PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(51) International Patent Classification 7 :
`A61K 47/10, C07D 498/18, A61K 3J.n15
`
`A2
`
`(11) International Publication Number:
`
`WO 00/33878
`
`(43) International Publication Date:
`
`15 June 2000 (15.06.00)
`
`(21) International Application Number:
`
`PCT/EP99/09521
`
`(22) International Filing Date:
`
`6 December 1999 (06.12.99)
`
`(30) Priority Data:
`9826882.4
`9904934.8
`
`7 December 1998 (07.12.98)
`4 March 1999 (04.03.99)
`
`GB
`GB
`
`(71) Applicant (for all designated States except AT US): NOV AR(cid:173)
`TIS AG [CH/CH]; Schwarzwaldallee 215, CH-4058 Basel
`(CH).
`
`(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB, BG,
`BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, EE, ES, FI,
`GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
`KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD,
`MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD,
`SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US,
`UZ, VN, YU, ZA, ZW, ARIPO patent (GH, GM, KE, LS,
`MW, SD, SL, SZ, TZ, UG, ZW), Eurasian patent (AM, AZ,
`BY, KG, KZ, MD, RU, TJ, TM), European patent (AT, BE,
`CH, CY, DE, DK, ES, Fl, FR, GB, GR, IE, IT, LU, MC,
`NL, PT, SE), OAPI patent (BF, BJ, CF, CG, Cl, CM, GA,
`GN, GW, ML, MR, NE, SN, TD, TG).
`
`(71) Applicant (for AT only): NOVARTIS-ERFINDUNGEN VER-
`WALTUNGSGESELLSCHAFf M.B.H. [AT/AT]; Brunner
`Strasse 59, A-1230 Vienna (AT).
`
`Published
`Without international search report and to be republished
`upon receipt of that report.
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): NAVARRO, Fran~ois
`[FR/FR]; 53, Rue Principale, F-68440 Bruebach (FR).
`PETIT, Samuel [FR/FR]; 11, Pare de la Risle, F-76130
`Mont-Saint-Aignan (FR). STONE, Guy [US/CH]; March(cid:173)
`bachstrasse 9, CH-4107 Ettingen (CH).
`
`(74) Agent: BECKER. Konrad; Novartis AG, Corporate Intellectual
`Property, Patent & Trademark Department, CH-4002 Basel
`(CH).
`
`(54) Title: MACROLIDES
`
`(57) Abstract
`
`The invention relates to the stabilization of poly-ene macrolides and to a particular macrolide obtained in crystalline form.
`
`West-Ward Exhibit 1003
`Navarro WO 00/33878
`Page 001
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`cu
`CZ
`DE
`DK
`EE
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cbte d'Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
`
`West-Ward Exhibit 1003
`Navarro WO 00/33878
`Page 002
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`WO 00/33878
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`- 1 -
`
`PCT/EP99/09521
`
`MACROLIDES
`
`The present invention relates to the stabilization of a pharmaceutically active ingredient
`
`sensitive to oxidation, e.g. a poly-ene macrolide, preferably a poly-ene macrolide having
`
`immunosuppressant properties, particularly rapamycins.
`
`The handling and storage particularly in the bulk form of pharmaceutically active ingredients
`
`which are sensitive to oxidation is difficult. Special handling is necessary and often the
`
`oxidation-sensitive ingredient is stored in air-tight packaging under protective gas.
`
`Substantial amounts of stabilizers are added during the formulating process of such
`
`pharmaceutically active ingredients.
`
`Poly-ene macrolides have satisfactory stability properties. However, it has now been found
`
`that their stability to oxygen may substantially be improved by the addition of a stabilizer,
`
`e.g. an antioxidant, during their isolation step.
`
`According to the invention, there is provided
`
`1.
`
`A process for stabilizing a poly-ene macrolide comprising adding an antioxidant to the
`
`purified macrolide, preferably at the commencement of its isolation step.
`
`This process is particularly useful for the production of a stabilized poly-ene macrolide
`
`in bulk. The amount of antioxidant may conveniently be up to 1 %, more preferably
`
`from 0.01 to 0.5 % (based on the weight of the macrolide). Such a small amount is
`
`referred to hereinafter as a catalytic amount.
`
`As alternatives to the above the present invention also provides:
`
`2.
`
`A mixture, e.g. a bulk mixture, comprising a poly-ene macrolide and an anti-oxidant,
`
`preferably a catalytic amount thereof, preferably in solid form.
`
`The mixture may be in particulate form e.g. cristallized or amorphous form. It may be
`
`in a sterile or substantially sterile condition, e.g. in a condition suitable for
`
`pharmaceutical use.
`
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`WO 00/33878
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`PCT/EP99/09521
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`- 2 -
`
`3.
`
`Use of a mixture as defined above in 2. in the manufacture of a pharmaceutical
`
`composition.
`
`Examples of poly-enes macrolides are e.g. molecules comprising double bonds, preferably
`
`conjugated double bonds, for example such having antibiotic and/or immunosuppressant
`
`properties, e.g. macrolides comprising a lactam or lactone bond and their derivatives, e.g.
`
`compounds which have a biological activity qualitatively similar to that of the natural
`
`macrolide, e.g. chemically substituted macrolides. Suitable examples include e.g.
`
`rapamycins and ascomycins. A preferred poly-ene macrolide is a macrolide comprising at
`
`least 2 conjugated double bonds, e.g. 3 conjugated double bonds.
`
`Rapamycin is a known lactam macrolide produceable, for example by Streptomyces
`
`hygroscopicus. The structure of rapamycin is given in Kessler, H. et al.; 1993; Helv. Chim.
`
`Acta, 76: 117. Rapamycin has antibiotic and immunosuppressant properties. Derivatives of
`
`rapamycin are known, e.g. 16-0-substituted rapamycins, for example as disclosed in WO
`
`94/02136 and WO 96/41807, 40-0-substituted rapamycins, for example as disclosed in WO
`
`94/09010, WO 92/05179, WO 95/14023, 94/02136, WO 94/02385 and WO 96/13273, all of
`
`which being incorporated herein by reference. Preferred rapamycin derivatives are e.g.
`
`rapamycins wherein the hydroxy in position 40 of formula A illustrated at page 1 of WO
`
`94/09010 is replaced by -OR wherein R is hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl
`
`or aminoalkyl, e.g. 40-0-(2-hydroxy)ethyl-rapamycin, 40-0-(3-hydroxy)propyl-rapamycin,
`
`and 40-0-[2-(2-hydroxy)ethoxy]ethyl-rapamycin.
`
`Ascomycins, of which FK-506 and ascomycin are the best known, form another class of
`
`lactam macrolides, many of which have potent immunosuppressive and anti-inflammatory
`
`activity. FK506 is a lactam macrolide produced by Streptomyces tsukubaensis. The
`
`structure of FK506 is given in the Appendix to the Merck Index, 11th ed. (1989) as item AS.
`
`Ascomycin is described e.g. in USP 3,244,592. Ascomycin, FK506, other naturally occurring
`
`macrolides having a similar biological activity and their derivatives, e.g. synthetic analogues
`
`and derivatives are termed collectively "Ascomycins". Examples of synthetic analogues or
`
`derivatives are e.g. halogenated ascomycins, e.g. 33-epi-chloro-33-desoxy-ascomycin such
`
`as disclosed in EP-A-427,680, tetrahydropyran derivatives, e.g. as disclosed in EP-A-
`
`626,385.
`
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`WO 00/33878
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`PCT/EP99/09521
`
`- 3 -
`
`Particularly preferred macrolides are rapamycin and 40-0-(2-hydroxy)ethyl-rapamycin.
`
`Preferred antioxidants are for example 2,6-di-tert.-butyl-4-methylphenol (hereinafter BHT),
`
`vitamin E or C, BHT being particularly preferred.
`
`A particularly preferred mixture of the invention is a mixture of rapamycin or 40-0-(2-
`
`hydroxy)ethyl-rapamycin and 0.2% (based on the weight of the macrolide) of antioxidant,
`
`preferably BHT.
`
`The antioxidant may be added to the poly-ene macrolide at the commencement of the
`
`isolation steps, preferably the final isolation step, more preferably just prior to the final
`
`precipitation step. The macrolide is preferably in a purified state. It may be dissolved in an
`
`inert solvent and the antioxidant is added to the resulting solution, followed by a
`
`precipitation step of the stabilized macrolide, e.g. in an amorphous form or in the form of
`
`crystals. Preferably the mixture of the invention is in amorphous form.
`
`The resulting stabilized macrolide exhibits surprisingly an improved stability to oxidation and
`
`its handling and storage, e.g. in bulk form prior to its further processing for example into a
`
`galenic composition, become much easier. It is particularly interesting for macrolides in
`
`amorphous form.
`
`The macrolide stabilized according to the invention may be used as such for the production
`
`of the desired galenic formulation. Such formulations may be prepared according to
`
`methods known in the art, comprising the addition of one or more pharmaceutically
`
`acceptable diluent or carrier, including the addition of further stabilizer if required.
`
`Accordingly there is further provided:
`
`4.
`
`A pharmaceutical composition comprising, as active ingredient, a stabilized mixture as
`
`disclosed above, together with one or more pharmaceutically acceptable diluent or
`
`carrier.
`
`The composition of the invention may be adapted for oral, parenteral, topical (e.g. on
`
`the skin), occular, nasal or inhalation (e.g. pulmonary) administration. A preferred
`
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`WO 00/33878
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`PCT/EP99/09521 .
`
`-4-
`
`composition is one for oral administration, preferably a water-free composition when
`
`the active ingredient is a lactone macrolide.
`
`The pharmaceutical compositions of the invention may comprise further excipients, e.g. a
`
`lubricant, a disintegrating agent, a surfactant, a carrier, a diluent, a flavor enhancer, etc. It
`
`may be in liquid form, e.g. solutions, suspensions or emulsions such as a microemulsions,
`
`e.g. as disclosed in USP 5,536,729, or in solid form, e.g. capsules, tablets, dragees,
`
`powders (including micronized or otherwise reduced particulates), solid dispersions,
`
`granulates, etc.,e.g. as disclosed in WO 97/03654, the contents of which being
`
`incorporated herein by reference, or semi-solid forms such as ointments, gels, creams and
`
`pastes. They are preferably adapted to be in a form suitable for oral administration.
`
`Preferably they are in solid form. The pharmaceutical compositions of the invention may be
`
`prepared according to known methods, by mixing the macrolide stabilized according to the
`
`invention with the additional ingredients under stirring; the ingredients may be milled or
`
`ground and if desired compressed, e.g into tablets.
`
`This invention is particularly interesting for rapamycin compositions in liquid or solid form. A
`
`particularly preferred composition is a solid dispersion, e.g. comprising a stabilized
`
`rapamycin according to the invention and a carrier medium, e.g. a water-soluble polymer
`
`such as hydroxypropylmethylcellulose, e.g. as disclosed in WO 97/03654.
`
`The compositions of the invention are useful for the indications as known for the macrolide
`
`they contain at e.g. known dosages. For example, when the macrolide has
`
`immunosuppressant properties, e.g. rapamycin or a rapamycin derivative, the composition
`
`may be useful e.g. in the treatment or prevention of organ or tissue acute or chronic allo- or
`
`xeno-transplant rejection, autoimmune diseases or inflammatory conditions, asthma,
`
`proliferative disorders, e.g tumors, or hyperproliferative vascular disorders, preferably in the
`
`prevention or treatment of transplant rejection.
`
`The amount of macrolide and of the composition to be administered depend on a number of
`
`factors, e.g. the active ingredient used, the conditions to be treated, the duration of the
`
`treatment etc. For e.g. rapamycin or 40-0-(2-hydroxy)ethyl-rapamycin, a suitable daily
`
`dosage form for oral administration comprise from 0.1 to 1 O mg, to be administered once or
`in divided form.
`
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`WO 00/33878
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`PCT/EP99/09521
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`- 5 -
`
`In another aspect, this invention also provides 40-0-(2-hydroxy)ethyl-rapamycin in a
`
`crystalline form, particularly in a substantially pure form. Preferably the crystal form is
`
`characterized by the absence or substantial absence of any solvent component; it is in non(cid:173)
`
`solvate form.
`
`40-0-(2-hydroxy)ethyl-rapamycin in crystalline form belongs to the monoclinic sytem. The
`
`resulting crystals have a m.p. of 146°-147°C, especially 146.5°C. To assist identification of
`
`the new crystalline form, X-ray diffraction analysis data are provided. The conditions under
`
`which these data are obtained are as follows:
`
`Temperature
`
`Wavelength
`
`Space group
`
`Unit cell dimensions
`a
`b
`c
`
`Volume
`z
`Density (calculated)
`
`Absorption coefficient
`
`F(OOO)
`
`Crystal size
`
`293(2)K
`1.54178 A
`P21
`
`14.378.(2) d
`11.244(1) A
`18.310(2) A
`108.58(1 )0
`2805.8(6) d3
`2
`1.134 g/cm3
`0.659 mm·1
`1040
`
`0.59x0.11 x0.03 mm
`
`0 range for data collection
`
`2.55 to 57 .20°
`
`Reflections collected
`
`Independent reflections
`
`Intensity decay
`
`Refinement method
`
`Data/restraints/parameters
`Goodness-of-fit on F2
`Final R indices [1>2 sigma(I)]
`
`Largest diff. peak and hole
`
`4182
`
`4037 [R(int) = 0.0341]
`
`32%
`Full-matrix least-squares on F2
`3134/1/613
`
`1.055
`
`R1=0.057 4, WR2=0.1456
`0.340 and -0.184 e!d3
`
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`WO 00/33878
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`PCT/EP99/09521
`
`- 6 -
`
`40-0-(2-hydroxy)ethyl-rapamycin in crystalline form may be prepared by dissolving the
`
`amorphous compound in a solvant e.g. ethyl acetate and adding an aliphatic hydrocarbon
`
`CnH2n+2 (n=5, 6 or 7). After addition of the hydrocarbon, the resulting mixture may be
`warmed e.g. at a temperature of 25 to 50°C, e.g. up to 30-35°C. Storing of the resulting
`
`mixture may conveniently take place at a low temperature, e.g. below 25°C, preferably from
`
`O to 25°C. The crystals are filtered and dried. Heptane is preferred as an aliphatic
`
`hydrocarbon. If desired, nucleation procedures may be commenced e.g. by sonication or
`
`seeding.
`
`The present invention also provides a process for purifying 40-0-(2-hydroxy)ethyl(cid:173)
`
`rapamycin comprising crystallizing 40-0-(2-hydroxy)ethyl-rapamycin from a crystal bearing
`
`medium, e.g. as disclosed above, and recovering the crystals thus obtained. The crystal
`
`bearing medium may include one or more components in addition to those recited above. A
`
`particularly suitable crystal bearing medium has been found to be one comprising ca. 2
`
`parts ethyl acetate and ca. 5 parts aliphatic hydrocarbon, e.g. heptane.
`
`40-0-(2-hydroxy)ethyl-rapamycin in crystalline form has been found to possess in vitro and
`
`in vivo immunosuppressive activity comparable to that of the amorphous form. In the
`
`localized GvHD, maximal inhibition (70-80%) of lymph node swelling is achieved with a
`
`dosage of 3 mg with 40-0-{2-hydroxy)ethyl-rapamycin in crystalline form.
`
`40-0-(2-hydroxy)ethyl-rapamycin may be useful for the same indications as known for the
`
`amorphous compound, e.g. to prevent or treat acute and chronic allo- or xeno-transplant
`
`rejection, autoimmune diseases or inflammatory conditions, asthma, proliferative disorders,
`
`e.g tumors, or hyperproliferative vascular disorders, e.g as disclosed in WO 94/09010 or in
`
`WO 97/35575, the contents thereof being incorporated herein by reference. In general,
`
`satisfactory results are obtained on oral administration at dosages on the order of from 0.05
`
`to 5 or up to 20 mg/kg/day, e.g. on the order of from 0.1 to 2 or up to 7.5 mg/kg/day
`
`administered once or, in divided doses 2 to 4x per day. Suitable daily dosages for patients
`
`are thus on the order of up to 1 O mg., e.g. 0.1 to 1 O mg.
`
`40-0-(2-hydroxy)ethyl-rapamycin in crystalline form may be administered by any
`
`conventional route, e.g. orally, for example tablets or capsules, or nasallly or pulmonary (by
`
`inhalation). It may be administered as the sole active ingredient or together with other
`
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`WO 00/33878
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`PCT/EP99/09521
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`- 7 -
`
`drugs, e.g. immunosuppressive and/or immunomodulatory and/or anti-inflammatory agents,
`
`e.g. as disclosed in WO 94/09010.
`
`In accordance with the foregoing, the present invention also provides:
`
`5.
`
`A method for preventing or treating acute or chronic allo- or xeno-transplant rejection,
`
`autoimmune diseases or inflammatory conditions, asthma, proliferative disorders, or
`
`hyperproliferative vascular disorders, in a subject in need of such treatment, which
`
`method comprises administering to said subject a therapeutically effective amount of
`
`40-0-(2-hydroxy)ethyl-rapamycin in crystalline form;
`
`6.
`
`40-0-(2-hydroxy)ethyl-rapamycin in crystalline form for use as a pharmaceutical;, e.g.
`
`in a method as disclosed above;
`
`7.
`
`A pharmaceutical composition comprising 40-0-(2-hydroxy)ethyl-rapamycin in
`
`crystalline form together with a pharmaceutically acceptable diluent or carrier therefor;
`
`8.
`
`A kit or package for use in immunosuppression or inflammation, including a
`
`pharmaceutical composition as disclosed above and a pharmaceutical composition
`
`comprising an immunosuppressant or immunomodulatory drug or an anti-inflammatory
`
`agent.
`
`The following examples illustrate the invention without limiting it.
`
`Example 1: Crystallisation
`
`0.5 g amorphous 40-0-(2-hydroxy)ethyl-rapamycin is dissolved in 2.0 ml ethyl acetate at
`
`40°C. 5.0 ml heptane is added and the solution becomes "milky". After warming to 30°C, the
`
`solution becomes clear again. Upon cooling to 0°C and with scratching an oil falls out of the
`
`solution. The test tube is closed and stored at 10°C overnight. The resulting white
`
`voluminous solid is then filtered and washed with 0.5 ml of a mixture of ethyl
`
`acetate/hexane (1 :2.5) and the resulting crystals are dried at 40°C under 5 mbar for 16
`
`hours. 40-0-(2-hydroxy)ethyl-rapamycin in crystalline form having a m.p. of 146.5°C is thus
`
`obtained.
`
`Crystallisation at a larger scale may be performed as follows:
`
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`- 8 -
`
`250 g amorphous 40-0-(2-hydroxy)ethyl-rapamycin is dissolved in 1.0 I ethyl acetate under
`
`argon with slow stirring. This solution is heated at 30°C and then during 45 minutes, 1.5 I
`
`heptane is added dropwise. 0.25 g of seed crystals prepared as disclosed above are added
`
`under the same conditions in portions. The mixture is further stirred at 30°C over a period of
`
`2 hours and the crystallisation mixture is cooled to 25°C over 1 hour and then to 10°c for 30
`
`minutes and filtered. The crystals are washed with 100 ml of a mixture ethyl acetate/hexane
`
`(2:3). Subsequent drying is performed at 50°C and ca 5 mbar. m.p. 146.5°C
`
`IRinKBr: 3452,2931, 1746, 1717, 1617, 1453, 1376, 1241, 1191, 1163, 1094, 1072,
`1010, 985, 896 cm·1
`
`Single X-ray structure with coordinates are indicated in Figures 1 to 3 below.
`
`Example 2: Production of stabilized 40-0-(2-hydroxy)ethyl-rapamycin
`
`1 OOg 40-0-(2-hydroxy)ethyl-rapamycin are dissolved in 6001 abs. ethanol. After addition of
`
`0.2g BHT, the resulting solution is added dropwise with stirring to 3.0 I water within 1 hour.
`
`The resulting suspension is stirred for an additional 30 minutes. Filtration with subsequent
`
`washing (3x200 ml water/ethanol at a v/v ratio of 5:1) results in a moist white product which
`
`is further dried under vacuum (1 mbar) at 30°C for 48 hours. The resulting dried product
`
`contains 0.2% (w/w) BHT.
`
`The resulting product shows improved stability on storage. The sum of by-products and
`
`degradation products in % after 1 week storage are as follows:
`
`Compound
`
`50°C in open flask
`
`Ex. 2 (0.2% BHT)
`
`Without BHT
`
`1.49
`
`>10
`
`The procedure of above Example may be repeated but using, as active ingredient,
`
`rapamycin.
`
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`WO 00/33878
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`PCT/EP99/09521
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`- 9 -
`
`CLAIMS
`
`1.
`
`2.
`
`A mixture comprising a poly-ene macrolide and an antioxidant.
`
`A mixture according to claim 1, wherein the antioxidant is present in an amount of up
`
`to 1 % based on the macrolide weight.
`
`3.
`
`A mixture according to claim 1 , wherein the antioxidant is present in an amount of
`
`0.2% based on the macrolide weight.
`
`4.
`
`A mixture according to claim 1, wherein the antioxidant is 2,6-di-tert.-butyl-4-
`
`methylphenol.
`
`5.
`
`A mixture according to claim 1, wherein the poly-ene macrolide is rapamycin or 40-0-
`
`6.
`
`7.
`
`(2-hydroxy)ethyl-rapamycin.
`
`A mixture according to claim 1, in solide form.
`
`A pharmaceutical composition comprising, as active ingredient, a mixture according to
`
`claim 1 together with one or more pharmaceutically acceptable carrier or diluent.
`
`8.
`
`A process for stabilizing a poly-ene macrolide comprising adding an antioxidant to the
`
`purified macrolide.
`
`9.
`
`40-0-(2-hydroxy)ethyl-rapamycin in crystalline form.
`
`10. The compound according to claim 9, in crystalline non-solvate form.
`
`11. The compound according to claim 9, having a crystal lattice
`a = 14.37 A., b = 11.24A., c = 18.31 A., the volume being 2805 A. 3
`12. A pharmaceutical composition comprising a compound according to claim 11 or 12,
`
`together with one or more pharmaceutically acceptable diluents or carriers therefor.
`
`13. A process for purifying 40-0-(2-hydroxy)ethyl-rapamycin, comprising crystallizing 40-
`
`0-(2-hydroxy)ethyl-rapamycin from a crystal bearing medium and recovering the
`
`crystals thus obtained.
`
`West-Ward Exhibit 1003
`Navarro WO 00/33878
`Page 0011
`
`

`

`WO 00/33878
`
`1 I 4
`FIGURE 1/3
`
`PCT /EP99/09521
`
`Atomic coordinates and equivalent isotropic displacement parameters (A 2
`(U(eq) is defined as one third of the trace of the orthogonalized Uij tensor)
`
`)
`
`x/a
`
`y/b
`
`zlc
`
`U(eq)
`
`C(l)
`0(1)
`C(2)
`C(3)
`C(4)
`C(5)
`C(6)
`N(7)
`C(8)
`0(8)
`C(9)
`0(9)
`C(lO}
`0(10)
`C(ll)
`C(llM)
`C(l2)
`C(l3)
`C(l4)
`0(14)
`C(l5)
`C(l6)
`0(16)
`C(l6M)
`C(17)
`C(l7M)
`C(l8)
`C(19)
`C(20)
`C(21)
`C(22)
`C(23)
`C(23M)
`C(24)
`C(25)
`C(25M)
`C(26)
`0(26)
`C(27)
`0(27)
`C(27M)
`C(28)
`0(28)
`
`.9065(6)
`.9239(4)
`.8041(5)
`.7847(7)
`.7627(7)
`.6795(7)
`.7005(6)
`.7272(4)
`.6781(5)
`.6965(4)
`.5940(6)
`.6074(4)
`.4962(5)
`.5045(4)
`.4079(6}
`.4107(7)
`.3135(6)
`.3099(6)
`.4002(6)
`.4868(4)
`.4070(6)
`.4953(7)
`.4841(5)
`.5697(8)
`.5056(6)
`.4268(7)
`.5806(7)
`.6018(7)
`.6768(8)
`.7032(8)
`.7771(8)
`.8086(8)
`.7254(9)
`.8912(8)
`.9826(9)
`1.0348(12)
`1.0512(10)
`1.1132(8)
`1.0375(8)
`1.0877(7)
`1.0445(17)
`1.0824(7)
`1.1827(4)
`
`.0121(9)
`-.0736(6)
`.0615(8)
`.1748(10)
`.1515(10)
`.0653(11)
`-.0496(9)
`-.0269(6)
`-.0693(7)
`-.0432(6)
`-.1566(8)
`-.2513(6)
`-.1136(8)
`-.1009(6)
`-.1951(8}
`-.3114(9)
`-.1252(10)
`-.0061(10)
`.0651(9)
`-.0019(5)
`.01811(10)
`.2564(8)
`.3639(6)
`.4308(10)
`.2802(9)
`.3541(11)
`.2368(10)
`.2458(11)
`.1937(12)
`.2069(13)
`.1565(15)
`.1781(16)
`.2152(23)
`.2643(18)
`.2329(20)
`.1245(20)
`.3412(22)
`.3601(21)
`.4278(16)
`.5366(13)
`.6202(22)
`.3750(11)
`.3501(7)
`
`.5077(5)
`.5482(4)
`.4625(4)
`.4984(6)
`.5725(7)
`.5610(6)
`.5256(5)
`.4567(4)
`.3883(5)
`.3287(3)
`.3784(5)
`.4074(4)
`.3223(5)
`.2486(3)
`.3160(5)
`.2776(6)
`.2738(6)
`.3115(7)
`.3156(6)
`.3559(3)
`.3592(6)
`.3624(6)
`.4015(4)
`.4288(7)
`.2841(6)
`.2307(6)
`.2680(6)
`.1964(6)
`.1809(6)
`.1094(7)
`.0948(7)
`.0240(6)
`-.0474(7)
`.0406(6)
`.1069(6)
`.0884(8)
`.1293(7)
`.0998(7)
`.1891(7)
`.1901(7)
`.1382(13)
`.2699(6)
`.2818(4)
`
`.060(2)
`.076(2)
`.060(2)
`.087(3)
`.098(3)
`.094(3)
`.074(3)
`.059(2)
`.055(2)
`.074(2)
`.056(2)
`.084(2)
`.057(2)
`.075(2)
`.068(3)
`.088(3)
`.088(3)
`.099(4)
`.078(3)
`.065(2)
`.082(3)
`.079(3)
`.095(2)
`.102(3)
`.073(3)
`.103(4)
`.079(3)
`.092(3)
`.097(3)
`.111(4)
`.121(5)
`.128(5)
`.184(9)
`.140(6)
`.141(6)
`.178(8)
`.157(8)
`.281(11)
`.118(5)
`.185(5)
`.256(13)
`.091(3)
`.108(2)
`
`West-Ward Exhibit 1003
`Navarro WO 00/33878
`Page 0012
`
`

`

`WO 00/33878
`
`2 I 4
`FIGURE 1/3 (Cont.)
`
`PCT/EP99/09521
`
`Atomic coordinates and equivalent isotropic displacement parameters (A2
`(cont.)
`
`)
`
`x/a
`
`rib
`
`zlc
`
`U(eq)
`
`C(29)
`C(29M)
`C(30)
`C(31)
`C(31M)
`C(32)
`0(32)
`C(33)
`C(34)
`0(34)
`C(35)
`C(35M)
`C(36)
`C(37)
`C(38)
`C(39)
`0(39)
`C(39M)
`C(40)
`0(40)
`C(41)
`C(42)
`C(43)
`C(44)
`0(45)
`
`1.0329(7)
`.9318(6)
`1.0764(7)
`1.0376(7)
`1.0198(9)
`1.1046(7)
`1.1436(7)
`1.1271(6)
`1.0764(5)
`.9735(3)
`1.1115(5)
`1.1060(7)
`1.2149(6)
`1.2650(6)
`1.2091(7)
`1.2680(9)
`1.2082(8)
`1.2099(20)
`1.3640(9)
`1.4177(7)
`1.4221(7)
`1.3653(6)
`1.4272(14)
`1.5146(20)
`1.4956(12)
`
`.2733(10)
`.2995(10)
`.1700(10)
`.0581(10)
`-.0385(13)
`.0210(10)
`-.0747(9)
`.1025(9)
`.0601(8)
`.0853(5)
`.1217(9)
`.2562(10)
`.0757(9)
`.1298(9)
`.1198(14)
`.1650(16)
`.1584(20)
`.2512(47)
`.0982(13)
`.1412(10)
`.1138(13)
`.0697(11)
`.0621(20)
`-.0307(24)
`-.1215(13)
`
`.2922(5)
`.2984(6)
`.3100(5)
`.3340(5)
`.2723(7)
`.4103(6)
`.4183(5)
`.4776(5)
`.5342(5)
`.4967(3)
`.6132(5)
`.6069(6)
`.6578(5)
`. 7370(5)
`. 7935(5)
`.8735(6)
`.9206(6)
`.9702(17)
`.9048(6)
`.9790(5)
`.8506(6)
`.7702(5)
`1.0408(9)
`1.0549(10)
`.9899(7)
`
`.073(3)
`.094(3)
`.077(3)
`.081(3)
`.124(4)
`.079(3)
`.132(3)
`.071(3)
`.062(2)
`.071(2)
`.064(2)
`.092(3)
`.072(3)
`.074(3)
`.110(4)
`.128(5)
`.243(9)
`.498(36)
`.. 0116(4)
`.151(4)
`.110(4)
`.096(3)
`.171(7)
`.238(12)
`.215(5)
`
`West-Ward Exhibit 1003
`Navarro WO 00/33878
`Page 0013
`
`

`

`WO 00/33878
`
`PCT/EP99/09521
`
`3 I 4
`FIGURE2/3
`
`Bond lengths (A)
`
`C(l)-0(1)
`C(l)-0(34)
`C(1)-C(2)
`C(2)-N(7)
`C(2)-C(3)
`C(3 )-C(4)
`C(4)-C(5)
`C(5)-C(6)
`C(6)-N(7)
`N(7)-C(8)
`C(8)-0(8)
`C(8)-C(9)
`C(9)-0(9)
`C(9)-C(10)
`C(l0)-0(10)
`C(l0)-0(14)
`C(10)-C(11)
`C(11)-C(11M)
`C(11)-C(12)
`C(12)-C(13)
`C(13)-C(14)
`C(14)-0(14)
`C(14)-C(15)
`C(15)-C(16)
`C(16)-0(16)
`C(16)-C(17)
`0(16)-C(16M)
`C(17)-C(18)
`C(17)-C(17M)
`C(18)-C(19}
`C(19)-C(20)
`C(20)-C(21)
`C(21 )-C(22)
`C(22 )-C(23)
`C(23 )-C(24)
`C(23 )-C(23M)
`
`1.193(10)
`1.329(10)
`1.545(11)
`1.465(10)
`1.500(13)
`1.511(14)
`1.502(13)
`1.518(14)
`1.453(10)
`1.315(9)
`1.237(9)
`1.523(11)
`1.178(9)
`1.532(11)
`1.398(9)
`1.425(10)
`1.540(11)
`1.491(13)
`1.546(12)
`1.51(2)
`1.506(13)
`1.441(10)
`1.516(14)
`1.511(12)
`1.439(11)
`1.512(14)
`1.392(11)
`1.301(12)
`1.491(13)
`1.441(14)
`1.333(14)
`1.48(2)
`1.30(2)
`1.52(2)
`1.49(2)
`1.52(2)
`
`C(24 )-C(25)
`C(25)-C(25M)
`C(25)-C(26)
`C(26)-0(26)
`C(26)-C(27)
`C(2 7)-0(2 7)
`C(27)-C(28)
`0(2 7)-C(2 7M)
`C(28)-0(28)
`C(28)-C(29)
`C(29)-C(30)
`C(29)-C(29M)
`C(30)-C(31)
`C(31)-C(32)
`C(31)-C(31M)
`C(32)-0(32)
`C(32)-C(33)
`C(33)-C(34)
`C(34)-0(34)
`C(34 )-C(35)
`C(35)-C(35M)
`C(35)-C(36)
`C(36)-C(37)
`C(37)-C(38)
`C(37)-C(42)
`C(38)-C(39)
`C(39)-0(39)
`C(39)-C(40)
`0(39)-C(39M)
`C(40)-0(40)
`C(40)-C(41)
`0(40)-C(43)
`C(41 )-C(42)
`C(43)-C(44)
`C(44)-0(45)
`
`1.52(2)
`1.53(2)
`1.54(3)
`1.20(2)
`1.53(2)
`1.42(2)
`1.533(14)
`1.34(2)
`1.415(10)
`1.471(14)
`1.311(13)
`1.523(12)
`1.497(14)
`1.482(13)
`1.53(2)
`1.201(11)
`1.487(13)
`1.521(11)
`1.447(9)
`1.537(11)
`1.517(13)
`1.540(11)
`1.525(12)
`1.503(11)
`1.532(12)
`1.526(14)
`1.399(13)
`1.51 (2)
`1.38(4)
`1.417(13)
`1.50(2)
`1.41(2)
`1.521(14)
`1.59(3)
`1.52(2)
`
`West-Ward Exhibit 1003
`Navarro WO 00/33878
`Page 0014
`
`

`

`WO 00/33878
`
`Bond angles (0
`
`)
`
`0( 1)-C(1)-0(34)
`O(l)-C(l)-C(2)
`0(34)-C(1)-C(2)
`N(7)-C(2)-C(3)
`N(7)-C(2)-C(l)
`C(3 )-C(2)-C(l)
`C(2)-C(3)-C(4)
`C(5)-C(4)-C(3)
`C(4 )-C(5 )-C( 6)
`N(7)-C(6)-C(5)
`C(8)-N(7)-C(6)
`C( 8)-N(7)-C(2)
`C( 6)-N(7)-C(2)
`0(8)-C(8)-N(7)
`0(8)-C(8)-C(9)
`N(7)-C(8)-C(9)
`0(9)-C(9)-C(8)
`0(9)-C(9)-C(10)
`C(8)-C(9)-C(10)
`0(10)-C(l0)-0{14)
`0( 10 )-C(l O)-C{9)
`0( 14 )-C(l O)-C{9)
`0(10)-C(lO)-C{l l)
`0(14 )-C(l 0)-C{l l)
`C{9)-C(10)-C{l 1)
`C{11M)-C(l l )-C(lO)
`C(l lM)-C(l 1 )-C(12)
`C(lO)-C(l 1 )-C(12)
`C{13)-C(12)-C(l 1)
`C{14)-C{13)-C(12)
`0{14)-C(14)-C(13)
`0(14)-C(14)-C(15)
`C(13 )-C(l 4)-C(15)
`C( 10)-0(14)-C(14)
`C( 16)-C( 15)-C( 14)
`0(16)-C(l 6)-C(15)
`0(16)-C(16)-C(17)
`C(l5)-C( 16)-C(l 7)
`C( 16M)-0( 16 )-C(l 6)
`C( 18)-C( 17)-C(l 7M)
`C(l8)-C(l 7)-C(16)
`C(17M)-C(17)-C(16)
`C(l 7)-C(18)-C(19)
`C(i.O)-C(19)-C(18)
`C(l9)-C(20)-C(21)
`C(22)-C(21)-C(20)
`C(21)-C(22)-C(23)
`C(24 )-C(23 )-C(23M)
`C(24)-C(23)-C(22)
`C(23M)-C(23 )-C(22)
`
`125.1(7)
`126.8(8)
`108.0(8)
`111.5(6)
`111.3(7)
`110.4(7)
`111.6(9)
`111.8(9)
`110.6(7)
`111.4(8)
`123.5(7)
`118.6(7)
`117.3(6)
`123.6(7)
`115.6(7)
`120.8(8)
`121.3(7)
`124.8(8)
`113.6(7)
`112.1(7)
`109.7(6)
`100.5(6)
`108.1(6)
`111.6(6)
`114.9(7)
`114.3(7)
`111.2(8)
`107.9(7)
`111.9(8)
`109.9(9)
`109.8(8)
`106.2(7)
`113.2(8)
`115.1(6)
`114.5(7)
`105.4(7)
`112.5(8)
`113.4(8)
`114.0(7)
`124.9(9)
`119.2(9)
`115.9(8)
`127.7(10)
`125.6(11)
`126.6(11)
`126.3(12)
`126.0(13)
`111(2)
`111.4(10)
`114.2(10)
`
`4 I 4
`FIGURE 3/3
`
`PCT /EP99/0952 l
`
`C(23 )-C(24 )-C(25)
`C(24 )-C(25)-C(25M)
`C(24 )-C(25)-C(26)
`C(25M)-C(25)-C(26)
`0(26)-C(26)-C(27)
`0(26)-C(26)-C(25)
`C(27)-C(26)-C(25)
`0(27)-C(27)-C(26)
`0(27)-C(27)-C(28)
`C(26)-C(27)-C(28)
`C(27M)-0(27)-C(27)
`0(28)-C(28)-C(29)
`0(28)-C(28)-C(27)
`C(29)-C(28)-C(27)
`C(30)-C(29)-C(28)
`C( 30 )-C(29)-C(29M)
`C(28)-C(29)-C(29M)
`C(29)-C(30)-C(31)
`C(32)-C(31)-C(30)
`C(32)-C(31)-C(31M)
`C(30)-C(31)-C(31M)
`0{32)-C(32)-C(31)
`0(32)-C(32)-C(33)
`C(31)-C(32)-C(33)
`C(32)-C(33)-C(34)
`0( 34 )-C( 34 )-C( 33)
`0(34 )-C(34 )-C(35)
`C(33 )-C(34 )-C(35)
`C(1)-0(34)-C(34)
`C(35M)-C(35)-C(34)
`C( 35 M )-C( 3 5 )-C( 3 6)
`C(34 )-C(35)-C(36)
`C(37)-C(36)-C(35)
`C(38)-C(37)-C(36)
`C(38)-C(37)-C(42)
`C(36)-C(37)-C(42)
`C(37)-C(38)-C(39)
`0(39)-C(39)-C(40)
`0(39)-C(39)-C(38)
`C(40)-C(39)-C(38)
`C(39)-0(39)-C(39M)
`0(40)-C(40)-C(41)
`0(40)-C(40)-C(39)
`C(41)-C(40)-C(39)
`C(43)-0(40)-C(40)
`C(40)-C(41)-C(42)
`C( 41)-C(42)-C(37)
`0(40)-C(43)-C(44)
`0(45)-C(44)-C(43)
`
`116(2)
`111.7(14)
`110(2)
`111.9(12)
`120(2)
`122(2)
`118.5(12)
`112.2(12)
`105.4(12)
`109.5(12)
`118.5(14)
`111.3(9)
`108.7(8)
`118.4(10)
`121.5(9)
`122.9(10)
`115.4(9)
`128.7(9)
`108.8(8)
`113.7(10)
`111.8(8)
`120.3(11)
`118.8(10)
`120.8(9)
`110.2(8)
`104.8(6)
`109.8(6)
`114.5(7)
`119.2(7)
`112.6(8)
`113.2(8)
`108.6(7)
`116.9(8)
`115.6(7)
`109.6(8)
`107.5(8)
`112.5(8)
`113.9(13)
`108.2(10)
`111.0(11)
`119(2)
`110.3(10)
`110.2(12)
`108.9(10)
`115.9(12)
`111.2(9)
`112.8(9)
`114(2)
`112.2(14)
`
`West-Ward Exhibit 1003
`Navarro WO 00/33878
`Page 0015
`
`