EP1189939A1 - Bh3 modified peptides - Google Patents

Abstract

The present invention is directed to a peptide of formula (I), as defined herein, or a pharmaceutically acceptable salt thereof, pharmaceutical compositions comprising said peptide of formula (I), and the use thereof to restore apoptosis in human tumor cells. A compound of formula (I) of the present invention is R-A?1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-NHR1¿ or a pharmaceutically acceptable salt thereof wherein, R is (C¿1?-C5)acyl; A?1¿ is absent or an aliphatic amino acid; A2 is absent or is selected from the group consisting of Gly, Ala, Ser, Thr and Asn; A?3 and A4¿ are each absent or are each a coded amino acid; A5 is an aliphatic amino acid; A6 is Ala or a basic amino acid; A7 is selected from the group consisting of Ile, Leu, Val, Ser, Thr, Lys and Arg; A8 is an aliphatic amino acid; A9 is Gly; A?10 and A11¿ are each independently Asp or Glu; A12 is an aliphatic amino acid Ile, Leu, Met and Val; A13 is Asp, Glu or Asn; A?14 and A15¿ are each independently absent or selected from the group consisting of Ile, Leu, Val, Met, Ser, Thr, Asn, Lys and Arg; and R1 is (C¿1?-C5)alkyl.

Description

BH3 MODIFIED PEPTIDES

Background of the Invention The present invention is directed to a peptide of formula (I), as defined herein, or a pharmaceutically acceptable salt thereof, pharmaceutical compositions comprising said peptide of formula (I), and the use thereof to restore apoptosis in human tumor cells.

The proliferation rate of a cell population reflects a balance between cell division, cell cycle arrest, differentiation, and programmed cell death or apoptosis (Rudin, CM. and Thompson, Annu. Rev. Med., 48:267-81 , 1997). The regulation of these processes is central to development and tissue homeostasis, whereas dysregulation may lead to overt pathological outcomes, notably cancer and neurodegenerative disorders (Spengler, D., et al., EMBO J., 16: 2814-2825, 1997). Apoptosis comprises an intrinsic cellular defense against tumorigenesis, which, when suppressed, may contribute to the development of malignancies (Reed, J.C., Cancer J. Sci. Am., 4 Suppl 1 :S8-14, 1998. The Bcl-2 oncogene product functions as a potent suppressor of apoptosis under diverse conditions (Kroemer, G. (published erratum appears in Nat Med 1997 Aug; 3(8):934), Nat.Med., 3: 614-620, 1997). Bcl-2 inhibits apoptosis induced by a wide variety of stimuli. The Bcl-2 protein is found to be over-expressed in many types of human tumors. Protein-protein interaction between members of the Bcl-2 family of proteins seems to be a key event in the regulation of apoptosis .

The family of Bcl2-related proteins is constituted by survival proteins such as Bcl2, and Bcl-xL (Antonawich, F.J., et al., J. Cereb. Blood Flow Metab., 18: 882- 886, 1998), and death-promoting proteins such as Bad, Bak, Bax, Bip1 , Bik, Bcl-xS etc. (Boyd, J.M., et al., Oncogene, 11 : 1921-1928, 1995; Jurgensmeier, J.M., et al., Proc. Natl. Acad. Sci. U.S.A., 95: 4997-5002, 1998; and Chittenden, T., et al., Nature, 374: 733-736, 1995). Using complementary DNA cloning and functional analysis, Bak was identified to promote cell death, and counteract the protection from apoptosis provided by Bcl-2. Moreover, enforced expression of Bak induces rapid and extensive apoptosis of serum- deprived fibroblasts (Chittenden, T., et al. EMBO J., 14: 5589-5596, 1995). Mutations in Bak that disrupt either type of interaction inhibit the ability of Bak to heterodimerize with Bcl-xL (Sattler, M., Science, 275: 983-986, 1997).

Bax antagonizes Bcl-2's death protecting function. Bcl-2 can formhomodimers with itself and heterodimers with Bax and it has been shown that point mutations in Bcl-2 can abrogate Bax binding while leaving homodimerization intact (Diaz, J. L, et al., J. Biol. Chem., 272: 11350-11355, 1997). The results from mutagenesis studies have led to the proposal that Bcl-2 has separate binding sites that are responsible for homodimer and heterodimer formation. Results from yeast two-hybrid studies have also suggested that homodimerization and heterodimerization reflect distinct modes of interaction. Similar assays demonstrate that Bcl-xL can form both homodimers and heterodimers and that these interactions are also inhibited by Bax . These results demonstrate that the same binding motifs are responsible for both homodimerization and heterodimerization between Bcl-2 family members.

One domain in Bak, termed BH-3, was identified to be both necessary and sufficient for cytotoxic activity and binding to Bcl-xL (Chittenden, T., et al., EMBO J., 14: 5589-5596, 1995). Sequences similar to this domain were identified in Bax and Bip1 , two other proteins that promote apoptosis and interact with Bcl-xL, and were likewise critical for their capacity to kill cells and bind Bcl-xL.

Thus, the BH3 domains of pro-apoptotic proteins are sufficient to trigger apoptosis accompanied by the release of cytochrome C from mitochondria and caspase activation. The ability of synthetic peptides to reproduce the effect of pro- apoptotic BH3 domains suggests that such peptides may be useful in the diagnosis and treatment of proliferative disease, and may provide the basis for engineering reagents to control the initiation of apoptosis.

Summary of the Invention The present invention is directed to a peptide of formula (I) which disrupt the physical interaction between pro-apoptotic and anti-apoptotic proteins leading to induction of apoptosis in human tumoral prostate cells. Thus, in one aspect, the present invention is directed to a compound of the formula (I):

R-A¹-A²-A³-A⁴-A⁵-A⁶-A⁷-A⁸-A⁹-A¹⁰-A¹¹-A¹²-A¹³-A¹⁴-A ⁵-NHR¹ or a pharmaceutically acceptable salt thereof wherein, R is (C C₅) acyl;

A¹ is absent or an aliphatic amino acid;

A² is absent or is selected from the group consisting of Gly, Ala, Ser, Thr and Asn;

A³ and A⁴ are each absent or are each a coded amino acid ;

A⁵ is an aliphatic amino acid; A⁶ is Ala or a basic amino acid;

A⁷ is selected from the group consisting of lie, Leu, Val, Ser, Thr, Lys and Arg;

A⁸ is an aliphatic amino acid;

A⁹ is Gly;

A¹⁰ and A¹¹ are each independently Asp or Glu; A¹² is an aliphatic amino acid lie, Leu Met and Val;

A¹³ is Asp, Glu or Asn;

A¹⁴ and A¹⁵ are each independently absent or selected from the group consisting of lie, Leu, Val, Met, Ser, Thr, Asn, Lys and Arg; and

R¹ is (d-Cs) alkyl. A preferred group of compounds of formula (I), are those compounds that have

A¹ is absent or lie, Leu, Met or Val;

A² is absent or is Gly, Ala, Ser, Thr and Asn;

A⁵ is lie, Leu, Met or Val; A⁸ is lie, Leu, Met and Val;

A⁹ is Gly;

A¹² is lie, Leu Met and Val; and

A¹³ is Asp, Glu or Asn, and the other variables of formula (I) are as defined above. A preferred group of compounds of formula(l), are those compounds wherein A⁶ is Ala, Lys or Arg and the other variables of formula (I) are as defined above.

Especially preferred compounds of formula (I) are Ac-Leu-Ser-Glu-Cys-Leu-Lys-Arg-lle-Gly-Asp-Glu-Leu-Asp-Ser-Asn-NH₂, and Ac-Leu-Ser-Glu-Ser-Leu-Lys-Arg-lle-Gly-Asp-Glu-Leu-Asp-Ser-Asn-NH₂.

The standard three letter abbreviations known in the art are used to represent the amino acids in formula (I). For example, Leu = leucine. Detailed Description

A peptide of formula (I) can be synthesized by any standard solid phase peptide synthesis. See, e.g., Stewart, J.M., et al., Solid Phase Synthesis (Pierce Chemical Co., 2d ed. 1984). The substituent R of a peptide of formula (I) can be attached to the free amine of the N-terminal amino acid by standard methods known in the art. For example, acyl groups, may be attached by coupling the corresponding free acid of the acyl group, to the free amine of the N-terminal amino acid by mixing the completed resin with 3 molar equivalents of both the free acid and diisopropylcarbodiimide in methylene chloride for about one hour.

The following describes a synthetic method for making a peptide of this invention, which method is well-known to those skilled in the art. Other methods are also known to those skilled in the art.

Peptides of the present invention can be and were synthesized on Rink Amide MBHA resin, (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)- phenoxyacetamido-norleucyl-MBHA resin), using a standard solid phase protocol for FMOC chemistry and cleaved from the resin with a TFA/Phenol/H₂0/triisopropylsilane (83ml/5g/10ml/2ml) mixture. Peptides were cyclized in CH₃CN/H₂0 (5ml/5ml) using EKATHIOX™ resin (EKAGEN Corporation, San Carlos, CA) and purified on C₁₈ silica (Rainin Instruments Co., Wobum, MA now Varian Analytical, Walnut Creek, CA), using acetonitrile/0.1 % trifluoroacetic acid buffers. Homogeneity was assessed by analytical HPLC and were determined to be >95% for each peptide. Peptides were characterized by HPLC and mass spectrometry.

Using the appropriate FMOC protected amino acid in a method of the foregoing process, the following compounds were made. Example 1 : Ac-Leu-Ser-Glu-Cvs-Leu-Lvs-Arg-lle-Gly-Asp-Glu-Leu-Asp-Ser-Asn-

HPLC Conditions

Column- Zorbax 300SB-C8, 5u, 4.6x250mm (Hewlett Packard) Eluants- A= 0.1 % Trifluoroacetic acid (TFA) in water

B= 20/80/0.1 Water/Acetonitrile/TFA Elution conditions:

20 to 80%B in 30 min. Flow Rate - 1.0 ml/min. Detection - UV at 220nm Observed retention time ~ 15.0 min. MS

Finnigan SSQ-7000 equipped with an electrospray (ESI) source Operating parameters

Sheath gas - 30 psi

Scan range/rate - 200 to 2200 amu in 4 sec. Capillary temp - 220 C ESI voltage - 4.5 kV Mode - infusion

Expected exact mass = 1731.8 amu Observed mass = 1732.1 amu

Example 2: Ac-Leu-Ser-Glu-Ser-Leu-Lvs-Arq-lle-Glv-Asp-Glu-Leu-Asp-Ser-Asn- NH HPLC Conditions

Column- Zorbax 300SB-C8, 5u, 4.6x250mm (Hewlett Packard)

Eluants- A= 0.1% Trifluoroacetic acid (TFA) in water

B= 20/80/0.1 Water/Acetonitrile/TFA Elution conditions: 20 to 80%B in 30 min.

Flow Rate - 1.0 ml/min. Detection - UV at 220nm Observed retention time - 12.4 min. MS Finnigan SSQ-7000 equipped with an electrospray (ESI) source Operating parameters

Sheath gas - 30 psi

Scan range/rate - 200 to 2200 amu in 4 sec. Capillary temp - 220 C ESI voltage - 4.5 kV

Mode - infusion

Expected exact mass = 1715.9 amu Observed mass = 1716.1 amu

The peptides of this invention can be provided in the form of pharmaceutically acceptable salts. Examples of such salts include, but are not limited to, those formed with organic acids (e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, methanesulfonic, toluenesulfonic, or pamoic acid), inorganic acids (e.g., hydrochloric acid, sulfuric acid, or phosphoric acid), and polymeric acids (e.g., tannic acid, carboxymethyl cellulose, polylactic, polyglycolic, or copolymers of poly lactic-g lycol ic acids). A typical method of making a salt of a peptide of the present invention is well known in the art and can be accomplished by standard methods of salt exchange. Accordingly, the TFA salt of a peptide of the present invention (the TFA salt results from the purification of the peptide by using preparative HPLC, eluting with TFA containing buffer solutions) can be converted into another salt, such as an acetate salt by dissolving the peptide in a small amount of 0.25 N acetic acid aqueous solution. The resulting solution is applied to a semi-prep HPLC column (Zorbax, 300 SB, C-8). The column is eluted with (1) 0.1 N ammonium acetate aqueous solution for 0.5 hrs., (2) 0.25N acetic acid aqueous solution for 0.5 hrs. and (3) a linear gradient (20% to 100% of solution B over 30 min.) at a flow rate of 4 ml/min (solution A is 0.25N acetic acid aqueous solution; solution B is 0.25N acetic acid in acetonitrile/water, 80:20). The fractions containing the peptide are collected and lyophilized to dryness. The ability of a peptide of formula (I) to induce apoptosis and mimic the effect of pro-apoptotic BH3 domains indicates that such peptides provide the basis for the development of assay procedures for the discovery of novel structures which initiate apoptosis in transformed cells.

In addition, a peptide of formula (I) can be used in a method for the biochemical and molecular characterization of tumor cells obtained from cancer patients. Such methods can help in the design of the best curative treatment for each patient. Examples of such methods, are a method for the analysis of relevant molecular determinants of tumor cell growth such as oncogenes and growth factor receptors, among others, and a method for the analysis of tumor cell apoptosis, such as p53 gene status and Bcl-2 family proteins, among others.

However, for BcI2 family members, the activity of these proteins (pro- or anti- apoptosis) is linked to their status of binding with other members by BH3 domains. Pro-apoptotic proteins, such as Bax and Bak, bind to the anti-apoptotic Bcl-2 via these domains, leading to the inactivation of the normal ability of Bcl-2 to suppress apoptosis. Bcl2 protein may be said to prevent apoptosis by neutralizing the pro-apoptotic members. Thus, competitive dimerization between family members is thought to regulate the apoptosis rate. For tumor characterization under such conditions, it would be important to evaluate not only the "total" amount of BcI2, but also the amount of Bcl2 protein which is free, and which is bound to pro-apoptotic proteins. In the case when BcI2 is not associated with other members (i.e., Bak or Bax), a peptide of formula (I) could interact with the "free" Bcl2 protein. Thus in another aspect, the present invention provides a method of using a peptide of formula (I) to evaluate the levels of free Bcl2 or bound Bcl2 in tumor cells from biopsies. For this utility, it may be advantageous for the BH3 analogs to be coupled to a species which allows improved cell permeation.

Similarly, the use of a labeled BH3 analog (radioactive, coupled to colorimetric system, coupled to fluorescence system) on tumor section, could define the state of the interactions and get a more relevant analysis than the measure of the amounts of Bcl2 as presently done. This methodology could assist in both diagnostic procedures, identification of treatment protocol, and in determination of prognosis, by identifying the potential for restoration of apoptosis. Restoration of apoptosis in human tumoral cells should be a way to block tumorigenesis. To achieve this goal, electroporation assisted delivery, allowing the cell penetration of a peptide of formula (I) has been employed . Cells were harvested during the exponential growth phase by trypsination and resuspended at a concentration of 0.625 10⁶ cells/ ml of Phosphate buffered saline. A 0.8 ml aliquot was taken from the cell suspension and mixed with the compounds or the vehicle (DMSO), allowed to stand for 10 minutes and added to a disposable 0.4 cm electroporation cuvette (Bioraad). Electroporation was carried out in a Gene Pulser (Biorad) with cells exposed to one pulse. The capacitance of 25μF and the voltage of 0.6kV were the preferred settings for electroporation.

Cell survival was analysed by flow cytometry using the propidium iodide uptake method. The uptake is only observed with dead cells. Table I shows that the percentage of PC3 cell survival levels decreased following electroporation in the presence of 50μg/ml of Example 2. Similar results were obtained with prostate cancer cell line DU145 (Table II).

To confirm that a compound of formula (I) is specifically mediated by preventing the molecular interaction between pro-apoptotic proteins and anti- apoptotic proteins, immunoprecipitation of protein complexes were performed.

The Bak/Bcl2 complex is detected in the prostatic cell line PC3. However, when cells were previously pretreated with Example 2 (with electroporation), the Bak/Bcl2 complex is not detectable. The results are a clear indication that a peptide of formula (I) prevents the formation of Bak/Bcl2 complex. One of the downstream effects of apoptosis induction is the activation of the caspase family of proteases, transforming an inactive pro-caspase to an active caspase by internal proteolytic process. As shown in Table III, Example 2 did not induce apoptosis in the presence of caspase inhibitor z-VAD.fmk, which supports the mechanism that the apoptotic effect was via caspase activation. A peptide of formula (I) of the instant application may be linked to or administered with a carrier which allows a peptide of formula (I) to cross the cell membrane. A compound of the instant application can be linked to carriers such as biotin (Chen, L.L., et al., Anal. Biochem., 227: 168-175, 1995), Tat (Fawell, S., et al., Proc. Natl. Acad. Sci. U.S.A., 91: 664-668, 1994) penetratin (Derossi, D., et al., Trends Cell Biol., 8: 84-87, 1998), growth factors for receptor which are able to be internalized (e.g. EGF-R) (Deshpande, D., et al., Pharm. Res., 13: 57-61 , 1996). Chitosan, which is a high molecular weight cationic polysaccharide has been reported to enhance the absorption of peptides such as insulin across the nasal epithelium has also to be mentioned (Felt, O., et al., Drug Dev. Ind. Pharm., 24: 979-993, 1998).

A peptide of formula (I) can be tested for its pro-apoptotic according to the following method. Material and methods: Cell lines: The cell lines DU145 (HTB-81 ) and PC3 (CRL-1435) were established from human prostatic tumors and were issued from American Tumor Tissue Collection (Maryland, USA).

Cells were cultured as recommended by the seller. Detection of apoptosis by FITC-labelled Annexin V binding:

Following the treatment of PC-3 and DU145 cells with a peptide of formula (I), apoptosis levels were assessed by flow cytometry for up to 48 hours on a Becton Dickinson FACScan by propidium iodide (PI) (Sigma, Ireland) uptake and annexin V binding (Bender Medsystem, Ireland). In this assay, phosphotidylserine translocation from the inner to the outer leaflet of the plasma membrane, an early feature of apoptotic cells, was measured by its ability to bind annexin V. Control and treated cells were washed once with PBS by centrifugation at 400 x g, resuspended in HEPES buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCI, 5 mM KCI, 12 mM MgCI₂ and 1.8 mM CaCI₂), incubated with FITC-conjugated annexin V (Fluorescein iothiocyanate) (1 μg/ml) (Bender MedSystems, Ireland) and propidium iodide (10 μg/ml) (Sigma Ireland) for about 5 min at room temperature and immediately analyzed by flow cytometry to quantitate annexin V binding. A minimum of 10,000 events were collected for each sample.

Immunoprecipitation and Western Blotting: Following treatment with peptides, cells were trypsinized and washed once with PBS. 0.5x10⁶ PC-3 cells were used for each sample. Cells were lysed in 500 μl of cold NP-40 isotonic lysis buffer (150 mM NaCI, 1.0% NP-40 Non ionic detergent 50 mM Tris at pH 8.0) containing phenylmethanesulfonyl fluoride 0.1 mM PMSF, sodium orthovanadate (1 mM), aprotinin (1 μg/ml), pepstatin (1 μg/ml), antipain (1 μg/ml), chymoststin (1 μg/ml) and leupeptin (0.1 μg/ml), incubated on ice with shaking for 40 min, passed through a 25G needle (x5) and centrifuged at 14,000 x g for 15 min. For immunoprecipitation of Bcl-2, a monoclonal anti-Bcl-2 antibody (6 μg) coupled to agarose beads (Santa Cruz, Ireland) was added to 300 μg of protein from each sample, PBS was added to make the final volume up to 1 ml, mixed and incubated on ice for 90 min. Immunoprecipitates were collected by centrifugation at 14,000 x g for 5 min and washed three times with 1 ml of NP-40 lysis buffer. For immunoprecipitation of Bak and Bax from tumor cells, lysates were pre-cleared with 30 μl of goat anti-mouse IgG agarose (Sigma, Ireland) for about 30 min, which was removed by centrifugation at 14,000 x g for 10 mins. Specific antibodies anti- Bak (2.5 μg/ml) (Calbiochem, San Diego, CA) or anti-Bax (8 μg/ml)) Immunotech, (Coulter, Miami, FL) were added to each sample and incubated on ice with shaking for 90 min. Immune complexes were captured by adding 80 μl of goat anti-mouse IgG agarose to each sample and left to shake at 4°C for 90 min. Samples were centrifuged at 14,000 x g for 15 min and washed three times with 1 ml each of cold lysis buffer.

For western blot analysis either immunoprecipitates (divided into equal portions) or direct cell lysates were separated on 10% minigels (Bio-Rad, Ireland) and transferred electrophoretically to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk in PBS solution [Gibco, Ireland for 1 h. The membranes were then probed overnight with relevant primary antibodies, washed and probed with species-specific secondary antibodies coupled to horseradish peroxidase (1:1000) for 1 h, followed by washing three times in PBS for 10 min. Immunoreactive material was detected by an enhanced chemiluminescence (ECL) system (Amersham) with exposure to x-ray film.

Table I

PC3 CELL SURVIVAL %

Time after electroporation

(hours)

Example 2 0 3 6

No 41.9 79.7 77.1 electroporation

Electroporation 30.9 16.7 5.6

The effects of electroporation of Example 2 (50μg/ml in DMSO) on cell PC3 survival levels of cells as assessed by Propidium uptake over time using flux cytometry. Table II

DU145 CELL SURVIVAL

%

Time after electroporation

(hours)

Example 2 0 3 6 !

No 50.5 76.9 68.4 electroporation

Electroporation 46.0 7.9 3.8

The effects of electroporation of Example 2 (50μg/ml in DMSO) on cell DU145 survival levels of cells as assessed by Propidium uptake over time using flux cytometry.

Table III

Apoptosis %

DU145 PC3

Control 2.6 11.1

Example 2 (50μg/ml) 19.1 21

Example 2 + Zvad.fmk 6.9 9.7

(50μM)

Effects of z-VAD.fmk on the levels of apoptosis in human prostate cell lines DU145 and PC3 following electroporation with Example 2. Cells were electroporated without or with the inhibitor of caspases z-VAD.fmk (50μM) and apoptosis was measured 10 hours after by assessment of uptake of FITC-annexin V and propidium iodine. Accordingly, the present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, at least one of the peptides of formula (I) in association with a pharmaceutically acceptable carrier.

In general an effective dosage for the activities of this invention, for example the treatment of acromegaly, is in the range of 0.01 to 200 mg/kg/day, preferably 0.5 to 100 mg/kg/day.

A peptide of this invention can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual or topical routes of administration and can be formulated with pharmaceutically acceptable carriers to provide dosage forms appropriate for each route of administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch. Such dosage forms can also comprise, as is normal practice, additional substances other than such inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, the elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents. Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene giycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.

Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as coca butter or a suppository wax.

Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.

Further, a compound of this invention can be administered in a sustained release composition such as those described in the following patents and patent applications. U.S. Patent No. 5,672,659 teaches sustained release compositions comprising a bioactive agent and a polyester. U.S. Patent No. 5,595,760 teaches sustained release compositions comprising a bioactive agent in a gelable form. U.S. Patent No. 5,821 ,221 , teaches polymeric sustained release compositions comprising a bioactive agent and chitosan. U.S. Application No. 08/740,778 filed November 1 , 1996, teaches sustained release compositions comprising a bioactive agent and cyclodextrin. U.S. Application No. 09/015,394 filed January 29, 1998, teaches absorbable sustained release compositions of a bioactive agent. U.S. Application No. 09/121 ,653 filed July 23, 1998, teaches a process for making microparticies comprising a therapeutic agent such as a peptide in an oil-in-water process. U.S. Application No.09/131 ,472 filed August 10, 1998, teaches complexes comprising a therapeutic agent such as a peptide and a phosphorylated polymer. U.S. Application No. 09/184,413 filed November 2, 1998, teaches complexes comprising a therapeutic agent such as a peptide and a polymer bearing a non- polymerizable lactone. The teachings of the foregoing patents and applications are incorporated herein by reference.

The dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment. Generally, dosage levels of between 0.0001 to 100 mg/kg of body weight daily are administered to humans and other animals, e.g., mammals.

A preferred dosage range is 0.01 to 5.0 mg/kg of body weight daily which can be administered as a single dose or divided into multiple doses. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference.

Claims

CLAIMSWhat is claimed is:

1. A compound of the formula (I): R-A¹-A²-A³-A⁴-A⁵-A⁶-A⁷-A⁸-A⁹-A¹⁰-A¹¹-A¹²-A¹³-A¹⁴-A¹⁵-NHR¹ or a pharmaceutically acceptable salt thereof wherein,

R is (C C₅) acyl;

A¹ is absent or an aliphatic amino acid;

A² is absent or is selected from the group consisting of Gly, Ala, Ser, Thr and Asn;

A³ and A⁴ are each absent or are each a coded amino acid ; A⁵ is an aliphatic amino acid;

A⁶ is Ala or a basic amino acid;

A⁷ is selected from the group consisting of lie, Leu, Val, Ser, Thr, Lys and Arg;

A⁸ is an aliphatic amino acid;

A⁹ is Gly; A¹⁰ and A¹¹ are each independently Asp or Glu;

A¹² is an aliphatic amino acid lie, Leu Met and Val;

A¹³ is Asp, Glu or Asn;

A¹⁴ and A¹⁵ are each independently absent or selected from the group consisting of lie, Leu, Val, Met, Ser, Thr, Asn, Lys and Arg; and R¹ is (Cι-C₅) alkyl.

2. A compound according to claim 1 : wherein

A¹ is absent or lie, Leu, Met or Val;

A² is absent or is Gly, Ala, Ser, Thr and Asn; A⁵ is lie, Leu, Met or Val;

A⁸ is lie, Leu, Met and Val;

A⁹ is Gly;

A¹² is lie, Leu Met and Val; and

A¹³ is Asp, Glu or Asn.

3. A compound according to claim 2 wherein A⁶ is Ala, Lys or Arg.

4. A compound according to claim 3 wherein said compound is

Ac-Leu-Ser-Glu-Cys-Leu-Lys-Arg-lle-Gly-Asp-Glu-Leu-Asp-Ser-Asn-NH₂ or

Ac-Leu-Ser-Glu-Ser-Leu-Lys-Arg-lle-Gly-Asp-Glu-Leu-Asp-Ser-Asn-NH₂.