`
`(12) United States Patent
`Navarro et a].
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 7,297,703 B2
`Nov. 20, 2007
`
`(54) MACROLIDES
`
`(75) Inventors: Francois Navarro, Bruebach (FR);
`Samuel Petit, Mom Saint-Aignan (FR);
`Guy Stone, Ettingen (CH)
`
`(73) Ass1gnee: Novartls AG, Basel (CH)
`
`(*) Notice?
`
`Subjec?o any disclaimer, IheIeIm Ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(52) US. Cl. ..................................... .. 514/291; 540/456
`58
`Field of Classi?cation Search .............. .. 540/456;
`(
`)
`514/291
`See application ?le for complete search history.
`
`(56)
`
`References Cited
`Us PATENT DOCUMENTS
`
`4,079,128 A
`4,080,445 A
`
`3/1978 Lin et a1. .................. .. 424/181
`3/1978 Lin et a1. .................. .. 424/227
`
`(21) Appl NO . 11/020 860
`
`.
`
`..
`
`,
`
`FOREIGN PATENT DOCUMENTS
`
`_
`Flledl
`
`Dec. 23,
`
`Prior Publication Data
`Us 2005/0107418 A1
`May 19, 2005
`
`Related US. Application Data
`
`EP
`EP
`EP
`WO
`WO
`WO
`WO
`
`0 041 795
`0 329 460
`0 423 714
`94/09010
`97/03654
`98/04279
`98/33482
`
`12/1981
`8/1989
`4/1991
`4/1994
`2/1997
`2/1998
`8/1998
`
`(63) Continuation of application No. 10/393,795, ?led on
`Mar. 21, 2003, HOW Pat. NO. 6,852,729, which is a
`continuation of application No. 09/866,977, ?led on
`May 29, 2001, HOW Pat. NO. 6,605,613, which is a
`continuation of application No. PCT/EP99/09521,
`?led 011 Dec. 6, 1999.
`
`_
`_
`_
`_
`Prlmary ExammeriBruck Kl?e
`(74) Attorney’ Agent’ Or F W miThomas R‘ savltsky;
`Gregory C Houghton
`
`(57)
`
`ABSTRACT
`
`(30)
`
`Foreign Application Priority Data
`
`A mixture comprising a poly-ene macrolide and an antioxi
`dant. Preferably the poly-ene macrolide is rapamycin and
`’
`the antioxidant is 2, 6-di-tert.-butyl-4-methylphenol. The
`presence of the antioxidant improves the stability of the
`poly-ene macrolide to oxidation.
`
`12 Claims, 4 Drawing Sheets
`
`EGB;
`
`D . 7 1998
`
`M261, 4’ 1999
`'
`’
`(51) Int_ CL
`C07D 498/18
`A61K 31/715
`
`GB ............................... .. 9826882.4
`
`990493 4 8
`'
`
`""""""""""""""""" "
`
`(2006.01)
`(2006.01)
`
`Par Pharm., Inc.
`Exhibit 1001
`Page 001
`
`
`
`U.S. Patent
`
`Nov. 20, 2007
`
`Sheet 1 0f 4
`
`US 7,297,703 B2
`
`FIGURE 1/ 3
`
`Atomic coordinates and equivalent isotropic displacement paramclcrs (A1)
`(U(eq) is de?ned as one third of the trace of the orthogonalized Uij tensor)
`
`x/a
`
`y/b
`
`71c
`
`U(eq)
`
`C(1)
`0(1)
`C(2)
`C(3)
`0(4)
`0(5)
`0(6)
`19(7)
`C(8)
`0(8)
`C(9)
`0(9)
`C(10)
`0(10)
`C(11)
`C(1 1114)
`C(12)
`C(13)
`C(14)
`0(14)
`C(15)
`C(16)
`0(16)
`C(16M)
`C(17)
`C(17M)
`C(18)
`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)
`.804l(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)
`5099(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)
`-.O432(6)
`-.1566(8)
`-.2513(6)
`-.1 136(8)
`-.1009(6)
`-.1951(8)
`-.31 14(9)
`-.1252(10)
`-.0061(10)
`0651(9)
`-.0019(5)
`.0181 1(10)
`2564(8)
`3639(6)
`.4308(10)
`2802(9)
`.3541(11)
`.2368(l0)
`2458(11)
`.1937(12)
`.2069(13)
`.1565(15)
`.1781(16)
`2152(23)
`2643118)
`.2329(20)
`.1245(20)
`.3412(22)
`.3601(21)
`.4278(16)
`5366(13)
`.6202(22)
`3750(1 1)
`5501(1)
`
`5077(5)
`5482(4)
`.4625(4)
`4984(6)
`5725(7)
`5610(6)
`5256(5)
`.4567(4)
`3883(5)
`3287(3)
`3784(5)
`4074(4)
`5223(5)
`2486(3)
`3160(5)
`2776(6)
`2738(6)
`3115(7)
`5156(6)
`3559(3)
`5592(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)
`2318(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)
`.08 8(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)
`.1 18(5)
`.185(5)
`.256(13)
`091(3)
`.108(2)
`
`Par Pharm., Inc.
`Exhibit 1001
`Page 002
`
`
`
`U.S. Patent
`
`Nov. 20, 2007
`
`Sheet 2 0f 4
`
`US 7,297,703 B2
`
`FIGURE 1/ 3 (Com)
`
`Atomic coordinates and cquivalent isotropic displacement parameters (A2)
`(c0m.)
`
`x/a
`
`)-'/b
`
`21C
`
`Uleq)
`
`C(29)
`C(29M)
`C(30)
`C(31)
`C(31M)
`C(32)
`0(32)
`C(33)
`C(34)
`C(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)
`3318(6)
`1.0764(7)
`1.0376(7)
`1.0198(9)
`1.1046(7)
`1.1436(7)
`1.1271(6)
`1.0764(5)
`3735(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)
`
`1922(5)
`2984(6)
`3100(5)
`3340(5)
`1723(7)
`.4103(6)
`.4183(S)
`.4776(5)
`.5342(5)
`.4967(3)
`.6132(5}
`~.6069(6)
`.6S78(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(S)
`143(9)
`.498(36)
`.0116(4)
`.151(4)
`.110(4)
`096(3)
`.171(7)
`.238(12)
`.215(5)
`
`Par Pharm., Inc.
`Exhibit 1001
`Page 003
`
`
`
`Par Pharm., Inc.
`Exhibit 1001
`Page 004
`
`
`
`Par Pharm., Inc.
`Exhibit 1001
`Page 005
`
`
`
`US 7,297,703 B2
`
`1
`MACROLIDES
`
`This application is a continuation of US. patent applica
`tion Ser. No. 10/393,795, ?led Mar. 21, 2003, now US. Pat.
`No. 6,852,729, Which is a continuation of US. patent
`application Ser. No. 09/866,977, ?led May 29, 2001, now
`US. Pat. No. 6,605,613, Which is a continuation of Inter
`national Application No. PCT/EP99/09521, ?led Dec. 6,
`1999, the contents of Which are incorporated herein by
`reference.
`The present invention relates to the stabilization of a
`pharmaceutically active ingredient sensitive to oxidation,
`eg a poly-ene macrolide, preferably a poly-ene macrolide
`having immunosuppressant properties, particularly rapamy
`c1ns.
`The handling and storage particularly in the bulk form of
`pharmaceutically active ingredients Which are sensitive to
`oxidation is dif?cult. Special handling is necessary and often
`the oxidation-sensitive ingredient is stored in air-tight pack
`aging under protective gas. Substantial amounts of stabiliZ
`ers are added during the formulating process of such phar
`maceutically 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,
`eg 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 puri?ed macrolide, prefer
`ably 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 pref
`erably from 0.01 to 0.5% (based on the Weight of the
`macrolide). Such a small amount is referred to herein
`after as a catalytic amount.
`As alternatives to the above the present invention also
`provides:
`2. A mixture, eg 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. cristailiZed or
`amorphous form. It may be in a sterile or substantially
`sterile condition, eg in a condition suitable for phar
`maceutical use.
`3. Use of a mixture as de?ned 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 immuno
`suppressant properties, eg 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, eg 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 immunosup
`pressant properties. Derivatives of rapamycin are knoWn,
`e.g. 16-O-substituted rapamycins, for example as disclosed
`in US. Pat. Nos. 5,728,710 5,985,890 and 6,200,985, 40-O
`substituted rapamycins, for example as disclosed in US. Pat.
`Nos. 5,665,772, 6,440,990, 5,130,307, 5,221,670, 5,358,
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`944, 5,378,696, 5,527,907, 5,583,139, 5,672,605, and 5,728,
`710, 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 US. Pat, Nos. 5,665,772 and 6,440,990 is
`replaced by ‘GR Wherein R is hydroxyalkyl, hydroxy
`alkoxyalkyl, acylaminoalkyl or aminoalkyl, e.g. 40-O-(2
`hydroxy)ethyl-rapamycin,
`40-O-(3-hydroxy)propyl-rapa
`mycin, and 40-O-[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-in?amma
`tory activity. FK506 is a lactam macrolide produced by
`Streptomyces Zsukubaensis. The structure of FK506 is given
`in the Appendix to the Merck Index, 11 th ed. (1989) as item
`A5. Ascomycin is described e.g. In US. Pat. No. 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 deriva
`tives are e.g. halogenated ascomycins, e.g. 33-epi-chloro
`33-desoxy-ascomycin such as disclosed in EP-A427,680,
`tetrahydropyran derivatives, eg as disclosed in EP-A-626,
`385.
`Particularly preferred macrolides are rapamycin and
`40-O-(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-O-(2-hydroxy)ethyl-rapamycin
`and 0.2% (based on the Weight of the macrolide) of anti
`oxidant, preferably BHT.
`The antioxidant may be added to the poly-ene macrolide
`at the commencement of the isolation steps, preferably the
`?nal isolation step, more preferably just prior to the ?nal
`precipitation step. The macrolide is preferably in a puri?ed
`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, eg 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,
`eg 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 (eg on the skin), occular, nasal or
`inhalation (e.g. pulmonary) administration. A preferred
`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, eg a lubricant, a disintegrating
`agent, a surfactant, a carrier, a diluent, a ?avor enhancer, etc.
`
`Par Pharm., Inc.
`Exhibit 1001
`Page 006
`
`
`
`US 7,297,703 B2
`
`3
`It may be in liquid form, eg solutions, suspensions or
`emulsions such as a microemulsions, eg as disclosed in
`Us. Pat. No. 5,536,729, or in solid form, eg capsules,
`tablets, dragees, poWders (including microniZed or other
`Wise reduced particulates), solid dispersions, granulates,
`etc., eg as disclosed in WO 97/03654, the contents ofWhich
`being incorporated herein by reference, or semi-solid forms
`such as ointments, gels, creams and pastes. They are pref
`erably adapted to be in a form suitable for oral administra
`tion. Preferably they are in solid form. The pharmaceutical
`compositions of the invention may be prepared according to
`knoWn methods, by mixing the macrolide stabiliZed accord
`ing 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 stabi
`liZed rapamycin according to the invention and a carrier
`medium, eg a Water-soluble polymer such as hydroxypro
`pylmethylcellulose, eg 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, eg rapamycin or a rapamy
`cin derivative, the composition may be useful eg in the
`treatment or prevention of organ or tissue acute or chronic
`allo or xeno-transplant rejection, autoimmune diseases or
`in?ammatory 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, eg the active
`ingredient used, the conditions to be treated, the duration of
`the treatment etc. For eg rapamycin or 40-O-(2-hydroxy)
`ethyl-rapamycin, a suitable daily dosage form for oral
`administration comprise from 0.1 to 10 mg, to be adminis
`tered once or in divided form.
`In another aspect, this invention also provides 40-O-(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-solvate form.
`40-O-(2-hydroxy)ethyl-rapamycin in crystalline form
`belongs to the monoclinic system. The resulting crystals
`have a m.p. of 146°-147o C., especially 146.50 C. To assist
`identi?cation 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
`
`293(2)K
`1.54178 A
`P2l
`
`a
`b
`c
`[3
`Volume
`Z
`Density (calculated)
`Absorption coef?cient
`F(000)
`Crystal size
`0 range for data collection
`Re?ections collected
`
`14.378.(2) A
`11.244(1) A
`18.310(2) A
`108.58(1)°
`2805.8(6) A3
`2
`1.134 gem3
`0.659 mm’1
`1040
`0.59 x 0.11 x 0.03 mm
`2.55 to 57.200
`4182
`
`4
`
`-continued
`
`Independent re?ections
`Intensity decay
`Re?nement method
`Data/restraints/paralneters
`Goodness-of-?t on I:2
`Final R indices
`[1 > 2 sigma(l)]
`Largest diff. peak and hole
`
`4037 [R(int) = 0.0341]
`32%
`Full-matrix least-squares on I:2
`3134/1/613
`1.055
`R1 = 0.0574, WR2 = 0.1456
`
`0.340 and —0.184 e/A3
`
`40-O-(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 hydro
`carbon CnH2n+2 (n:5, 6 or 7). After addition of the hydro
`carbon, the resulting mixture may be Warmed eg at a
`temperature of 25 to 500 C., e.g. up to 30-350 C. Storing of
`the resulting mixture may conveniently take place at a loW
`temperature, eg below 250 C., preferably from 0 to 250 C.
`The crystals are ?ltered and dried. Heptane is preferred as an
`aliphatic hydrocarbon. If desired, nucleation procedures
`may be commenced eg by sonication or seeding.
`The present invention also provides a process for purify
`ing 40-O-(2-hydroxy)ethyl-rapamycin comprising crystal
`liZing 40-O-(2-hydroxy)ethyl-rapamycin from a crystal
`bearing medium, eg 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-O-(2-hydroxy)ethyl-rapamycin in crystalline form has
`been found to possess in vitro and in vivo immunosuppres
`sive 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-O-(2-hydroxy)ethyl-rapamycin in crystalline form.
`40-O-(2-hydroxy)ethyl-rapamycin may be useful for the
`same indications as knoWn for the amorphous compound,
`eg to prevent or treat acute and chronic allo- or xeno
`transplant rejection, autoimmune diseases or in?ammatory
`conditions, asthma, proliferative disorders, eg tumors, or
`hyperproliferative vascular disorders, eg as disclosed in
`WO 94/09010 or in WO 97/35575, the contents thereof
`being incorporated herein by reference. In general, satisfac
`tory results are obtained on oral administration at dosages on
`the order of from 0.05 to 5 or up to 20 mg/kg/day, eg 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 10 mg.,
`e.g. 0.1 to 10 mg.
`40-O-(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 ingre
`dient or together With other drugs, eg immunosuppressive
`and/or immunomodulatory and/ or anti-in?ammatory agents,
`eg 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 alto
`or xeno-transplant rejection, autoimmune diseases or
`in?ammatory conditions, asthma, proliferative disorders,
`or hyperproliferative vascular disorders, in a subject in
`need of such treatment, Which method comprises admin
`istering to said subject a therapeutically effective amount
`of 40-O-(2-hydroxy)ethyl-rapamycin in crystalline form;
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Par Pharm., Inc.
`Exhibit 1001
`Page 007
`
`
`
`US 7,297,703 B2
`
`5
`6. 40-0-(2-hydroxy)ethyl-rapamycin in crystalline form for
`use as a pharmaceutical; eg in a method as disclosed
`above;
`7. A pharmaceutical composition comprising 40-0-(2-hy
`droxy)ethyl-rapamycin in crystalline form together With a
`pharmaceutically acceptable diluent or carrier therefor;
`8. A kit or package for use in immunosuppression or
`in?ammation, including a pharmaceutical composition as
`disclosed above and a pharmaceutical composition com
`prising an immunosuppressant or immunomodulatory
`drug or an anti-in?ammatory, 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 ?ltered 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: 250 g amorphous 40-0-(2-hydroxy)ethyl-rapamy
`cin is dissolved in 1.01 ethyl acetate under argon With sloW
`stirring. This solution is heated at 30° C. and then during 45
`minutes, 1.51 heptane is added dropWise. 0.25 g of seed
`ciystals 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 crystalliZation
`mixture is cooled to 25° C. over 1 hour and then to 10° C.
`for 30 minutes and ?ltered. The crystals are Washed With 100
`ml of a mixture ethyl acetate/hexane (2:3). Subsequence
`drying is performed at 50° C. and ca 5 mbar. m.p. 146.5° C.
`
`IR in KBr: 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
`Tables 1-3 beloW.
`
`EXAMPLE 2
`
`Production of Stabilized
`40-0-(2-hydroxy)ethyl-rapamycin
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`10 g 40-0-(2-hydroxy)ethyl-rapamycin are dissolved in
`600 l abs. ethanol. After addition of 0.2 g BHT, the resulting
`solution is added dropWise With stirring to 3.0 1 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 stor
`65
`age. The sum of by-products and degradation products in %
`after 1 Week storage are as folloWs:
`
`60
`
`Compound
`Ex. 2 (0.2% BHT)
`Without BHT
`
`50° C. in open ?ask
`1.49
`>10
`
`The procedure of above Example may be repeated but
`using, as active ingredient, rapamycin.
`
`TABLE 1
`
`Atomic Coordinates and equivalent isotropic displacement parameters (A2)
`
`x/a
`
`y/b
`
`Z/c
`
`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(10)
`0(10)
`C(11)
`C(llM)
`C(12)
`C(13)
`C(14)
`0(14)
`C(15)
`C(16)
`0(16)
`C(16M)
`C(17)
`C(17M)
`C(18)
`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)
`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)
`
`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)
`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)
`
`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)
`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)
`
`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)
`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)
`
`.060(2)
`.076(2)
`.060(2)
`087(3)
`098(3)
`094(3)
`074(3)
`059(2)
`055(2)
`074(2)
`056(2)
`084(2)
`059(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)
`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)
`
`Par Pharm., Inc.
`Exhibit 1001
`Page 008
`
`
`
`Par Pharm., Inc.
`Exhibit 1001
`Page 009
`
`
`
`US 7,297,703 B2
`
`9
`(b) adding an antioxidant to the resulting solution in an
`amount up to 1% based on the rapamycin or l6-O or
`40-O-substituted rapamycin, Weight, and
`(c) isolating the resulting mixture of the rapamycin or
`l6-O or 40-O-substituted rapamycin and the antioxi
`dant.
`9. The process of claim 8 Wherein the antioxidant is
`selected from the group consisting of Vitamin E, Vitamin C,
`2,6-di-tert.-butyl-4-methylphenol (BHT) and combinations
`thereof.
`
`5
`
`10
`10. The process of claim 8 Wherein the antioxidant is
`present in an amount of up to 1% based on the macrolide
`Weight.
`11. The process of claim 8 Wherein the antioxidant is
`present in an amount of from 0.01 to 0.5% based on the
`macrolide Weight.
`12. The process of claim 8 Wherein the antioxidant is
`present in an amount of 0.2% based on the macrolide Weight.
`
`*
`
`*
`
`*
`
`*
`
`*
`
`Par Pharm., Inc.
`Exhibit 1001
`Page 010