I lllll llllllll Ill lllll lllll lllll lllll lllll 111111111111111111111111111111111
`US007297703B2
`
`c12) United States Patent
`Navarro et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,297,703 B2
`Nov. 20, 2007
`
`(54) MACROLIDES
`
`(75)
`
`Inventors: Francois Navarro, Bruebach (FR);
`Samuel Petit, Mont Saint-Aignan (FR);
`Guy Stone, Ettingen (CH)
`
`(73) Assignee: Novartis AG, Basel (CH)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(52) U.S. Cl. ....................................... 514/291; 540/456
`(58) Field of Classification Search ................ 540/456;
`514/291
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,079,128 A
`4,080,445 A
`
`3/1978 Lin et al. .................... 424/181
`3/1978 Lin et al. .................... 4241227
`
`(21) Appl. No.: 111020,860
`
`(22) Filed:
`
`Dec. 23, 2004
`
`(65)
`
`Prior Publication Data
`
`US 2005/0107418 Al May 19, 2005
`
`Related U.S. Application Data
`
`FOREIGN PATENT DOCUMENTS
`
`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, filed on
`Mar. 21, 2003, now Pat. No. 6,852,729, which is a
`continuation of application No. 09/866,977, filed on
`May 29, 2001, now Pat. No. 6,605,613, which is a
`continuation of application No. PCT/EP99/09521,
`filed on Dec. 6, 1999.
`
`(30)
`
`Foreign Application Priority Data
`
`Dec. 7, 1998
`Mar. 4, 1999
`
`(GB)
`(GB)
`
`................................. 9826882.4
`................................. 9904934.8
`
`(51)
`
`Int. Cl.
`C07D 498118
`A61K 311715
`
`(2006.01)
`(2006.01)
`
`Primary Examiner-Bruck Kifle
`(7 4) Attorney, Agent, or Firm-Thomas R. Savitsky;
`Gregory C. Houghton
`
`(57)
`
`ABSTRACT
`
`A mixture comprising a poly-ene macrolide and an antioxi(cid:173)
`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
`
`West-Ward Exhibit 1073
`Navarro USP '703
`Page 001
`
`

`

`U.S. Patent
`
`Nov. 20, 2007
`
`Sheet 1 of 4
`
`US 7,297,703 B2
`
`FIGURE 1/3
`
`Atomic coordinates and equivalent isolropic displacement parameters (A
`(U(eq) is defined as one third of the trace of the onhogonalized Uij tensor)
`
`2
`)
`
`xf 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(JO)
`0(10)
`C(ll)
`C(J JM)
`C(12)
`C(13)
`C(14)
`0(14)
`C(l3)
`C(16)
`0(16)
`C(I6M)
`C(l7)
`C(17M)
`C{18)
`C{l9)
`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)
`.5~06(7)
`.6018(7)
`.6768(8)
`.7032(8)
`.7771(8)
`.8086(8)
`.7254(9)
`.8912(8)
`.9826(9)
`1.0348(12)
`l.05l2(10)
`1.1132(8)
`1.0375(8)
`1.0877(7)
`1.0445(17)
`1.0824(7)
`J.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)
`JSOJ(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)
`.)382(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)
`.H0(6)
`.141(6)
`.178(8)
`.1S7(8)
`.281(11)
`.118(5)
`.185(5)
`.256(13)
`.091(3}
`.108{2)
`
`West-Ward Exhibit 1073
`Navarro USP '703
`Page 002
`
`

`

`U.S. Patent
`
`Nov.20,2007
`
`Sheet 2 of 4
`
`US 7,297, 703 B2
`
`FIGURE 1/3{Cont.)
`
`Atomic coordinates and equivalent isotropic displacement parameters (A 2
`(cont.)
`
`)
`
`xJa
`
`y/b
`
`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)
`l.4221(7)
`1.3653(6)
`l.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 1073
`Navarro USP '703
`Page 003
`
`

`

`U.S. Patent
`
`Nov. 20, 2007
`
`Sheet 3 of 4
`
`US 7,297,703 B2
`
`FlGURE 2/3
`
`Bond lengths {A)
`
`C(l)-0(1)
`C(1)-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(l 0)-0(14)
`C(lO}-C{ll)
`C(ll)-C(llM)
`C(l 1)-C(12)
`C(l2)-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(l 7)-C(l 7M)
`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).
`J.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(2-5)-C(26)
`C(26)-0(26)
`C(26)-C(27)
`. C(27)-0(27)
`C(27)-C(28)
`0(27)-C(27M)
`C(2.8)-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(3 7)
`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(i 1)
`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 1073
`Navarro USP '703
`Page 004
`
`

`

`U.S. Patent
`
`Nov. 20, 2007
`
`Sheet 4 of 4
`
`US 7,297,703 B2
`
`Bond angles (0
`
`)
`
`0( 1)-C(1 )-0(34)
`0( 1}-C(1 )-C(2)
`0(34)-C(l )-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)-~(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(l0)
`C(8)-C(9)-C( 10)
`0(10)-C(l0)-0(14)
`0(10)-C(l0)-C(9)
`0(14)-C(10)-C(9)
`0(10)-C(l 0)-C(l l}
`0(14}-C(lO)-C(l 1)
`C(9)-C(10)-C(l 1)
`C{llM)-C(tt)-C(IO}
`C(l lM)-C(l l )-C(12}
`C(10}-C(l 1)-C(12)
`C(l3 )-C(12)-C(11)
`C(H)-C(l3)-C(l2)
`0( 14)-C(14)-C(13)
`0(14)-C(14)-C(15)
`C( 13)-C(14)-C(15)
`C(l0)-0(14)-C(l4)
`C(16)-C(1S)-C(l4)
`0(16)-C(16)-C(15)
`0(16)-C(16)-C(17}
`C(15)-C{ 16)-C(l7)
`C( 16M)-0(16)-C(l 6)
`C{ 18)-C( 171-C( 17M)
`C{18)-C(17)-C(l6)
`C( 17M)-C( 17)-C( J 6)
`C(l7)-C(l8)-C( 19)
`C(iO)-C(19)-C(18)
`C( 19)-C(20)-C(2 I)
`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)
`] }1.6(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)
`]] 1.6(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)
`
`FIGURE 3/3
`
`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(2 7)-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(31)-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(35M)-C(35)-C(36)
`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(J9)-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(4 l)
`0(40}-C(40)~C(39)
`C(41)·C(40)-Q39)
`C(43)-0(40)-C(40)
`C(40)-C(4l)·C(42)
`C(4 l )-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)
`1H(2)
`112.2(14)
`
`West-Ward Exhibit 1073
`Navarro USP '703
`Page 005
`
`

`

`US 7,297,703 B2
`
`1
`MACRO LID ES
`
`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 U.S. Pat, Nos. 5,665,772 and 6,440,990 is
`replaced by -OR wherein R is hydroxyalkyl, hydroxy(cid:173)
`alkoxyalkyl, acylaminoalkyl or aminoalkyl, e.g. 40-0-(2-
`hydroxy)ethyl-rapamycin,
`40-0-(3-hydroxy)propyl-rapa-
`mycin, 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-inflamma(cid:173)
`tory activity. FK506 is a lactam macrolide produced by
`Streptomyces tsukubaensis. The structure ofFK506 is given
`15 in the Appendix to the Merck Index, 11 th ed. (1989) as item
`AS. Ascomycin is described e.g. In U.S. 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
`20 "Ascomycins". Examples of synthetic analogues or deriva(cid:173)
`tives are e.g. halogenated ascomycins, e.g. 33-epi-chloro-
`33-desoxy-ascomycin such as disclosed in EP-A427,680,
`tetrahydropyran derivatives, e.g. as disclosed in EP-A-626,
`385.
`Particularly preferred macrolides are rapamycin and
`40-0-(2-hydroxy )ethy 1-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 anti(cid:173)
`oxidant, 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,
`45 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
`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.
`
`10
`
`30
`
`This application is a continuation of U.S. patent applica(cid:173)
`tion Ser. No. 10/393,795, filed Mar. 21, 2003, now U.S. Pat.
`No. 6,852,729, which is a continuation of U.S. patent
`application Ser. No. 09/866,977, filed May 29, 2001, now
`U.S. Pat. No. 6,605,613, which is a continuation of Inter(cid:173)
`national Application No. PCT/EP99/09521, filed 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,
`e.g. a poly-ene macrolide, preferably a poly-ene macrolide
`having immunosuppressant properties, particularly rapamy(cid:173)
`cms.
`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 pack(cid:173)
`aging under protective gas. Substantial amounts of stabiliz(cid:173)
`ers are added during the formulating process of such phar(cid:173)
`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, 25
`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, prefer(cid:173)
`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(cid:173)
`erably from 0.01 to 0.5% (based on the weight of the
`macrolide). Such a small amount is referred to herein- 35
`after 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 40
`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, e.g. in a condition suitable for phar(cid:173)
`maceutical use.
`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 immuno- 50
`suppressant 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. 55
`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 60
`rapamycin is given in Kessler, H. et al.; 1993; Helv. Chim.
`Acta, 76: 117. Rapamycin has antibiotic and immunosup(cid:173)
`pressant properties. Derivatives of rapamycin are known,
`e.g. 16-0-substituted rapamycins, for example as disclosed
`in U.S. Pat. Nos. 5,728,710 5,985,890 and 6,200,985, 40-0- 65
`substituted rapamycins, for example as disclosed in U.S. Pat.
`Nos. 5,665,772, 6,440,990, 5,130,307, 5,221,670, 5,358,
`
`West-Ward Exhibit 1073
`Navarro USP '703
`Page 006
`
`

`

`US 7,297,703 B2
`
`4
`
`-continued
`
`4037 [R(int) ~ 0.0341]
`32%
`Full-matrix least-squares on F2
`3134/1/613
`1.055
`R 1 ~ 0.0574, wR2 ~ 0.1456
`
`3
`It may be in liquid form, e.g. solutions, suspensions or
`emulsions such as a microemulsions, e.g. as disclosed in
`U.S. Pat. No. 5,536,729, or in solid form, e.g. capsules,
`Independent reflections
`tablets, dragees, powders (including micronized or other-
`Intensity decay
`wise reduced particulates), solid dispersions, granulates,
`Refinement method
`etc., e.g. as disclosed in WO 97 /03654, the contents of which
`Data!restraints/parameters
`Goodness-of-fit on F2
`being incorporated herein by reference, or semi-solid forms
`Final R indices
`such as ointments, gels, creams and pastes. They are pref-
`[l > 2 sigma(!)]
`erably adapted to be in a form suitable for oral adminis~ra-
`Largest cliff. peak and hole
`0.340 and -0.184 e/A3
`tion. Preferably they are in solid form. The pharmaceutical 10 __ __:. __ __ __ ___ __ ____ ____ _
`compositions of the invention may be prepared according to
`known methods, by mixing the macrolide stabilized accord(cid:173)
`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(cid:173)
`lized rapamycin according to the invention and a carrier
`medium, e.g. a water-soluble polymer such as hydroxypro(cid:173)
`pylmethylcellulose, 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 rapamy(cid:173)
`cin 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 _o~al
`administration comprise from 0.1 to 10 mg, to be adm1ms(cid:173)
`tered once or in divided form.
`In another aspect, this invention also provides 40-0-(2-
`hydroxy )ethyl-rapamycin in a crystalline form, particularl_Y
`in a substantially pure form. Preferably the crystal form 1s
`characterized by the absence or substantial absence of any
`solvent component; it is in non-solvate form.
`40-0-(2-hydroxy)ethyl-rapamycin in crystalline form
`belongs to the monoclinic system. 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
`
`293(2)K
`1.54178 A
`P2 1
`
`a
`b
`
`13
`Volume
`z
`Density (calculated)
`Absorption coefficient
`F(OOO)
`Crystal size
`8 range for data collection
`Reflections collected
`
`14.378.(2) A
`11.244(1) A
`18.310(2) A
`108.58(1) 0
`2805.8(6) A3
`2
`1.134 g/cm3
`0.659 = - !
`1040
`0.59 x 0.11 x 0.03 mm
`2.55 to 57.20°
`4182
`
`65
`
`w
`
`30
`
`35
`
`40-0-(2-hydroxy)ethyl-rapamycin in crystalline fo1:11
`may be prepared by dissolving the amorphous compound m
`a solvant e.g. ethyl acetate and adding an aliphatic hydro-
`15 carbon CnH2n+2 (n=5, 6 or 7). After addition of the hydro(cid:173)
`carbon the resulting mixture may be warmed e.g. at a
`temper~ture 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 0 to 25° C.
`The crystals are filtered and dried. Heptane is preferred as an
`d
`aliphatic hydrocarbon. If desired, nucleation proce ures
`may be commenced e.g. by sonication or seeding.
`The present invention also provides a process for purify(cid:173)
`ing 40-0-(2-hydroxy)ethyl-rapamycin comprising crystal-
`25 lizing 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 immunosuppres(cid:173)
`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
`40-0-(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,
`40 e.g. to prevent or treat acute and chronic allo- or xeno(cid:173)
`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
`45 being incorporated herein by reference. In general, satisfac(cid:173)
`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, 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
`50 dosages for patients are thus on the order of up to 10 mg.,
`e.g. 0.1 to 10 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
`55 inhalation). It may be administered as the sole active ingre(cid:173)
`dient or together with other 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
`60 also provides:
`5. A method for preventing or treating acute or chronic alto(cid:173)
`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 admin(cid:173)
`istering to said subject a therapeutically effective amount
`of 40-0-(2-hydroxy)ethyl-rapamycin in crystalline form;
`
`West-Ward Exhibit 1073
`Navarro USP '703
`Page 007
`
`

`

`US 7,297,703 B2
`
`5
`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-hy-
`droxy )ethy 1-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 com- 10
`prising an immunosuppressant or immunomodulatory
`drug or an anti-inflammatory, agent.
`The following examples illustrate the invention without
`limiting it.
`
`6
`
`Compound
`Ex. 2 (0.2% BHT)
`Without BHT
`
`50° C. in open flask
`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)
`
`EXAMPLE 1
`
`Crystallisation
`
`Single X-ray structure with coordinates are indicated in
`Tables 1-3 below.
`
`EXAMPLE 2
`
`Production of Stabilized
`40-0-(2-hydroxy )ethy 1-rapamycin
`
`C(l)
`0(1)
`C(2)
`C(3)
`C(4)
`C(5)
`C(6)
`0.5 g amorphous 40-0-(2-hydroxy)ethyl-rapamycin is
`N(7)
`dissolved in 2.0 ml ethyl acetate at 40° C. 5.0 ml heptane is
`C(8)
`added and the solution becomes "milky". After warming to
`0(8)
`C(9)
`30° C., the solution becomes clear again. Upon cooling to 0°
`0(9)
`C. and with scratching an oil falls out of the solution. The 25 C(lO)
`test tube is closed and stored at 10° C. overnight. The
`0(10)
`C(ll)
`resulting white voluminous solid is then filtered and washed
`C(llM)
`with 0.5 ml of a mixture of ethyl acetate/hexane (1 :2.5) and
`C(12)
`the resulting crystals are dried at 40° C. under 5 mbar for 16
`C(13)
`hours. 40-0-(2-hydroxy)ethyl-rapamycin in crystalline form 30 C(14)
`0(14)
`having a m.p. of 146.5° C. is thus obtained.
`C(15)
`Crystallisation at a larger scale may be performed as
`C(16)
`0(16)
`follows: 250 g amorphous 40-0-(2-hydroxy)ethyl-rapamy-
`C(16M)
`cin is dissolved in 1.01 ethyl acetate under argon with slow
`35 C(17)
`stirring. This solution is heated at 30° C. and then during 45
`C(17M)
`minutes, 1.51 heptane is added dropwise. 0.25 g of seed
`C(18)
`C(19)
`ciystals prepared as disclosed above are added under the
`C(20)
`same conditions in portions. The mixture is further stirred at
`C(21)
`30° C. over a period of 2 hours and the crystallization
`C(22)
`mixture is cooled to 25° C. over 1 hour and then to 10° C.
`C(23)
`C(23M)
`for 30 minutes and filtered. The crystals are washed with 100
`C(24)
`ml of a mixture ethyl acetate/hexane (2:3). Subsequence
`C(25)
`drying is performed at 50° C. and ca 5 mbar. m.p. 146.5° C.
`C(25M)
`C(26)
`IRinKBr: 3452, 2931, 1746, 1717, 1617, 1453, 1376, 1241, 45 0(26)
`1191, 1163, 1094, 1072, 1010, 985, 896 cm- 1
`C(27)
`0(27)
`C(27M)
`C(28)
`0(28)
`50 C(29)
`C(29M)
`C(30)
`C(31)
`C(31M)
`C(32)
`55 0(32)
`C(33)
`10 g 40-0-(2-hydroxy)ethyl-rapamycin are dissolved in
`C(34)
`600 1 abs. ethanol. After addition of 0.2 g BHT, the resulting
`0(34)
`solution is added dropwise with stirring to 3 .0 1 water within
`C(35)
`1 hour. The resulting suspension is stirred for an additional
`C(35M)
`C(36)
`30 minutes. Filtration with subsequent washing (3x200 ml 60
`C(37)
`water/ethanol at a v/v ratio of 5: 1) results in a moist white
`C(38)
`product which is further dried under vacuum (1 mbar) at 30°
`C(39)
`C. for 48 hours. The resulting dried product contains 0.2%
`0(39)
`C(39M)
`(w/w) BHT.
`C(40)
`The resulting product shows improved stability on star- 65 0(40)
`age. The sum of by-products and degradation products in%
`C(41)
`after 1 week storage are as follows:
`
`15
`
`20
`
`40
`
`.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)
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`.2680(6)
`.1964(6)
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`.1094(7)
`.0948(7)
`.0240(6)
`-.0474(7)
`.0406(6)
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`.0884(8)
`.1293(7)
`.0998(7)
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`.1382(13)
`.2699(6)
`.2818(4)
`.2922(5)
`.2984(6)
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`.3340(5)
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`.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)
`
`West-Ward Exhibit 1073
`Navarro USP '703
`Page 008
`
`

`

`US 7,297,703 B2
`
`7
`
`TABLE I-continued
`
`8
`
`TABLE 3-continued
`
`Bond Angles (0
`
`)
`
`113.6(7) C(32)-C(31)-C(30)
`112.1(7) C(32)-C(31)-C(31M)
`109.7(6) C(30)-C(31)-C(31M)
`100.5(6) 0(32)-C(32)-C(31)
`108.1(6) 0(32)-C(32)-C(33)
`111.6(6) C(31)-C(32)-C(33)
`114.9(7) C(32)-C(33)-C(34)
`114.3(7) 0(34)-C(34)-C(33)
`111.2(8) 0(34)-C(34)-C(35)
`107.9(7) C(33)-C(34)-C(35)
`111.9(8) C(l)-0(34)-C(34)
`109.9(9) C(35M)-C(35)-C(34)
`109.8(8) C(35M)-C(35)-C(36)
`106.2(7) C(34)-C(35)-C(36)
`113.2(8) C(37)-C(36)-C(35)
`115.1(6) C(38)-C(37)-C(36)
`114.5(7) C(38)-C(37)-C(42)
`105.4(7) C(36)-C(37)-C(42)
`112.5(8) C(37)-C(38)-C(39)
`113.4(8) 0(39)-C(39)-C(40)
`114.0(7) 0(39)-C(39)-C(38)
`124.9(9) C(40)-C(39)-C(38)
`119.2(9) C(39)-0(39)-C(39M)
`115.9(8) 0(40)-C(40)-C(41)
`127.7(10)0(40)-C(40)-C(39)
`125.6(11)C(41)-C(40)-C(39)
`126.6(11)C(43 )-0( 40)-C(40)
`126.3(12)C(40)-C(41)-C(42)
`126.0(13 )C( 41)-C(42)-C(37)
`0(40)-C(43)-C(44)
`111(2)
`111.4(10)0( 45)-C( 44 )-C(43)
`114.2(10)
`
`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)
`