrated to dryness at 40.degree. C. Crude
amorphous pimecrolimus was obtained as a brownish foam with a yield
of 33 g.
Example 3
[0069] 30 g ascomycin was converted to crude Pimecrolimus according
to Example 2. The crude product was introduced to a column of 600 g
silica gel 60 (0.040-0.063 mm) as a concentrated solution in
toluene. This was eluted with n-hexane-acetone-acetonitrile
(20:2:1) mixture.
[0070] The fractions that contained Pimecrolimus of sufficient
purity according to analysis by HPLC are combined and extracted
with 10 V/V % acetonitrile. The lower phase, that contains the
solution of Pimecrolimus in a mixture of acetonitrile and acetone
(with some n-hexane), was removed and the upper phase (mainly
n-hexane) was extracted twice with 5 V/V % acetonitrile. The lower
(acetonitrile-acetone) phases were combined and concentrated at
40.degree. C. to obtain a colorless resin. This was then dissolved
in 217 ml acetone at 40.degree. C. and concentrated. Residue: 38.76
g. The residue was diluted with 6 ml distilled water with stirring.
Finally 1 ml acetone was added. This solution was added slowly to 2
L chilled distilled water which was stirred efficiently. After the
addition had been completed, the suspension was stirred 20 min at
0.degree. C. Then the solid was filtered and dried at 45.degree. C.
in vacuum oven overnight. Product: 15.65 g yellowish solid.
Amorphous (XRD, DSC). Purity: 95.75% area by HPLC.
[0071] While it is apparent that the invention disclosed herein is
well calculated to fulfill the objects stated above, it will be
appreciated that numerous modifications and embodiments may be
devised by those skilled in the art. Therefore, it is intended that
the appended claims cover all such modifications and embodiments as
falling within the true spirit and scope of the present
invention.
* * * * *