WO1997047317A1

WO1997047317A1 - Combination of a somatostatin analogue and a rapamycin

Info

Publication number: WO1997047317A1
Application number: PCT/EP1997/003036
Authority: WO
Inventors: Gisbert Weckbecker
Original assignee: Novartis Ag
Priority date: 1996-06-11
Filing date: 1997-06-11
Publication date: 1997-12-18
Also published as: JP2000514415A; ATE233098T1; JP3784418B2; AU3257297A; ES2193379T3; DK0896544T3; EP0896544A1; CA2249439A1; US6362164B1; CA2249439C; DE69719320T2; EP0896544B1; DE69719320D1

Abstract

A combination of a compound of the somatostatin class and a rapamycin macrolide is useful for the prevention or treatment of cell hyperproliferation.

Description

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 compπsing 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 ammo acid units have been omitted and/or replaced by one or more other ammo radιcal(s) and/or wherein one or more functional groups have been replaced by one or more other functional groups and/or one or more groups have been replaced by one or several other isosteric groups.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- 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 91/09056; WO 97/01579; WO 97/14715, 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

wherein

A is C,.ι₂alkyl, C₇.₁₀phenylalkyl or a group of formula RCO-, whereby i) R is hydrogen, C, .,, alkyl, phenyl or C₇.,₀phenylalkyl, or ii) RCO- is

a) a D-phenylalanine residue optionally ring-substituted by halogen, NO₂, NH₂, OH, C,.₃alkyl and/or C,.₃alkoxy; or

b) the residue of a natural or a synthetic α-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 α-amino group of amino acid residues a) and b) and the N-terminal amino group of dipeptide residues c) being optionally mono- or di-Cι._I2alkylated or substituted by C,.₈alkanoyl;

A' is hydrogen or C,.₃alkyl,

Y, and Y₂ represent together a direct bond or each of Y, and Y₂ is hydrogen

B is -Phe- optionally ring-substituted by halogen, NO₂, NH₂, OH, C,.₃alkyl and /or C,.₃alkoxy (including pentafluoroalanine), naphthylalanine or pyridylalanine,

C is (L)-Trp- or (D)-Trp- optionally α-N-methylated and optionally benzene- ring-substituted by halogen, NO₂, NH₂, OH, C,_.3alkyl and/or C,.₃alkoxy,

D is Lys, 4-aminocyclohexylAla or 4-aminocyclohexylGly

E is Thr, Ser, Val, Tyr, He, Leu or an aminobutyric or aminoisobutyric acid residue

G is a group of formula

wherein

R₇ is hydrogen or C,.₃alkyl,

R,₀ is hydrogen or the residue of a physiologically acceptable, physiologically hydrolysable ester, e.g. formyl, C_2.,₂alkylcarbonyl, benzoyl, R_u is hydrogen, C,.₃alkyl, phenyl or C₇.₁₀phenyl-alkyl, R_l2 is hydrogen, C,.₃alkyl or a group of formula -CH(R_I3)-X,, R₁₃ is CH₂OH, -(CH₂)₂-OH, -(CH₂)₃-OH, -CH(CH₃)OH, isobutyl, butyl, benzyl, naphthyl-methyl or indol-3-yl-methyl, and

X, is a group of formula

wherein

R₇ and R_]0 have the meanings given above,

R_l4 is hydrogen or C, ₃alkyl and

R₁₅ is hydrogen, C,.₃alkyl, phenyl or C₇.₁₀phenylalkyl, and

R,₆ is hydrogen or hydroxy,

with the proviso that when R_ι: is -CH(R₁₃)-Xj then R,, is hydrogen or methyl,

wherein the residues B, D and E have the L-configuration, and the residues in the 2- and 7-posιtιon each independently have the (L)- or (D)- configuration,

in free form 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-dys-Phe-(D)Tφ-Lys-Thr-Cys-Thr-ol also known as octreotide

b. (D)Phe-Cys-Tyr-(D)Tφ-Lys-Val-Cys-ThrNH₂

c (D)Phe-Cys-Tyr-(D)Tφ-Lys-Val-Cys-TφNH₂ also known as vapreotide

d. (D)Tφ-Cys-Phe-(D)Tφ-Lys-Thr-Cys-ThrNH₂

r e (D)Phe-Cys-Phe-(D)Tφ-Lys-Thr- ICys-ThrNH 2

f. 3-(2-(Naphthyl)-(D)Ala-Cys-Tyr-(D)Tφ-Lys-Val-iys-ThrNH₂ also known as lanreotide g- (D)Phe-Cys-Tyr-(D)Tφ-Lys-Val-Cys-β-Nal-NH₂

h. 3-(2-naphthyl)-Ala- £Cys-Tyr-(D)Tφ-Lys-Val-Cys-β-Nal-NH₂

i . (D)Phe-Cys-β-Nal-(D)Tφ-Lys-Val-Cys-Thr-NH₂

(D)Phe-Cys-Tyr-(D)Tφ-Lys-Leu-Cys-Thr-NH₂

k. (D)Phe-Cys-Tyr-(D)Tφ-Lys S--CCΛys-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, and/or an addition of polymeric organic substances.

Further somatostatin analogues suitable for use in accordance with the present invention are: cyclo [-Asn-Phe-Phe-DTφ-Lys-Thr-Phe-Gaba-], cyclo(Asu-Lys-Asn-Phe-Phe-Tφ-Lys-Thr-Tyr-Thr-Ser), and

(D)Nal-Glu-Tyr-(D)Tφ-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) -(D/L)Tφ-Lys-X₂-X₃- (II)

wherein X₂ is a radical of formula (a) or (b)

-NH-CH-CO-

CH-0-CH₂-R, (a)

I

CH,

or -NH-CH-CO- I CH, (b)

wherein R, is optionally substituted phenyl,

R₂ is -Z,-CH₂-R„ -CH₂-CO-O-CH₂-R„

wherein Z, is O or S, and

X₃ is an α-ammo acid having an aromatic residue on the C_σ side chain, or an amino acid unit selected from Dab, Dpr, Dpm, Hιs,(Bzl)HyPτo, thienyl- 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, m particular with respect to the specifically exemplified compounds, being incoφorated herein by reference.

Preferably the sequence of formula LI as defined above corresponds to the residues at positions 8 through 1 1 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 α-carbonyl group of the residue at position 6 and the α-amino group of the residue at position 1.

While Lys, X₂ and X₃ in the sequence of formula LI have the L-configuration, Tφ may have the D- or L-configuration, preferably the D-configuration.

X₂ is preferably a residue of formula (a) or (b), R₂ being preferably -Z,-CH₂-R, or

When X₃ comprises an aromatic residue on the C_α side chain, it may suitably be a natural or unnatural α-amino acid, e.g. Phe, Tyr, Tφ, Nal, Pal, benzothienyl-Ala, Tic and thyronin, preferably Phe or Nal, more preferably Phe. X₃ is preferably an α-amino acid bearing an aromatic residue on the C_α side chain.

When R, 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, is unsubstituted phenyl. Z, is preferably O.

Representative somatostatin analogues comprising a residue of formula II are e.g compounds of formula (III)

cyclo[A - ZZ_a- Tip - Lys - X - X ] (II)

1 2 3 4 5 6 wherein

X₂ and X₃ are as defined above,

A, is a divalent residue selected from Pro,

(R₃-NH-CO-O)Pro-, R₅-N-R_5a-Pro-, HO-R_5a-Prp-,

R„

R_3aR_3bN-(CH₂)₁.₆-CO-NH-Pro- , R_3aR_3bN-(CH₂)₁.₆-S-Pro-

R₃-NH-CO-O-R_D-CH(NR₄)-CO-, R_l7-CH(NR₄)-CO- and -NR_4a-CH₂-CO-

wherein R₃ is NR_gR₀-C₂.₆alkylene, guanidino-C_{2 6}alkylene or C₂.₆alkylene-COOH, R_3a is H, C_Malkyl or has independently one of the significances given for R₃ R_3bis H or C_Malkyl, R_a is OH or NR₅R₆, R_b is -(CH₂)_{1 3}- or -CH(CH₃)-, R₄ is H or CH₃, R_4a is optionally ring-substituted benzyl, each of R₅ and R₆ independently is H, C_Malkyl, o>amino-C_Malkylene, ω-hydroxy-C_Malkylene or acyl, R_5l is a direct bond or C,.₆alkylene, each of R_g and R₉ independently is H, C,.₄alkyl, ω-hydroxy-C_walkylene, acyl or CH₂OH-(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,₇ is optionally ring-substituted benzyl, -(CH₂),.₃-OH, CH₃-CH(OH)- or -(CH₂),.₅-NR₅R₆, and ZZ_a is a natural or unnatural α-amino acid unit.

ZZ_a may have the D- or L-configuration. When ZZ_a is a natural or unnatural α-amino acid unit, it may suitably be e.g. Thr, Ser, Ala, Val, He, Leu, Nle, His, Arg, Lys, Nal, Pal, Tyr, Tφ, optionally πng-substituted Phe or N^α-benzyl-Gly. When ZZ_a is Phe, the benzene ring thereof may be substituted by e.g. NH₂, NO₂, CH₃, OCH₃ or halogen, preferably in para position When ZZ_a is Phe, the benzene πng thereof is preferably unsubstituted

When A, 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

as well as in the trans form The present invention covers each geometric isomer individually as well as mixtures thereof.

When A, is (NR₈R₉-C₂.₆alkylene-NH-CO-O)Pro- where NR₈R₉ forms a heterocyclic group, such group may be aromatic or saturated and may comprise one nitrogen or one nitrogen and a second heteroatom selected from nitrogen and oxygen Preferably the heterocyclic group is e.g. pyπdyl or moφhohno. C₂.₆Alkylene in this residue is preferably -CH₂-CH₂-.

Any acyl as R₅, R₆, R_g and R₉ in A, may be e.g. R_lgCO- wherein R_lg is H, C_Malkyl, C_2u)alkenyl, C₃.₆cycloalkyl or benzyl, preferably methyl or ethyl. When R_4a or R_]7 in A, is πng-substituted benzyl, the benzene ring may be substituted as indicated above for ZZ_.

A preferred group of compounds of formula LTJ are such wherein A, is free of a lateral -NH-CO-O- moiety. A further group of preferred compounds of formula LTJ are such wherein A, comprises a basic lateral radical, e.g. a R₃-NH-CO-O- or R₅-N-R_5a- moiety

A still further group of preferred compounds of formula III are such wherein the N- terminal amino acid comprises a substituted Pro, particularly 4-substιtuted Pro, e.g. compounds of formula III wherein A, is 4-substιtuted Pro.

Preferably A, is 4-(R₃-NH-CO-O)Pro.

Examples of somatostatin analogues comprising a residue of formula II include e.g. cyclo[4-(NH₂-C₂H₄-NH-CO-0-)Pro-Phe-DTφ-Lys-Ser(Benzyl)-Phe]

The term "macrolide" as used herein, refers to a macrocyclic lactone, for example a compound having a 12-membered or larger lactone πng Of particular interest are the "lactam macrohdes", 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 lactam macrolide that is produced by Streptomyces hygroscopicus. and having the structure depicted in Formula A

See, e.g., McAlpine, J.B., et al., J. Antibiotics (1991) 44: 688; Schreiber, S.L.. et al., J. Am. Chem. Soc. (1991 ) 113: 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:

wherein

X₄ is (H,H) or O;

Y₃ is (H,OH) or O;

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)- triazolyl-alkyl and hydroxy alkoxy-alkyl; wherein "alk-" or "alkyl" refers to C,_.6alkyl, branched or linear, preferably C,.₃alkyl,; "aryl" is phenyl or tolyl; and acyl is a radical derived from a carboxylic acid; and

R₂₂ is methyl or R₂₂ and R₂₀ together form C₂.₆alkyl; provided that R₂₀ and R₂₁ are not both H; and hydroxyalkoxyalkyl is other than hydroxy alkoxy methyl .

Such compounds are disclosed in WO 94/09010 the contents of which, in particular with respect to the specifically exemplified compounds, are incoφorated herein by reference.

A preferred compound is e.g. 40-O-(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 incoφorated herein by reference. Particularly preferred are 32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-40-O-(2-hydroxyethyl)-rapamycin,

16-0-pent-2-ynyl-32-(S)-dihydro-rapamycin and 16-O-pent-2-ynyl-32-(S)-dihydro-40-O- (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 1 18 677, carbamates such as described in US 5 1 18 678, 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 sulfamates, 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 93/1 1 130, WO 94/02136, WO 94/02385 and WO 95/14023, all incoφorated 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. 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 suφrisingly 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 deπvative thereof, can be combined and synergistically inhibit cell hypeφroliferation.

In accordance with the particular findings of the present invention, there is provided in a first aspect:

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 hypeφroliferation 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 hypeφroliferation.

3 A method for preventing or treating cell hypeφroliferation 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 hypeφroliferation, 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 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 hypeφiasia, 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 % CO₂. 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 10O00 cells per well in 180 μl). After a 48-hr incubation period (Day O), the number of cells in a separate control plate is determined both by counting cells in a Coulter counter and by the sulforhodamine 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 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 (OD) 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. Biol. 1 14: 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 10^"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.

B. In Vivo Assay

The AR42J (AR4-2J) rat pancreatic tumor cell line is derived from an azaserine- induced exocrine pancreatic tumor (Jessop and Hay, 1980). It was obtained from ATCC. Cultures are propagated m DMEM supplemented with 10% fetal calf serum (FCS) at 5% CO_:. Cells are grown in the absence of antibiotics or antifungal agents. Female nude mice (nu/nu Balbc-A from Iffa Credo, Lyon, France) weighing 19-22 g, are kept in groups of 5 animals in macrolon cages (type ITJ, 16 x 22 x 1 1 cm). The cages are placed in ventilated cabinets (Iffa Credo) that are maintained at 24 ± 1 ° 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 lOxlO⁶ 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 mg/kg 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 Volume SE mm³

Control 4020 579

A) Compound B, 5 mg/kg p.o. 3685 263

B) Rapamycin, 5 mg/kg p.o. 2748 325

C) Octreotide pamoate (biodegradable, sustained release formulation),

30 mg/kg, single inj. 2205 339 Compound B + octreotide (C) 130 75

Rapamycin + octreotide (C) 106 44

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 pnmitive 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 syπnge 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. m the form of tablets, capsules, drink solutions, emulsions or microemulsion preconcentrates, nasally, pulmonary (by inhalation), 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 2,265,31 IB. 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 mg 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 μg 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 10% 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. Formulation Examples:

A. Somatostatin Formulations:

1. Ampoules

Octreotide 0.5 mg

Mannitol 45.0 mg

Lactic acid (88%) 3.4 mg

Sodium hydrogeno- carbonate to pH 4

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. Rapamvcin (or derivative thereof) formulation: e.g. capsules

Ethanol 20.0 mg

1 ,2-propylene glycol 81.0mg

Refined oil 121.5mg

Cremophor RH40 202.5mg

Rapamycin or Compound B 20.0mg

Total . 500 mg

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 hypeφroliferation 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 effective amounts with a rapamycin macrolide for the prevention or treatment of cell hypeφroliferation.

3. Use according to claim 1 or 2, wherein the compound of the somatostatin class is a compound of formula I

wherein

A is C,_.,₂alkyl, C₇.₁₀phenylalkyl or a group of formula RCO-, whereby i) R is hydrogen, C,.,, alkyl, phenyl or C₇.₁₀phenylalkyl, or ii) RCO- is

a) a D-phenylalanine residue optionally ring-substituted by halogen, NO₂, NH₂, OH, C,._?alkyl and/or C^alkoxy; or b) the residue of a natural or a synthetic α-ammo-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 α-amino group of ammo acid residues a) and b) and the N-terminal amino group of dipeptide residues c) being optionally mono- or di-C, ₁₂alkylated or substituted by Cι_₈alkanoyl;

A' is hydrogen or C,_.3alkyl,

Y, and Y₂ represent together a direct bond or each of Y, and Y₂ is hydrogen

B is -Phe- optionally ring-substituted by halogen, NO₂, NH₂, OH, C,.₃alkyl and /or C,_.3alkoxy (including pentafluoroalanine), naphthylalanine or pyridylalamne,

C is (L)-Tφ- or (D)-Tφ- optionally α-N-methylated and optionally benzene- πng-substituted by halogen, NO₂, NH₂, OH, C,.₃alkyl and/or C,.₃alkoxy,

D is Lys, 4-amιnocyclohexylAla or 4-aminocyclohexylGly

E is Thr, Ser, Val, Tyr, He, Leu or an aminobutyπc or aminoisobutyric acid residue

G is a group of formula

wherein

R₇ is hydrogen or C,.₃alkyl,

R₎₀ is hydrogen or the residue of a physiologically acceptable, physiologically hydrolysable ester, R_n is hydrogen, C, ,alkyl, phenyl or C₇.₁₀phenyl-alkyl, R_l2 is hydrogen, C,.₃alkyl or a group of formula -CH(R_I3)-X,, R_π is CH₂OH, -(CH₂)₂-OH, -(CH₂)₃-OH, -CH(CH₃)OH, isobutyl, butyl, benzyl, naphthyl-methyl or ιndol-3-yl-methyl, and

X, is a group of formula

-COOR_r -CH₂OR₎₀or -CO-N

wherein

R₇ and R₁₀ have the meanings given above,

R_l4 is hydrogen or C,.₃alkyl and

R,, is hydrogen, C,.₃alkyl, phenyl or C₇.₁₀phenylalkyl, and

R₁₆ is hydrogen or hydroxy,

with the proviso that when R₁₂ is -CH(R_π)-X, then R_M is hydrogen or methyl.

wherein the residues B, D and E have the L-configuration, and the residues in the 2- and 7-posιtιon each independently have the (L)- or (D)- configuration

or a somatostatin analogue comprising the amino acic sequence of formula II

-(D/L)Tφ-Lys-X₂-X₃- (II)

wherein X₂ is a radical of formula (a) or (b) or -NH-CH-CO-

I CH-O-CH^R, (a)

I CH₃

-NH-CH-CO- I CH, (b) wherein R, is optionally substituted phenyl,

R, is -Z_rCH₂-R„ -CH₂-CO-O-CH₂-R„

wherein Z, is O or S, and

X₃ is an α-amino acid having an aromatic residue on the C_α side chain, or an amino acid unit selected from Dab, Dpr, Dpm, His,(Bzl)HyPro, thienyl- Ala, cyclohexyl-Ala and t. -butyl-Ala, the residue Lys of said sequence corresponding to the residue Lys⁹ of the native somatostatin- 14,

in free form or in pharmaceutically acceptable salt form.

4. Use according to claim 3, wherein the compound of the somatostatin class is octreotide, lanreotide or vapreotide.

5. A method for preventing or treating cell hypeφroliferation 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.

6. A kit or package for the treatment or prevention of cell hypeφroliferation, 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 pharmaceutical composition comprising a rapamycin macrolide, together with instructions for use.

7. A kit or package according to claim 6, wherein the compound of the somatostatin class is a compound of formula I

wherein

A is C_M2alkyl, C_7.10phenylalkyl or a group of formula RCO-, whereby i) R is hydrogen, C,., , alkyl, phenyl or C₇.₁₀phenylalkyl, or ii) RCO- is

a) a D-phenylalanine residue optionally ring-substituted by halogen, NO₂, NH₂, OH, C,.₃alkyl and/or C^alkoxy; 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 α-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_M2alkylated or substituted by C,.₈alkanoyl; A' is hydrogen or C alkyl,

Y, and Y₂ represent together a direct bond or each of Y, and Y₂ is hydrogen

B is -Phe- optionally ring-substituted by halogen, NO₂, NH₂, OH, C,_.3alkyl and /or C,.₃alkoxy (including pentafluoroalanine), naphthylalanine or pyridylalanine,

C is (L)-Tφ- or (D)-Tφ- optionally α-N-methylated and optionally benzene- ring-substituted by halogen, NO₂, NH₂, OH, C,.₃alkyl and/or C,_.3alkoxy,

D is Lys, 4-aminocyclohexylAla or 4-aminocyclohexylGly

G is a group of formula

-COOR_T

wherein

R₇ is hydrogen or C,.₃alkyl,

R₁₀ is hydrogen or the residue of a physiologically acceptable, physiologically hydrolysable ester, R_n is hydrogen, C,.₃alkyl, phenyl or C₇.₁₀phenyl-alkyl, R₁₂ is hydrogen, C,.₃alkyl or a group of formula -CH(R,₃)-X,, R₁₃ is CH₂OH, -(CH₂)₂-OH, -(CH₂)₃-OH, -CH(CH₃)OH, isobutyl, butyl, benzyl, naphtyl- methyl or indol-3-yl-methyl, and

X, is a group of formula -COOR_r -CH₂OR₁₀or

wherein

R₇ and R₁₀ have the meanings given above,

R₁₄ is hydrogen or C,.₃alkyl and

R₁₅ is hydrogen, C,.₃alkyl, phenyl or C₇.₁₀phenylalkyl, and

R₁₆ is hydrogen or hydroxy,

with the proviso that when R₁₂ is -CH(R₁₃)-X, then R_n 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 (D)- configuration

or a somatostatin analogue comprising the amino acid sequence of formula II

-(D/L)Tφ-Lys-X₂-X₃- (II)

wherein X₂ is a radical of formula (a) or (b)

-NH-CH-CO- I

CH-0-CH₂ (a) I CH,

-NH-CH-CO-

CH, (b)

R₂ or wherein R, is optionally substituted phenyl,

R₂ is -Z,-CH₂-R„ -CH₂-CO-O-CH₂-R„

wherein Z, is O or S, and

X₃ is an α-ammo acid having an aromatic residue on the C_α side chain, or an amino acid unit selected from Dab, Dpr, Dpm, Hιs,(Bzl)HyPro, thienyl- Ala, cyclohexyl-Ala and t.-butyl-Ala, the residue Lys of said sequence corresponding to the residue Lys⁹ of the native somatostatin- 14,

in free form or in pharmaceutically acceptable salt form.

8. A kit or package according to claim 7, wherein the compound of the somatostatin class is octreotide, lanreotide or vapreotide.

9. A kit or package according to claim 6 for simultaneous, separate or sequential use in synergistically effective amounts.