PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`wo 97/47317
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(51) International Patent Classification 6 :
`A61K 38/31
`
`Al
`
`(11) International Publication Number:
`
`( 43) International Publication Date:
`
`18 December 1997 ( 18.12.97)
`
`(21) International Application Number:
`
`PCTIEP97/03036
`
`(22) International Filing Date:
`
`II June 1997 ( 11.06.97)
`
`(30) Priority Data:
`9612171.0
`9619310.7
`
`II June 1996 (11.06.96)
`GB
`16 September 1996 (16.09.96) GB
`
`(71) Applicant (for all designated States except US); NOV ARTIS
`AG [CHICH]; Schwarzwaldallee 215, CH-4058 Basel (CH).
`
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, Fl, GB, GE,
`GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR,
`LS, LT. LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ,
`PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TI,
`UA, UG, US, UZ, VN, YU, ZW, ARIPO patent (GH, KE,
`LS, MW, SD, SZ, UG, ZW), Eurasian patent (AM, AZ, BY,
`KG, KZ, MD, RU, TJ, TM), European patent (AT, BE, CH,
`DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT,
`SE), OAPI patent (BF, BJ, CF, CG, Cl, CM, GA, GN, ML,
`MR. NE, SN, TD, TG).
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): WECKBECKER, Gisbert
`[DEICH]; Loeliring 31, CH-4105 Biel-Benken (CH).
`
`Published
`With international search report.
`
`(74) Agent: ROTH, Bernhard, M.; Novartis AG, Patent- und
`Markenabteilung, Klybeckstrasse 141, CH-4002 Basel (CH).
`
`(54) Title: COMBINATION OF A SOMATOSTATIN ANALOGUE AND A RAPAMYCIN
`
`(57) Abstract
`
`A combination of a compound of the somatostatin class and a rapamycin macrolide is useful for the prevention or treatment of cell
`hyperproliferation.
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 001
`
`

`
`Codes used to identify States party to the Per on the front pages of pamphlets publishing international applications under the Per.
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`AL
`AM
`AT
`AU
`AZ
`BA
`DB
`BE
`BF
`BG
`BJ
`DR
`BY
`CA
`CF
`CG
`CH
`Cl
`CM
`CN
`cu
`cz
`DE
`OK
`EE
`
`Albania
`Annenia
`Austria
`Australia
`Arerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cllte d'Jvoire
`Cameroon
`China
`Cuba
`Czech Republic
`Gennany
`Denmark
`Estonia
`
`ES
`Fl
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`u
`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
`Kazak51an
`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
`Lalvia
`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
`
`Sl
`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
`VietNam
`Yugoslavia
`Zimbabwe
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 002
`
`

`
`wo 97/47317
`
`PCT/EP97/03036
`
`COMBINATION OF A SOMATOSTATIN ANALOGUE AND A RAPAMYCIN
`
`The present invention relates to a pharmaceutical combination and its use in the
`
`treatment of disorders associated with excess benign and malignant cell proliferation, e.g.
`
`tumors or intimal cell proliferation.
`
`There is a continuing need for the development of drugs having increased
`
`effectiveness in inhibiting or slowing down undesired cell proliferation, particularly in the
`
`cancer field and in vasculopathies.
`
`Accordingly, there is provided a pharmaceutical combination comprising a
`
`compound of the somatostatin class, and a rapamycin macrolide.
`
`The somatostatin class is a known class of small peptides comprising the naturally
`
`occurring somatostatin-14 and analogues having somatostatin related activity, e.g. as
`
`disclosed by A.S. Dutta in Small Peptides, Vol.19, Elsevier (1993). By "somatostatin
`
`analogue" as used herein is meant any straight-chain or cyclic polypeptide having a
`
`structure based on that of the naturally occurring somatostatin-14 wherein one or more
`
`amino acid units have been omitted and/or replaced by one or more other amino radical(s)
`
`and/or wherein one or more functional groups have been replaced by one or more other
`
`functional groups and/or one or more groups have been replaced by one or several other
`
`isosteric groups. In general, the term covers all modified derivatives of the native
`
`somatostatin-14 which exhibit a somatostatin related activity, e.g. they bind to at least one
`
`somatostatin receptor (hSST-1, hSST-2, hSST-3, hSST-4 or hSST-5), preferably in the
`
`nMolar range, more preferably to at least the hSST-2 receptor in the nMolar range.
`
`Cyclic, bridge cyclic and straight-chain somatostatin analogues or derivatives are
`
`known and have been described together with processes for their production e.g. in US
`
`Patent Specifications 4,310,518 and 4,235,886, in European Patent Specifications EP(cid:173)
`
`A-1295; 23,192; 29,310; 29,579; 30,920; 31,303; 63,308; 70,021; 83,305; 215,171;
`
`203,031; 214,872; 143,307; 298,732; 277,419; 389,180; 395,417; 450,480A2; in Belgian
`
`Patent Specification BE-A-900,089; and in WO 91109056; WO 97/01579; WO 97/14715,
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 003
`
`

`
`W097/47317
`
`PCT/EP97/03036
`
`2
`
`the contents thereof, in particular with respect to the compounds, being incorporated
`
`herein by reference.
`
`Preferred somatostatin analogues are e. g. compounds of formula I
`
`Y2-S-1H 2
`CH -S-Y
`I 2
`A'
`I
`'N-CH-CO-B-C-D-E-NH- H-G
`A/
`
`(I)
`
`wherein
`
`A is C 1_12alkyl, C7_10phenylalkyl or a group of formula RCO-,
`whereby
`
`i)
`
`R is hydrogen, C 1_11alkyl, phenyl or C7_10phenylalkyl, or
`ii) RCO- is
`
`a)
`
`a D-phenylalanine residue optionally ring-substituted by halogen, N02, NH2, OH,
`C1_3alkyl and/or C 1_3alkoxy; or
`
`b)
`
`the residue of a natural or a synthetic a-amino-acid other than defined under a)
`
`above, or of a corresponding D-amino acid, or
`
`c)
`
`a dipeptide residue in which the individual amino acid residues are the same or
`
`different and are selected from those defined under a) and/or b) above,
`
`the a-amino group of amino acid residues a) and b) and the N-terminal amino group of
`
`dipeptide residues c) being optionally mono- or di-C 1•12alkylated or substituted by
`C 1_8alkanoyl;
`
`A'
`
`is hydrogen or CHalky!,
`
`Y 1 and Y 2 represent together a direct bo.nd or each of Y 1 and Y 2 is hydrogen
`
`B
`
`is -Phe- optionally ring-substituted by halogen, N02, NH2, OH, C 1_3alkyl and lor
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 004
`
`

`
`W097147317
`
`PCT/EP97/03036
`
`3
`
`C 1_3alkoxy (including pentafluoroalanine), naphthylalanine or pyridylalanine,
`
`C
`
`is (L)-Trp- or (D)-Trp- optionally a-N-methylated and optionally benzene(cid:173)
`
`ring-substituted by halogen, N02, NH2, OH, C1_3alkyl and/or C1_3alkoxy,
`
`D
`
`is Lys, 4-aminocyclohexylAla or 4-aminocyclohexylGly
`
`E
`
`is Thr, Ser, Val, Tyr, lie, Leu or an aminobutyric or aminoisobutyric acid residue
`
`G
`
`is a group of formula
`
`or
`
`wherein
`
`R7
`R 10
`
`R 11
`R 12
`R 13
`
`is hydrogen or C1_3alkyl,
`is hydrogen or the residue of a physiologically acceptable, physiologically
`
`hydrolysable ester, e.g. fonnyl, c2-12alkylcarbonyl, benzoyl,
`
`is hydrogen. C1_3alkyl, phenyl or C7_10phenyl-alkyl,
`is hydrogen, C1_3alkyl or a group of fonnula -CH(R 13)-X"
`is CH20H, -(CH2kOH, -(CH2kOH, -CH(CH3)0H, isobutyl, butyl, benzyl,
`naphthyl-methyl or indol-3-yl-methyl, and
`
`X 1 is a group of formula
`
`R
`-CO-N/ 14
`
`" R1s
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 005
`
`

`
`wo 97/47317
`
`wherein
`
`4
`
`PCT/EP97 /03036
`
`R7 and R 10 have the meanings given above,
`R14
`is hydrogen or C 1_3alkyl and
`R15
`is hydrogen, C 1_3alkyl, phenyl or C7_10phenylalkyl, and
`R 16
`is hydrogen or hydroxy,
`
`with the proviso that
`
`when R 1" is -CH(R 13)-X 1 then R 11 is hydrogen or methyl,
`
`wherein the residues B, D and E have the L-configuration, and the residues in the 2- and
`
`7-position each independently have the (L)- or (0)- configuration,
`
`in free fonn or in pharmaceutically acceptable salt or complex form.
`
`Individual compounds of formula I suitable in accordance with the present invention are
`
`the following somatostatin analogues:
`
`a. (D )Phe-2ys-Phe-(D )Trp-Lys-Thr-2ys-Thr-ol
`
`also known as octreotide
`
`b. (D)Phe-~ys-Tyr-(D)Trp-Lys-Val-~ys-ThrNH2
`
`c. (D)Phe-Cys-Tyr-(D)Trp-Lys-Vai-Cys-TrpNH2
`also known as vapreotide
`
`d. (D )Trp-Cys-Phe-(D )Trp-Lys-Thr-Cys-ThrNH 2
`
`e. (D)Phe-~ys-Phe-(D)Trp-Lys-Thr-2ys-ThrNH2
`
`f. 3-(2-(Naphthyl)-(D)Ala-~ys-Tyr-(D)Trp-Lys-Val-tys-ThrNH2
`also known as lanreotide
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 006
`
`

`
`W097/47317
`
`PCT!EP97/03036
`
`5
`
`g. (D)Phe-~ys-Tyr-(D)Trp-Lys-Val-tys-~-Nal-NH2
`
`h. 3-(2-naphthy 1 )-Ala-6ys-Tyr-(D)Trp-Lys-Val-tys-~-Nal-NH2
`
`1. (D)Phe-tys-13-Nal-(D)Trp-Lys-Val-tys-Thr-NH2
`
`J. (D)Phe-Cys-Tyr-(D)Trp-Lys-Leu-Cys-Thr-NH2
`
`k. (D)Phe-tys-Tyr-(D)Trp-Lys-tys-Thr-NH2•
`
`A preferred compound of formula I is octreotide.
`
`Compounds of formula I may exist e.g. in free form, salt form or in the form of
`
`complexes thereof. Acid addition salts may be formed with e.g. organic acids, polymeric
`
`acids and inorganic acids. Such acid addition salt forms include e.g. the hydrochlorides
`
`and acetates. Complexes are e.g. formed from compounds of the invention on addition of
`
`inorganic substances, e.g. inorganic salts or hydroxides such as Ca- and Zn-salts, andlor
`
`an addition of polymeric organic substances.
`
`Further somatostatin analogues suitable for use in accordance with the present
`
`invention are:
`
`cyclo [ -Asn-Phe-Phe-DTrp-Lys-Thr-Phe-Gaba-],
`
`cyclo(Asu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser), and
`
`I
`I
`(D)Nal-Glu-Tyr-(D)Trp-Lys-Val-Lys-Thr-NH2
`
`According to an alternatively preferred embodiment of the invention, the somatostatin
`
`component of the combination is a somatostatin analogue comprising the amino acid
`
`sequence of formula (II)
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 007
`
`

`
`wo 97/47317
`
`PCT/EP97/03036
`
`6
`
`(II)
`
`(a)
`
`(b)
`
`wherein X2 is a radical of formula (a) or (b)
`
`or
`
`-NH-CH-C0-
`1
`CH-O-CH2·R1
`I
`CH 3
`
`-NH-CH-C0-
`1
`CH2
`I
`R2
`
`wherein R 1 is optionally substituted phenyl,
`R2 is -Z1-CH2-R}t -CH2-CO-O-CH2-R 1,
`
`~0-CH-R ~ 21
`
`or
`
`wherein Z 1 is 0 or S,
`
`and
`
`X3
`
`is an a-amino acid having an aromatic residue on the Ca side chain, or an
`
`amino acid unit selected from Dab, Dpr, Dpm, His,(Bzl)HyPro, thienyl(cid:173)
`
`Ala, cyclohexyl-Ala and t.-butyl-Ala,
`
`the residue Lys of said sequence corresponding to the residue Lys9 of the native
`
`somatostatin-14.
`
`Such somatostatin analogues are e.g. disclosed in WO/ 97/01579, the contents
`
`thereof, in particular with respect to the specifically exemplified compounds, being
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 008
`
`

`
`WO 97147317
`
`PCT/EP97103036
`
`7
`
`incorporated herein by reference.
`
`Preferably the sequence of formula II as defined above corresponds to the residues
`
`at positions 8 through 11 of the somatostatin-14. More preferably the somatostatin
`
`analogue as disclosed above comprises a hexapeptide unit, the residues at positions 3
`
`through 6 of said hexapeptide unit comprising the sequence of formula II. More
`
`particularly the hexapeptide unit is cyclic, e.g. having a direct peptide linkage between the
`
`a.-carbonyl group of the residue at position 6 and the a.-amino group of the residue at
`
`position 1.
`
`While Lys, X" and X3 in the sequence of formula II have the L-configuration, Trp
`may have the D- or L-configuration, preferably the D-configuration.
`
`X~ is preferably a residue of formula (a) or (b), R2 being preferably -Z1-CH2-R 1 or
`~0-CH-R ~ 21 .
`
`When X 3 comprises an aromatic residue on the Ca side chain, it may suitably be a
`natural or unnatural a.-amino acid, e.g. Phe, Tyr, Trp, Nal, Pal, benzothienyl-Ala, Tic and
`
`thyronin, preferably Phe or Nal, more preferably Phe. X3 is preferably an a.-amino acid
`bearing an aromatic residue on the Ca side chain.
`
`When R 1 is substituted phenyl, it may suitably be substituted by halogen, methyl,
`ethyl, methoxy or ethoxy e.g. in ortho and/or para. More preferably R 1 is unsubstituted
`phenyl. zl is preferably 0.
`
`Representative somatostatin analogues comprising a residue of formula II are e.g
`
`compounds of formula (III)
`
`cvclo[A - Z:Z - Trp - Lys - X - X ]
`•
`a
`2
`3
`5
`I
`2
`3
`4
`6
`
`(II)
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 009
`
`

`
`WO 97/47317
`
`PCT/EP97/03036
`
`8
`
`wherein
`X2 and X3 are as defined above,
`a divalent residue selected from Pro,
`A 1 is
`
`(ICO(cid:173)
`I
`v -(CH) -N-N
`2 1-6
`.. "a
`
`wherein R3 is NR8R9-C 2_6alkylene, guanidino-C 2•6alkylene or C2_6alkylene-COOH,
`R33 is H, C 14alkyl or has independently one of the significances given for R3_ R3bis
`H or C 14alkyl, Ra is OH or NRsR6, Rb is -(CH2) 1-r or -CH(CH3)-, R4 is H or CH,,
`R4a is optionally ring-substituted benzyl, each of R5 and R6 independently is H,
`
`cl-4alkyl, (1)-amino-CI-4alkylene, (1)-hydroxy-CI-4alkylene or acyl, Rs. is a direct
`
`bond or C 1_6alkylene, each of R8 and R9 independently is H. C 1_4alkyl,
`())-hydroxy-C24alkylene, acyl or CH20H-(CHOH)c·CH2- wherein c is 0, 1, 2, 3 or
`4, or R8 and R9 form together with the nitrogen atom to which they are attached a
`heterocyclic group which may comprise a further heteroatom, and R 17 is optionally
`ring-substituted benzyl, -(CH2) 1•3-0H, CH3-CH(OH)- or
`-(CH2) 1.5-NR5R6 , and
`ZZa is a natural or unnatural a-amino acid unit.
`
`ZZa may have the D- or L-configuration. When ZZ1 is a natural or unnatural a-amino
`acid unit, it may suitably be e.g. Thr, Ser, Ala, Val, lie, Leu, Nle, His, Arg, Lys, Nal, Pal,
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 010
`
`

`
`wo 97/47317
`
`PCT/EP97/03036
`
`9
`
`Tyr, Trp, optionally ring-substituted Phe or N°-benzyl-Gly. When zz. is Phe, the benzene
`ring thereof may be substituted by e.g. NH2, N02, CH3, OCH3 or halogen, preferably in
`para position. When zz. is Phe, the benzene ring thereof is preferably unsubstituted.
`
`When A1 comprises a Pro amino acid residue, any substituent present on the proline
`ring, e.g. R3-NH-CO-O- etc., is preferably in position 4. Such substituted proline residue
`may exist in the cis form, e.g.
`
`-ob-c-
`
`N
`I
`
`II
`o
`
`as well as in the trans form. The present invention covers each geometric isomer
`
`individually as well as mixtures thereof.
`
`When A1 is (NR8R9-C2_6alkylene-NH-CO-O)P{o- where NR8R9 forms a
`
`heterocyclic group, such group may be aromatic or saturated and may comprise one
`
`nitrogen or one nitrogen and a second heteroatorn selected from nitrogen and oxygen.
`
`Preferably the heterocyclic group is e.g. pyridyl or morpholino. C2_6Aikylene in this
`residue is preferably -CH2-CH2-.
`
`Any acyl as R5, R6 , R8 and R9 in A 1 may be e.g. R 18CO- wherein R18 is H, C 1-4alkyl,
`C2-4alkenyl, C3_6cycloalkyl or benzyl, preferably methyl or ethyl. When R4• or R 17 in A1 is
`ring-substituted benzyl, the benzene ring may be substituted as indicated above for zz..
`
`A preferred group of compounds of formula m are such wherein A 1 is free of a
`lateral -NH-C0-0- moiety. A further group of preferred compounds of formula m are
`such wherein A 1 comprises a basic lateral radical, e.g. a R3-NH-CO-O- or R5-N-R5.-
`I
`
`~
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 011
`
`

`
`wo 97/47317
`
`PCT/EP97/03036
`
`10
`
`moiety.
`
`A still further group of preferred compounds of formula III are such wherein the N(cid:173)
`
`terminal amino acid comprises a substituted Pro, particularly 4-substituted Pro, e.g.
`
`compounds of formula III wherein A 1 is 4-substituted Pro.
`
`Preferably A 1 is 4-(R3-NH-CO-O)Pro.
`
`Examples of somatostatin analogues comprising a residue of formula II include e.g.
`
`cyclof 4-(NH 2-C 2H4- NH-C0-0- )Pro-Phe-DTrp-Lys-Ser(Benzy 1)-Phe].
`
`The term "macrolide" as used herein, refers to a macrocyclic lactone, for example a
`
`compound having a 12-membered or larger lactone ring. Of particular interest are the
`
`"lactam macrolides", i.e. macrocyclic compounds having a lactam (amide) bond in the
`
`macrocycle in addition to a lactone (ester) bond, for example rapamycin and its numerous
`
`derivatives and analogues. Rapamycin is an immunosuppressive Jactam macrolide that is
`
`produced by Streptomyces hy~roscopicus, and having the structure depicted in Formula
`
`A:
`
`sc;x;" ..... ~·· lS
`
`_ll
`ll4
`, ~ .
`
`'
`
`7
`N
`
`J
`
`2
`
`t
`J
`!
`
`(A)
`
`0
`II
`1 ~OH
`
`0~ 0
`0 '
`
`11
`
`I • • &
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 012
`
`

`
`W097/47317
`
`PCT/EP97/03036
`
`11
`
`See, e.g., McAlpine, J.B., et al., J. Antibiotics (1991) 44: 688; Schreiber, S.L.. eta!.,
`J. Am. Chern. Soc. (1991) ill: 7433; US Patent No.3 929 992. One group of rapamycin
`derivatives are 40-0-substituted derivatives of rapamycin having the structure of
`
`Formula IV:
`
`IV
`
`wherein
`
`X 4 is (H,H) or 0;
`Y 3 is (H,OH) or 0;
`R20 and R21 are independently selected from H, alkyl, arylalkyl, hydroxyalkyl,
`dihydroxyalkyl, hydroxyalkoxycarbonylalkyl, hydroxyalkylarylalkyl,
`
`dihydroxyalkylarylalkyl, acyloxyalkyl, aminoalkyl, alkylaminoalkyl,
`
`alkoxycarbonylaminoalkyl, acylaminoalkyl, arylsulfonamidoalkyl, allyl,
`
`dihydroxyalkylallyl, dioxolanylallyl, dialkyl-dioxolanylalkyl, di(alkoxycarbonyl)(cid:173)
`
`triazolyl-alkyl and hydroxyalkoxy-alkyl; wherein "alk-" or "alkyl" refers to C 1_6alkyl,
`branched or linear, preferably C 1_3alkyl,; "aryl" is phenyl or tolyl; and acyl is a radical
`derived from a carboxylic acid; and
`R22 is methyl or R22 and R 20 together form C2•6alkyl;
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 013
`
`

`
`W097/47317
`
`PCT/EP97/03036
`
`12
`
`provided that R20 and R21 are not both H; and hydroxyalkoxyalkyl is other than
`hydroxyalkoxymethyl.
`
`Such compounds are disclosed in WO 94/09010 the contents of which, in particular
`
`with respect to the specifically exemplified compounds, are incorporated herein by
`
`reference.
`
`A preferred compound is e.g. 40-0-(2-hydroxy)ethyl-rapamycin (referred thereafter as
`
`Compound B).
`
`Further preferred rapamycin derivatives are e.g. those disclosed in WO 96/41807, the
`
`contents thereof, in particular with respect to the specifically exemplified compounds of
`
`formula I disclosed therein, being incorporated herein by reference. Particularly preferred
`
`are 32-deoxo-rapamycin, 16-0-pent-2-ynyl-32-deoxo-rapamycin,
`
`16-0-pent-2-ynyl-32-deoxo-40-0-(2-hydroxyethyl)-rapamycin,
`
`I 6-0-pent-2-yny 1-32-(S)-dihydro-rapamycin and 16-0-pent-2-yny 1-32-(S )-dihydro-40-0-
`
`(2-hydroxyethyl )-rapamycin.
`
`Further rapamycin derivatives are known, e.g. carboxylic acid esters such as disclosed
`
`in WO 92/05179. amide esters such as disclosed in US 5 118 677, carbamates such as
`
`described in US 5 118 678, fluorinated esters such as disclosed in US 5 100 883, acetals,
`
`e.g. in US 5 151 413, silyl ethers, e.g. in US 5 120 842, arylsulfonates and su1famates,
`
`e.g. in US 5 177 203, derivatives wherein the methoxy group at the position 16 is
`
`replaced with alkynyloxy, e.g. in WO 95/16691 and further derivatives such as disclosed
`
`in WO 93111130, WO 94/02136, WO 94/02385 and WO 95/14023, all incorporated
`
`herein by reference.
`
`Rapamycin and above mentioned derivatives have been shown to have potent
`
`immunosuppressant properties. Rapamycin has also been shown to inhibit smooth muscle
`
`cell proliferation and to inhibit cancer growth.
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 014
`
`

`
`W097/47317
`
`PCT/EP97/03036
`
`13
`
`Somatostatin analogues, e.g. octreotide, vapreotide and lanreotide, have been disclosed
`
`i.a. to inhibit growth hormone secretion and to have an inhibiting effect on malignant
`
`tumor growth, e.g. in breast cancer. Octreotide and lanreotide have also been disclosed to
`
`inhibit smooth muscle cell proliferation.
`
`In accordance with the invention, it has now surprisingly been found that a
`
`combination of 2 active ingredients believed to act on basically different mechanisms
`
`such as a somatostatin analogue and rapamycin or a derivative thereof, can be combined
`
`and synergistically inhibit cell hyperproliferation.
`
`In accordance with the particular findings of the present invention, there is provided in
`
`a first aspect:
`
`I. Use of a compound of the somatostatin class, in free form or in pharmaceutically
`
`acceptable salt form, for manufacturing a pharmaceutical composition for use in
`
`synergistically effective amounts in the prevention or treatment of cell
`
`hyperproliferation in combination with a rapamycin macrolide, e.g. for the manufacture
`
`of a kit as disclosed hereinafter.
`
`2. Use of a compound of the somatostatin class, in free form or in pharmaceutically
`
`acceptable salt form, in combination in synergistically effective amounts with a
`
`rapamycin macrolide for the prevention or treatment of cell hyperproliferation.
`
`3. A method for preventing or treating cell hyperproliferation in a subject in need of such
`
`treatment which comprises administering to such subject a synergistically effective
`
`amount of a compound of the somatostatin class in free form or in pharmaceutically
`
`acceptable salt form, and a rapamycin macrolide.
`
`4. A kit or package for the treatment or prevention of cell hyperproliferation, said kit or
`
`package including a pharmaceutical composition comprising a compound of the
`
`somatostatin class in free form or in pharmaceutically acceptable salt form, and a
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 015
`
`

`
`wo 97/47317
`
`PCT/EP97/03036
`
`14
`
`pharmaceutical composition comprising a rapamycin macrolide. The kit or package
`
`may also contain instructions to use the pharmaceutical compositions in accordance
`
`with the present invention.
`
`According to the invention, the combination of a compound of the somatostatin class
`
`and a rapamycin macrolide is indicated for the prevention or treatment of malignant
`
`tumor growth, e.g. breast, lung, GEP tumors, pituitary adenomas. lymphomas, etc., for the
`
`prevention or treatment of proliferative vascular diseases, e.g. biologically or
`
`mechanically induced vascular injury causing intimal thickening, e.g. restenosis,
`
`atherosclerosis, vascular occlusion, injury following percutaneous transluminal coronary
`
`angioplasty, vascular surgery or transplantation surgery, transplant vasculopathies, for
`
`example chronic rejection of various tissues and organs such as heart, kidney, pancreas,
`
`lung, liver, bowel, trachea and combined heart-lung.
`
`The combination is particularly indicated for preventing intimal smooth muscle cell
`
`hyperplasia, restenosis and vascular occlusion in a mammal.
`
`Utility of the combination in the treatment of disorders and diseases as hereinbefore
`
`specified, may be demonstrated for example in accordance with the method hereinafter
`
`described.
`
`A. In vitro Assay
`
`AR42J cell cultures are propagated in DMEM supplemented with 10% fetal calf
`
`serum (FCS) at 5 % C02• Cells are grown in the absence of antibiotics or antifungal
`agents. Subconfluent AR42J cells growing in DMEM and supplemented with 10% FCS
`are trypsinized, diluted in DMEM + 2.5 % FCS and seeded in uncoated 96-well plates
`
`(5'000 to 10'000 cells per well in 180 J.ll). After a 48-hr incubation period (Day 0), the
`
`number of cells in a separate control plate is determined both by counting cells in a
`
`Coulter counter and by the sulforhoda~ine B (SRB) staining assay. The cells are then
`
`exposed either to the somatostatin analogue alone, e.g. octreotide, or to rapamycin or a
`
`derivative thereof alone or to a combination of the somatostatin analogue and rapamycin
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 016
`
`

`
`WO 97/47317
`
`PCTIEP97/03036
`
`15
`
`or its derivative up to 5 days at various concentrations. Total drug exposure lasts for up
`
`to 5 days following the first addition and SRB analysis as described above is performed
`
`e.g. on day 2 and day 5. Growth is determined as difference in absorbance (00) between
`
`day 0 and day x values (= delta OD). Calculations are made based on the fractional
`
`product method of Webb (Valeriote and Lin, 1975; Cory and Carter, 1986; Berenbaum,
`
`J. Theor. Bioi. 114: 413-431, 1985) and the method by Chou and Talalay
`
`(Adv. Enz. Regul. 22: 27-55, 1984). If the measured cell growth (%of control)
`
`is < to the calculated cell growth, this shows evidence for a synergistic effect. Under
`
`these conditions a combination of a somatostatin analogue at a concentration of from 10· 10
`to 1 o-6 M with a rapamycin macrolide thereof at a concentration of from 1 to 1000 nM
`significantly inhibits the growth of the tumor cells.
`
`In this assay, the following results are obtained with octreotide alone, Compound B
`
`alone and a combination of octreotide and Compound B. The synergy according to the
`
`Webb Method is confirmed by using the Chou-Talalay Method.
`
`Cell Growth (% of CONTROL)
`
`Cell Growth
`(~00)
`(%)
`
`664 ::t 9
`
`397 :!: 16
`
`420:!: 12
`
`103 ± 5
`
`Observed
`(%)
`
`Calculated
`(Webb Method)
`(%)
`
`100
`
`59.8
`
`63.3
`
`15.6
`
`37.9
`
`Concentration
`(nM)
`
`Control
`
`Octreotide
`
`Compound B
`
`Octreotide
`+
`Compound B
`
`1.2
`
`12.0
`
`1.2
`+
`12.0
`
`B. In Vivo Assay
`
`The AR42J (AR4-2J) rat pancreatic tumor cell line is derived from an azaserine(cid:173)
`
`induced exocrine pancreatic tumor (Jessop and Hay, 1980). It was obtained from ATCC.
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 017
`
`

`
`wo 97/47317
`
`PCT/EP97/03036
`
`16
`
`Cultures are propagated in DMEM supplemented with 10% fetal calf serum (FCS) at 59'c
`
`CO~. Cells are grown in the absence of antibiotics or antifungal agents. Female nude mice
`
`(nu/nu Balbc-A from lffa Credo, Lyon, France) weighing 19-22 g, are kept in groups of 5
`animals in macrolon cages (type m, 16 x 22 x 11 em). The cages are placed in ventilated
`cabinets (Iff a Credo) that are maintained at 24 ± I o C. The animals have free access to
`
`drinking water and a pathogen-free rodent diet (Diet A, Kliba, Basel, Switzerland). To
`
`initiate tumors from cultured cells, AR42J cells are trypsinized and lOx I 06 tumor cells (in
`
`0.2 ml) are injected subcutaneously (s.c.) into both flanks of nude mice. When tumors
`
`have reached a volume of 0.03 cm3
`
`, animals are randomized into control and treatment
`
`groups. Control animals receive placebo. Animals are treated as indicated below for 3
`
`weeks with single agents or the drug combination. The somatostatin analogue is given as
`
`a single injection of a slow release form at 30 mglkg s.c .. The size of the tumors is
`
`determined with a caliper. To calculate the tumor volume in ml the equation "volume
`
`(ellipsoid) = length x depth x height x 0.52" was used.
`
`Results
`
`After 4 weeks the following tumor size were determined.
`
`(Please note that values in the control group correspond to 3 week values, since animals
`
`were killed afterwards for tumors became excessively large.)
`
`Treatment
`
`Control
`
`Volume
`
`mm 3
`
`4020
`
`A) Compound B, 5 mglkg p.o.
`
`3685
`
`B) Rapamycin, 5 mglkg p.o.
`
`2748
`
`C) Octreotide pamoate (biodegradable,
`
`sustained release formulation),
`
`30 mg/kg, single inj.
`
`2205
`
`SE
`
`579
`
`263
`
`325
`
`339
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 018
`
`

`
`wo 97/47317
`
`PCT/EP97/03036
`
`17
`
`130
`
`106
`
`75
`
`44
`
`Compound B + octreotide (C)
`
`Rapamycin + octreotide (C)
`
`C. Clinical trial
`
`Patients are included who have breast cancer as evidenced by histological biopsy
`
`(glandular analysis - EOA). They present a metastatic illness and/or loco-regional
`
`localisation which is measurable and evaluable. If desired, patients may be included who
`
`are resistant to other treatment to conventional therapy such as surgery, radiotherapy.
`
`other chemotherapy and/or hormone therapy.
`
`The patients present at least one target, on X-ray analysis, which is measurable or
`
`evaluable such as a primitive metastatic tumour which is cutaneous or sub-cutaneous. It
`
`may be gangliar or visceral. Preferably the patients have lesions which have progressed
`
`within the month preceding the trial and have an estimated survival time of at least 3
`
`months.
`
`The rapamycin macrolide, e.g rapamycin or compound B is administered orally. The
`
`treatment is for at least 3 months or until complete remission. The response may be
`
`followed by conventional methodology, e.g. according to IUCC response criteria, e.g.
`
`progression. stabilization, partial or complete remission.
`
`The somatostatin analogue, e.g. octreotide, is administered parenterally, e.g.
`
`subcutaneous, particularly in a continuous subcutaneous way by means of a portable
`
`syringe pump (infusion pump).
`
`According to the invention, the somatostatin analogue and the rapamycin macrolide
`
`are preferably administered in the form of a pharmaceutical composition. Rapamycin and
`
`its derivatives, e.g. Compound B, may be administered by any conventional route, in
`
`particular enterally, e.g. orally, e.g. in the form of tablets, capsules, drink solutions,
`
`emulsions or microemulsion preconcentrates, nasally, pulmonary (by inhalation),
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 019
`
`

`
`wo 97/47317
`
`PCT!EP97/03036
`
`18
`
`parenterally, e.g. in the form of injectable solutions or suspensions. or topically.
`
`Rapamycin and its derivatives are preferably administered per
`
`os and the somatostatin analogue is preferably administered parenterally, e.g by infusion.
`
`The somatostatin analogue may also be administered in a slow release form, e.g. as
`
`disclosed in UK Patent Specification 2,265,311B. The administration of each component
`
`of the combination may take place either separately, simultaneously or sequentially, e.g.
`
`rapamycin or Compound B may be administered at first followed later, e.g. 8 to 24 hours
`
`later, by the somatostatin analogue.
`
`The amount of each component administered is determined taking into account various
`
`factors such as the etiology and severity of the disease, and the patient's condition.
`
`Rapamycin or its derivatives may conveniently be administered at doses which are in the
`
`range used in immunosuppressive applications such as prevention and treatment of
`
`graft vs. host disease, transplant rejection or autoimmune diseases e.g. at a daily dosage
`
`from about 0.5 to 500 ID6 as a single dose or in divided doses. Such doses may also be
`
`given intermittently, for example, every other day or every third day. The somatostatin
`
`analogue may be administered, e.g. subcutaneously, in a dosage range of about 100 ~g to
`
`10 mg per day as a single dose or in divided doses. Thus octreotide may be administered
`
`at a dose of from 0.2 mg to 10 mg twice or three times daily. When administered as a
`
`slow release form, such formulation may comprise the somatostatin peptide in a
`
`concentration from 2.0 to I 0% by weight. The release period of such a formulation may
`
`be from 1 week to about 2 months. The combination of the somatostatin analogue with
`
`rapamycin or its derivative allows to maximize the antiproliferative effect.
`
`The invention contemplates that the active ingredients discussed herein may be utilized
`
`in combination with pharmaceutically acceptable diluents and carriers.
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 020
`
`

`
`wo 97147317
`
`PCTIEP97/03036
`
`19
`
`Formulation Examples:
`
`A. Somatostatin Formulations:
`
`1. Ampoules
`
`Octreotide
`
`Mannitol
`
`0.5 mg
`
`45.0 mg
`
`Lactic acid (88%)
`
`3.4 mg
`
`Sodium hydrogeno-
`
`carbonate
`
`to pH 4.2
`
`Water (inject.grade)
`
`to 1 ml
`
`Carbon dioxide
`
`q.s.
`
`2. Biodegradable sustained release formulation:
`
`Octreotide Acetate
`
`4.65%
`
`(by weight)
`
`Poly(DL-lactide-co-glycolide)
`
`78.35%
`
`Sterile Mannitol
`
`17%
`
`Vehicle: Carboxymethylcellulose 0.5 %
`
`(by weight)
`
`Mannitol
`
`0.6%
`
`Water for injection
`
`98.9%
`
`B. Rapamycin (or derivative thereoO formulation: e.~. capsules
`
`Ethanol
`
`1 ,2-propylene glycol
`
`Refined oil
`
`Cremophor RH40
`
`Rapamycin or Compound B
`
`Total
`
`20.0 mg
`
`81.0mg
`
`121.5mg
`
`202.5mg
`
`20.0mg
`
`. 500 mg
`
`Par Pharm., Inc.
`Exhibit 1053
`Page 021
`
`

`
`WO 97/47317
`
`PCT/EP97/03036
`
`20
`
`CLAIMS
`
`1.
`
`Use of a compound of the somatostatin class, in free form or in pharmaceutically
`
`acceptable salt form, for manufacturing a pharmaceutical composition for use in
`
`synergistically effective amounts in the prevention or treatment of cell
`
`hyperproliferation in combination with a rapamycin macrolide.
`
`2.
`
`Use of a compound of the somatostatin class, in free form or in pharmaceutically
`
`acceptable salt form, in combination in synergistically