KR20010089538A - Controlled release formulation comprising gnrh-ii - Google Patents

Abstract

The present invention relates to a pharmaceutical formulation for controlled release of a therapeutic peptide or salt thereof, wherein the peptide has the following sequence:

pyroGlu-His-Trp-Ser- Xaa ¹ -Gly- Xaa ² - Xaa ³ -Pro-Gly-NH ₂

(In the sequence, Xaa ¹ is His or Tyr, Xaa ² is Trp or Leu, Xaa ³ is a case of a Tyr or Arg, stage, Xaa ¹ is Tyr and Xaa ² is Leu Xaa ³ is not an Arg),

The formulation further comprises a pharmaceutically acceptable biodegradable polymer. This formulation can be used for the treatment of bone and prostate disorders.

Description

CONTROLLED RELEASE FORMULATION COMPRISING GNRH-II}

The study of the physiology of the hypothalamic-pituitary-gonadal axis led to the recognition of one key regulatory hormone, the gonadotropin free hormone (GnRH, also known as the luteinizing hormone free hormone (LHRH)). GnRH is released by the hypothalamus and acts on the pituitary gland to stimulate the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). More recently, peptides homologous to GnRH have been identified (White et al . , Proc. Natl. Acad. Sci. USA 95 305-309, 1998). This peptide was called GnRH-II. The sequence of the two peptides is as follows:

GnRH pyroGlu-His-Trp-Ser- Tyr -Gly- Leu - Arg -Pro-Gly-NH ₂ (SEQ ID No. 5)

GnRH-II pyroGlu-His-Trp-Ser- His -Gly- Trp - Tyr -Pro-Gly-NH ₂ (SEQ ID No.6)

The name "GnRH-II" introduces some confusion. The new peptide is a separate gene product and is clearly distinguished from GnRH in tissue distribution. It is unlikely that GnRH-II acts as an endogenous releaser of LH and FSH. Since no clear evidence has been provided for the physiological role of GnRH-II, no one has ever paid attention to the practical aspects of using this peptide as a therapeutic.

The present invention relates to pharmaceutical formulations which release the therapeutic agent over a long period of time.

1 is a graph showing the effect of increasing GnRH-II dose on serum calcium concentration in ovarian excised mice.

We have found that GnRH-II plays an important role in the function of many organs. GnRH-II, for example, affects bone formation and regulates proliferation of prostate epithelial cells. The present inventors therefore studied a means by which this material and its analogs can be usefully delivered to the medical field, and an object of the present invention is to provide a formulation suitable for achieving this object. The formulation according to the invention utilizes a biodegradable polymer that holds the peptide in a depot, wherein the peptide is released from the reservoir into the systemic circulation at a controlled rate. These formulations comprise two key elements: biologically active peptides and biodegradable polymers. Biologically active peptides are decapeptides according to the following sequence:

pyroGlu-His-Trp-Ser- Xaa ¹ -Gly- Xaa ² - Xaa ³ -Pro-Gly-NH ₂ (SEQ ID No.7)

Wherein Xaa ¹ is His or Tyr, Xaa ² is Trp or Leu, Xaa ³ is Tyr or Arg,

However, when Xaa ¹ is Tyr and Xaa ² is Leu, Xaa ³ is not Arg. The polymer is any pharmaceutically acceptable biodegradable polymer and is preferably a copolymer of glycolic acid and lactic acid. The present invention also encompasses the use of such formulations for the treatment of human disease.

The abbreviations which refer to amino acids herein have the usual meaning and refer to the natural L-isomers (except the achiral amino acid glycine).

In a first aspect the present invention provides a pharmaceutical formulation for releasing a therapeutic peptide over an extended period of time (ie, at least one day, preferably several days, more preferably at least one week) at a controlled rate as described herein. And, in particular, formulations used for the treatment of bone and prostate related diseases. The therapeutic peptide is a decapeptide according to the following sequence:

pyroGlu-His-Trp-Ser- Xaa ¹ -Gly- Xaa ² - Xaa ³ -Pro-Gly-NH ₂ (7)

Wherein, Xaa ¹ is His or Tyr, Xaa ² is Trp or Leu, Xaa ³ is Tyr or Arg, and when, stage, Xaa ¹ is Tyr and Xaa ² is Leu Xaa ³ is not Arg. Preferably, Xaa ¹ is His, Xaa ² is Trp and Xaa ³ is Tyr. It will be appreciated that such peptides can be reacted with acids (eg, acetic acid, trifluoroacetic acid, benzoic acid, hydrochloric acid, phosphoric acid, etc.) to form salts. Such salts are included within the scope of the present invention in that such salts are formed by reaction with pharmaceutically acceptable acids.

An essential second element of the formulations of the invention is a biodegradable and pharmaceutically acceptable polymer. Such polymers are known to those skilled in the art. They can be either homopolymers (ie polymers of single monomers) and copolymers (ie formed from two or more different monomers). Suitable monomers include amino derivatives and hydroxy derivatives of carboxylic acids. In a preferred embodiment of the invention, the polymer consists of hydroxyacyl monomer units, more preferably α-hydroxyacyl units. Most preferably the polymer is poly (glycolic acid), poly (lactic acid) or a copolymer of glycolic acid and lactic acid. Such polymers have the following chemical structure:

Wherein R is hydrogen in poly (glycolic acid), methyl group in poly (lactic acid), and randomly hydrogen or methyl group in the copolymer.

Methods of making the formulations of the invention can be divided into two complementary methods. Peptides may be bound into a matrix of a polymer, or more preferably encapsulated by a polymer. In this second case, the encapsulated peptide may be solid or in solution. It is preferred that the peptide is a solid.

Formulations of the invention include bone growth disorders such as osteoporosis associated with age, osteoporosis associated with postmenopausal hormonal status, primary and secondary parathyroidism, disuse osteoporosis, diabetes related osteoporosis, and glucocorticoid related osteoporosis, and the like. Useful for the treatment of human diseases including prostate growth (including benign prostate hyperplasia and prostate cancer).

In a second aspect the invention described herein includes a method of treating a person suffering from a disorder of bone or prostate growth or a person determined to be at risk of such a disease. This method of treatment consists of the following sequences as active active ingredients in such a person:

pyroGlu-His-Trp-Ser- Xaa ¹ -Gly- Xaa ² - Xaa ³ -Pro-Gly-NH ₂ (7)

A therapeutically effective amount of a peptide or a pharmaceutically acceptable salt thereof as defined above, wherein Xaa ¹ , Xaa ² , Xaa ³ is as defined above, and a second component, containing a pharmaceutically acceptable polymer. Administering a formulation, wherein the formulation releases the peptide into the systemic circulation as the polymer corrodes. The method of treatment may involve administering the formulation once, but usually involves a repeated administration procedure. The frequency of administration can be from once a day to once a month. The amount of active peptide included in each dose will depend on the schedule of administration and the route of administration. Generally the amount will be between 1 milligram (1 mg) and 1 gram (1 g). The precise dosage will be determined by the practitioner according to variables generally considered relevant in the art. The formulations are administered by intramuscular injection or subcutaneous injection.

Peptides comprising the active agents of the compositions of the invention can be prepared by methods generally known to those skilled in the art. For example, the peptide may be prepared by solid phase synthesis. This involves sequentially adding amino acid residues to the resin binding intermediate according to the following method:

1. Formation of Resin Bonded First Intermediate

PG-Aaa-OH + FG-Res → PG-Aaa-L-Res

Aaa = amino acid

PG = protector

FG = functional group

Res = polymer resin

L = connector (-O- or -NH-)

2. Deprotection

PG-Aaa-L-Res → H-Aaa-L-Res

3. Chain extension

PG-Bbb-OH + H-Aaa-L-Res → PG-Bbb-Aaa-L-Res

4. Repeat Step 2 and Step 3 as needed.

PG-Bbb-Aaa-L-Res →→→ PG-Nnn -...- Bbb-Aaa-L-Res

5. Cleave / Protector Removal

PG-Nnn -...- Bbb-Aaa-L-Res → -H-Nnn -...- Bbb-Aaa-OH (or -NH ₂ )

In step 1, the protected amino acid is reacted with the functional group attachment resin. The protecting group (PG) is most commonly tert-butyloxycarbonyl (Boc) or 9-fluorenylmethyloxycarbonyl (Fmoc). The functional group (FG) attached to the resin is usually a chloroalkyl group, a hydroxy group or an amine group. When FG is a chloroalkyl group or a hydroxy group, the linking group (L) is an oxygen atom (-O-). When FG is an amine group, L is -NH-.

In step 2, the protecting group PG is removed from the α-amino group. If the PG is Boc, removal of the protecting group can be achieved by treating the resin with an acid such as hydrogen chloride in trifluoroacetic acid or dichloromethane. When PG is Fmoc, protecting group removal can be achieved by treating the resin with a base such as piperidine.

In step 3, the peptide chain is extended by one amino acid residue. The protected amino acid binds to the amine group released in step 2. Reagents for achieving this conversion are known in the art. One combination is dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBt). In general, a base is also required. Suitable bases include triethylamine and N, N-diisopropylethylamine. The solvent is generally dichloromethane, dimethylformamide, or mixtures thereof.

If the side chains (Aaa-Nnn) of amino acids contain reactive groups (eg amino groups, carboxylic acid groups, hydroxyl groups), it will be necessary to protect these side chains. The groups chosen for the protection of the side chains are generally those which are stable under the conditions necessary to remove the protecting group (PG) from the α-amino group. If the PG is Fmoc, the side chain protecting groups may conveniently be based on tert-butyl chemistry. Whereas when PG is Boc, the side chain protecting group may be based on fluorenylmethyl chemistry. Other protecting groups known in the art can also be used.

In step 4, the protecting group removal / chain extension cycle is repeated until the desired peptide sequence is constructed.

In step 5, the completed peptide is released from the resin. The protecting group is removed from the side chain before or after cleavage. If L is -NH-, the free peptide is usually a C-terminal free acid, so step 2 is necessary to form a C-terminal amide.

Peptides can also be prepared by solution-phase synthesis, which can be more convenient when large amounts of material are required.

The polymers required for the formulation are generally well known to those skilled in the art. As mentioned above, the formulation may take the form of simply dispersing the peptide in a polymer matrix, or the peptide may be microencapsulated into a polymer. Dispersions can be prepared by mixing the peptide (as a solid) and the polymer to homogenize and then compressing the mixture to form a solid mass. It may be necessary to add the binder to the mixture to obtain a suitable tacky composition. The solid mass can then be finely divided to obtain particles suitable for suspension and injection in biological compatibility solutions (eg water or isotonic saline).

Microencapsulated formulations can be prepared from solid peptides (as powders) or solutions of the peptides, in particular aqueous solutions. First, the polymer is dissolved in a suitable organic solvent. The peptide is then added to this solution and the mixture is vigorously stirred to disperse the peptide in the organic phase. Next, a second organic solvent is added. This second solvent is chosen to lower the solubility of the polymer in the organic phase. The polymer is released from the solution to form a film surrounding the solid peptide particles (or surrounding the droplets of the dispersed aqueous solution). The microcapsules obtained are then washed and hardened by removing trace organic solvents.

The general description as described above will be described in more detail with reference to several examples below. These examples are intended to illustrate aspects of the invention and are not intended to limit the invention.

Example 1 Synthesis of GnRH-II

1A. Preparation of Peptides Protected by Resin Bonds

pyroGlu-His (Bom) -Trp (CHO) -Ser (Bzl) -His (Bom) -Gly-Trp (CHO) -Tyr (Bzl) -Pro-Gly-Ores

This peptide was prepared using standard solid phase method using Boc-Gly-esterified Merrifield resin (60 g, 1 mmol / g) as starting material. Synthesis was carried out in a manual synthesizer using a total volume of 300 ml of solvent and reactants for each operation. Standard protector removal / cleaning / coupling protocols are summarized in Table 1.

TABLE 1

step Reactant Minutes Operation count Boc Protector Removal HCl / DCM ^* 60 One wash DCM 2 to 4 3 Chinese 10% DIPEA / DCM 4 2 wash DCM 2 to 4 One Coupling Activated ester 60-120 ^** 1 to 2 wash DCM 2 to 4 3 Gas phase hydrogen chloride was aerated through a suspension of resin in DCM. Completion of the reaction was determined by a negative ninhydrin test.

Throughout the synthesis, benzotriazolyl esters were used as activating esters. This ester was prepared by reacting the corresponding protective amino acid with 1-hydroxybenzotriazole (1 equiv) and dicyclohexylcarbodiimide (1 equiv). The amount used (in terms of resin replacement capacity) is listed in Table 2.

TABLE 2

Cycle no. Amino acid derivatives Excess mall One Boc-Pro-OH 1.8 2 Boc-Tyr (Bzl) -OH 1.8 3 Boc-Trp (CHO) -OH 1.8 4 Boc-Gly-OH 1.8 5 Boc-His (Bom) -OH 1.8 6 Boc-Ser (Bzl) -OH 2.0 7 Boc-Trp (CHO) -OH 2.0 8 Boc-His (Bom) -OH 2.0 9 pyroGlu-OH 2.0

Following the final coupling, the resin was washed with dichloromethane (3 × 3 L) and dried at + 40 ° C. under reduced pressure until no weight loss.

Amino Acid Analysis: consistent with proposed sequence.

1B. Cutting and Protector Removal

pyroGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH ₂ (6)

The peptidoresin prepared in Example 1A was placed in a linen bag in a pressure vessel. The vessel was then charged with gaseous ammonia until the final pressure reached 4 atm. After 72 hours excess ammonia was drained and the resin was extracted with acetic acid (3 x 100 ml) and ethanol (3 x 100 ml). The extracts were combined and degassed with nitrogen, 10% palladium supported carbon was added, and the mixture was stirred in a hydrogen atmosphere. When the reaction was complete (judged by HPLC) the mixture was filtered and the filtrate was evaporated. The residue was purified by reverse-phase HPLC to give the title compound.

Example 2 Microencapsulation of Peptides

Copoly (D, L-lactic acid, glycolic acid) is used with 50/50 ratio lactic acid / glycolic acid. To a solution of the polymer dissolved in dichloromethane (100 ml) in a reaction vessel equipped with a stirrer, GnRH-II acetate (0.15 g, prepared by dissolving the peptide of Example 1 in acetic acid and lyophilizing the resulting solution) is added. . The mixture is stirred at a speed of 500 rpm and then silicone oil (Dow Corning 360 Medical Fluid ^(R) , 45 g) is added over 10 minutes. The mixture is then introduced into caprylic acid-capric acid-triglyceride (Miglyol ^(R) 812, 3.3 L) in a thin jet while continuing stirring at a speed of 1000 rpm. After the addition is complete, stirring is continued for 1 hour, followed by filtration to collect microcapsules, washed twice with isopropanol and finally dried.

Example 3 In Vitro Analysis of the Effect of GnRH-II and Analogs on Osteoblastic Cell Populations

(a) Human osteoblasts were isolated from cancerous bone from orthopedic surgery (Nilsson et al., 1995) according to standard procedures known to those skilled in the art. The bone explants were chopped into small bone fragments and washed extensively in Dulbecco's modified Eagle's medium (DMEM) / F12 (1: 1 Gibco, Paisley, UK). These osteoblast-like cells, ie Murine osteoblastic MC3T3-E1 cells and human clonal osteosarcoma cell lines MG-63 (non-mineralized) and SaOS-2 (mineralized osteosarcoma), were treated with 10% fetal calf serum (FCS, Gibco), fun 37 ° C. in a humidified CO ₂ chamber in DMEM: F12, 1: 1 with fat (500 mg / l), gentamicin sulfate (50 mg / l), L-glutamine (2 mM) and l-ascorbic acid (100 mg / l) Incubated at the temperature of.

(b) Human bone marrow stromal cells were isolated from washed bone fragments in phosphate buffered saline. Bone marrow cells were collected and spun through a column of Ficoll Hypaque (Kimble et al. , J. Clin. Invest. 93 1959-1967, 1994). Cells at the interface were pelleted, counted and seeded into 75 cm 2 plisks. Cells were cultured in medium humidified CO ₂ chamber at 37 ° C. with weekly media exchange. Harvest cells using trypsin EDTA at confluence and supplement with 10% fetal calf serum (FCS, Gibco), penicillin (100 U / ml), streptomycin (100 mg / ml), punzione and L-glutamine (2 mM) -Inoculated in minimum essential medium (α-MEM).

(c) All cells were serum-starved for 48 hours prior to addition of GnRH-I and GnRH-II. Cells were maintained for 48 hours in 12 well plates in DMEM containing 10% activated carbon stripped serum without phenol red (to avoid the estrogen-like effect of phenol red). Dose dependent effects of GnRH-I, GnRH-II and peptide analogs were studied after addition of peptides in the final concentration range 10 ^-9 to 10 ^-6 M. 1 mM dibutyryl cAMP was used as a control. The cells were incubated for 24 hours, 48 hours and 96 hours, respectively, with peptide replacement every 24 hours.

(d) To assess the effect of the peptide on cell proliferation, [ ³ H] thymidine was added at 1 mCi / ml over an additional 24 hours and [ ³ H] thymidine incorporation was measured. Radioisotope incorporation was measured using a scintillation counter and the results calculated in cpm / mg total protein.

(e) The expression of osteoblast differentiation markers was also measured (Tintut Y. et al., J Biol Chem 273 7547-53, 1998). Total RNA was isolated in several steps before treatment at 24, 48, 72 and 96 hours after peptide addition. Expression of type I procollagen, osteopontin and 28S RNA (used as internal control) was measured by Northern blot assay. Alkaline phosphatase, matrix GLA protein, osteoblastin and GAPDH (as internal control) were measured by RT-PCR using specific primers designed for each gene.

The peptide of the present invention had a pronounced effect at concentrations of 100 μM or less.

Example 4- In vitro analysis of the effects of GnRH-II and analogs on the osteoclast population

(a) Human clonal cell line of osteoclast precursor (FLG 29.1) was used as an in vitro model for osteoclast differentiation (Gattei V et al., Cell Growth Differ 7 753-63, 1996). In addition, cocultures of FLG 29.1 and osteoblasts (Saos-2) were evaluated for mobility change, adhesion change, cytochemical change, morphological change, and biochemical change. Dose-dependent effects of GnRH-I, GnRH-II and peptide analogs were studied after adding peptides to FLG 29.1 cultures and cocultures with final concentrations ranging from 10 ⁻⁹ M to 10 ⁻⁶ M. Parathyroid hormone was added as a control. Synergy (or inhibition) of preosteoclast discrimination (melting into large multinucleated elements) and many other factors were measured (Orlandini et al., Cell Tissue Res . 281 33-42, 1995). This includes:

1. Positive staining for tartrate resistant acid phosphatase in FLG 29.1 cells

2. Reduced alkaline phosphatase activity expressed by Saos-2 cells

3. Development of General Microstructure of Mature Osteoclasts in FLG 29.1 Cells.

4. Release of granulocyte-macrophage colony stimulating factors into the medium

5. To assess the effect of the peptide on cell proliferation, as described above, [ ³ H] thymidine was added at 1 mCi / ml over an additional 24 hours and [ ³ H] thymidine incorporation was measured.

(b) Bone marrow cells detached from human bone fragments were cultured in the presence of 10 nM 1.25- (OH) ₂ vitamin D ₃ for 7 days to generate a multinucleated osteoclast using standard techniques known to those skilled in the art (Takahashi et al., Endocrinol 122 1473 -1482, 1988). Remove the medium (α-MEM) and replace with medium supplemented with 10% activated carbon strips and inactivated heat treated FCS and antibiotic as a fresh medium free of phenol red and supplemented with 10% activated carbon strips and cultures It was kept for an additional 24 hours. Suspended cells were harvested and stained for osteoclasts for tartrate resistant acid phosphatase (TRAP) expression as a differential marker for osteoclasts (Hughes et al . , Nat. Med. 2 1132-1135, 1996).

1.Put cells in 0.2M acetate buffer and pH 2 to 2% naphthol AS-BI phosphate (dissolved in ethylene glycol monomethyl ether at a concentration of 20 mg / ml) with pH 4.7-5.0 and 15 minutes at 37 ° C. Incubated. The cells were then subjected to 37 ° C. with a second solution consisting of the same buffer and the same concentration of tartaric acid and 0.1% pararosanoline chloride (equal volume of 4% sodium nitrite and hexazotised by mixing for 5 minutes at room temperature). Over 15 minutes. By this treatment, cells expressing TRAP stain the cytoplasm red. Harris hematoxylin was used as nuclear counterstain.

2. Apoptotic multinucleated osteoclasts were identified by strong expression of TRAP larger than the accompanying viable TRAP-positive cells. Death was confirmed using acridine orange staining. Viable osteoclasts were counted after fixation in 95% ethanol and stained with TRAP hematoxylin, and apoptotic osteoclasts were in percentage of the total number of multinucleated osteoclasts (viable and apoptosis) in each culture well. Indicated.

The peptide of the present invention had a pronounced effect at concentrations of 100 μM or less.

Example 5 Expression Analysis of GnRH mRNA in Osteoblastic and Osteoclast Populations

Total RNA was extracted from the cultured cells as described previously:

1. Osteoblast-like Cells Isolated from Cancerous Bone

2. Rodent Osteoblast-like MC3T3-E1 Cells

3.MG-63 (non-mineralized)

4. SaOS-2 (mineralized osteosarcoma)

5. Human Bone Marrow Stromal Cells

6. Human FLG 29.1 osteoclast precursor cells

7. Multinucleated Osteoclasts Produced from Bone Marrow

Expression of GnRH-I and GnRH-II is shown in SEQ I.D. It was measured by RT-PCR using the PCR primers shown in No 1-4. The total amount of cDNA generated was determined by assessing the relative levels of actin amplification.

Example 6 Effect of GnRH-II on Bone Mineral Density in Ovariectomized Rats

(a) Left and right ovaries of Sprague Dawley rats, grown females (8 weeks, 200-215 g), were extracted (OVX). The animals were stored for 4 weeks after delivery before starting treatment. Purina rat chow (1.00% calcium, 0.61% phosphorus) and water were fed frequently. Each study consisted of six groups (n = 8 / group) by weight.

(b) Treatment started 4 weeks after OVX. After 4 weeks, the OVX group of the reference control group was sacrificed (Group A). The remaining groups were vehicle (group B), GnRH-II 1 μg / weight 1 kg (group C), GnRH-II 10 μg / weight 1 kg (group D), GnRH-II 100 μg / weight 1 kg (group E) , And hPTH (1-34) 80 μg / kg body weight (group F) once daily.

(c) Body weights of all mice were measured four times a day and the dose was adjusted for an average weight gain of 50 g in the group. Rats were injected subcutaneously with alternating calcane (30 mg / kg) and tetracycline (30 mg / kg) dissolved in 2% sodium bicarbonate-saline and mineralized on days 10, 19 and 26 following drug treatment, respectively. Labeled on the surface. Bone mineral density was assessed by dual energy x-ray absorptometry (DEXA). On day 28, serum calcium levels were measured by colorimetric analysis using a commercial kit.

(d) The autopsy failed to detect ovarian tissue and significant atrophy of the uterine keratin was observed, confirming the successful OVX. Both legs were disarticulate at the buttocks. Excess muscle and flexible tissue were removed from the left tibia and femur and placed in 70% ethanol. The anterior ridge of the right tibia metaphyseal was cut with a razor to barely expose the bone marrow. Both the right femur and tibia were immersed in 10% phosphate buffered formalin for 24 hours and then transferred to 70% ethanol.

Ovariectomized animals showed significant hypercalcemia after 28 days of treatment with 10 μg and 100 μg GnRH-II per kg body weight and 80 μg PTH per kg body weight daily. The result is shown in FIG.

Example 7 Cellular Localization of GnRH-II in Paraffin Fragments of Normal Rat and Human Bone

(a) Frozen and / or paraffin-embedded human and rat bone sections were fixed for 3 to 36 hours (3 to 5 hours at room temperature, then about 24 hours at 4 ° C.) depending on size and decalcification It was immersed in 0.1M Tris + 5% EDTA (12.11g + 50g EDTA) until pH 7.3.

(b) The sections were then subjected to antibody staining (rabbit polyclonal anti-GnRH-II antibody) using standard techniques.

Staining for GnRH-II was observed in megakaryocytes (especially proliferating chondrocytes) in platelets and growth plates. Some staining also appeared in osteogenic cells, especially active osteoblasts and new immature bone.

Example 1 sets forth a method for preparing the peptides of the invention, which may then be formulated as illustrated in Example 2. Examples 3-7 show the biological activity of the peptide of interest. The scope of the present invention is not limited to these Examples. In particular, it will be appreciated that various controlled release formulations of these peptides may be prepared by changing the physical properties of the polymer and / or combination of peptide and polymer. However, these changes are considered to be within the scope of the present invention as defined in the claims that follow, as these formulations provide formulations with equivalent biological properties.

SEQ I.D., referenced in Example 5 Nos. 1 to 4 are as follows:

cTG CAG CTG CCT GAA GGA C (1)

GGG CGG GGC GGG GCT CTC G (2)

ATT CTA CTG ACT TGG TGC GTG (3)

GGA ATA TGT GCA ACT TGG TGT (4)

Claims (6)

A pharmaceutical formulation for controlled release of a therapeutic peptide or salt thereof, The peptide has the following sequence: pyroGlu-His-Trp-Ser- Xaa ¹ -Gly- Xaa ² - Xaa ³ -Pro-Gly-NH ₂ (In this sequence, Xaa ¹ is His or Tyr, Xaa ² is Trp or Leu, Xaa ³ is Tyr or Arg, Provided that when Xaa ¹ is Tyr and Xaa ² is Leu, then Xaa ³ is not Arg), Wherein said formulation further comprises a pharmaceutically acceptable biodegradable polymer. The method of claim 1, The peptide pyroGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH ₂ Pharmaceutical formulation. The method of claim 1, A pharmaceutical formulation wherein the polymer is a polymer of a hydroxy derivative of carboxylic acid or a copolymer of a derivative thereof. The method of claim 3, A pharmaceutical formulation wherein the polymer is a polymer of glycolic acid, a polymer of lactic acid, or a copolymer of lactic acid and glycolic acid. The method of claim 1, A pharmaceutical formulation in which the peptide is microencapsulated by the polymer. In the method of treating a human medical condition, A method of treatment comprising administering to the individual in need thereof a therapeutically effective amount of a controlled release formulation of a peptide according to any one of claims 1 to 5.