ZA200104530B - Controlled release formulation comprising GNRH-II. - Google Patents

Description

py A WO 00/32218 PCT/GB99/04045

CONTROLLED RELEASE FORMULATION COMPRISING GNRH-II

FIELD OF INVENTION -

The present invention relates to a pharmaceutical preparation that releases a therapeutic agent over an extended period.

BACKGROUND TO THE INVENTION

Studies on the physiology of the hypothalamic-pituitary-gonadal axis have resulted in the recognition of gonadotropin releasing hormone (GnRH. otherwise known as luteinizing hormone releasing hormone, LHRH) as a key regulatory hormone. GnRH is released by the hypothalamus and acts on the pituitary to stimulate the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). More recently, a peptide with homology to GnRH has been identified (White et al, Proc. Natl. Acad.

Sci. USA 95 305-309, 1998). This peptide has been called GnRH-Il. The sequences of the two peptides are compared below.

GnRH pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH, (SEQ 1.D. No.5)

GnRH-Il pyroGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH, (SEQ 1.D. No.6)

The name "GnRH-II" is, to some extent, misleading. The new peptide is a separate gene product, and is clearly distinguishable from GnRH in its tissue distribution. It seems unlikely that GnRH-I! acts as an endogenous releaser of LH and FSH. Since no clear evidence for a physiological role for GnRH-Il has been presented, no attentionhas been paid to the practical aspects of using this peptide as a therapeutic agent.

SUMMARY OF THE INVENTION

We have now found that GnRH-Ii has an important role in the function of a number of organs. For example, it influences osteogenesis and it modulates the proliferation of prostatic epithelial cells. Accordingly, we have considered the means by which this agent and its analogues might usefully be delivered in a clinical situation. and it is an object of the present invention to provide suitable formulations for achieving this purpose. The formulations according to the present invention rely on the use ofa

SUBSTITUTE SHEET (RULE 26)

biodegradable polymer to hold the peptide in a depot. from which it is released into the ) or systemic circulation at a controlled rate. These formulations comprise two key elements, the biologically active peptide and the biodegradable polymer. The biologically active peptide is a decapeptide according to the sequence pyroGlu-His-Trp-Ser-Xaa'-Gly-Xaa*-Xaa’-Pro-Gly-NH, (SEQ [.D. No.7) wherein Xaa' is His or Tyr,

Xaa?is Trp or Leu, and

Xaa®is Tyr or Arg, provided that when Xaa' is Tyr and Xaa? is Leu, then Xaa® is not Arg. The polymer is any pharmaceutically acceptable biodegradable polymer, and preferably a co- polymer of glycolic and lactic acids. The invention further comprises the use of the formulations for the treament of human pathologies.

DESCRIPTION OF THE FIGURE

Figure 1 shows the effect of increasing doses of GnRH-il on serum calcium concentrations in ovariectomised rats.

DESCRIPTION OF THE INVENTION

As used herein, abbreviations referring to amino acids have their conventional meanings and indicate the natural L-isomer (except for the achiral amino acid glycine).

In a first aspect, the invention as disclosed herein comprises a pharmaceutical formulation that releases a therapeutic peptide at a controlled rate and for an extended period of time (i.e. for a period of at least one day, preferably several days, and more preferably at least one week), particularly for the treatment of diseases of the bone and prostate. The therapeutic peptide is a decapeptide according to the sequence pyroGlu-His-Trp-Ser-Xaa'-Gly-Xaa?’-Xaa’-Pro-Gly-NH, (7)

SUBSTITUTE SHEET (RULE 26)

‘ A ~ wooos228 PCT/GBO9/04045 ) wherein Xaa' is either His or Tyr, Xaa® is either Trp or Leu. and Xaa® is either Tyr or

Arg, provided that when Xaa' is Tyr and Xaa® is Leu. then Xaa® is not Arg.

Preferably, Xaa' is His, Xaa?is Trp, and Xaa®’ is Tyr. It willbe recognised that such a peptide can form salts with acids (for example, acetic acid, trifluoroacetic acid. benzoic acid, hydrochloric acid, phosphoric acid and the like). To the extent that such salts are formed with pharmaceutically acceptable acids. they are included within the scope of the invention.

A second essential component of the formulation is a biodegradable, pharmaeutically acceptable polymer. Such polymers are known in the art. They can either be homopolymers (i.e. polymers of a single monomer) or copolymers (i.e. formed from two or more different monomers). Suitable monomers include amino and hydroxy dervatives of carboxylic acids. In a preferred embodiment of the present invention, the polymer is composed of hydraxyacyl monomeric units, and mare preferably of a- hydroxyacy! units. Most preferably, the polymer is a poly(glycolic acid), a poly(lactic acid) or a copolymer of glycolic and lactic acids. Such a polymer has the foliowing chemical structure.

R oO R i

Ye YY

Oo R ©) R where R is hydrogen in poly(glycolic acid), methyl in poly(lactic acid), and randomly hydrogen or methyl in the copolymer.

Two complementary methods for making the formulation of the present invention can be distinguished. The peptide can either be incorporated into a matrix of the polymer, or, more preferably, it can be encapsulated by the polymer. In this second case, the peptide that is encapsulated may be either a solid or in solution. It is preferred for the peptide to be a solid.

This formulation is useful in the treatment of human pathologies, including disorders of bone growth (including age-related osteoporosis and osteoporosis associated with post-menopausal hormone status, primary and secondary hyperparathyroidism, disuse osteoporosis. diabetes-related osteoporosis, and glucocorticoid-related

SUBSTITUTE SHEET (RULE 26)

osteoporosis) and prostate growth (including benign prosiatic hyperplasia and } prostate cancer).

In a second aspect. the invention as disclosed herein comprises a method for the treatment of an individual suffering from a disorder of bone or prostate growth, or considered to be at risk of so suffering. This method of treatment comprises the administration to said individual of a therapeutically effective amount of a formulation containing, as an active principal, a peptide according to the sequence pyroGlu-His-Trp-Ser-Xaa'-Gly-Xaa?-Xaa*-Pro-Gly-NH, (7) or a pharmaceutically acceptable salt thereof, wherein Xaa®. Xaa® and Xaa® are as defined above, and as a second component, a pharmaceutically acceptable biodegradable polymer, which formulation releases the peptide into the systemic circulation as the polymer is eroded. The method of treatment may comprise a single administration of the formulation, but is more likely to comprise a course of repeated administrations. The frequency of the administrations may be from once per day to once per month. The amount of active peptide in each dose will depend on the dosing schedule and the route of administration. Generally, it will be between one milligram (1mg) and one gram (1g). The supervising physician will determine the precise dose depending on the parameters generally considered in the art to be relevant. The formulation is administered by intramuscular or subcutaneous injection.

The peptides that comprise the active agents of the compositions of the present invention can be prepared by the methods generally known in the art. For example, the peptides may be preparéd by Solid-phase synthesis. THIS IRVOIves the sequential addition of amino acid residues to a resin-bound intermediate according to the following strategy. 1. Formation of resin-bound first intermediate

PG-Aaa-OH + FG-Res - PG-Aaa-L-Res

Aaa = amino acid

PG = protecting group

FG = functional group

Res = polymeric resin 4

SUBSTITUTE SHEET (RULE 26)

a Wo eeaR PCT/GBYY/04045 ) L = linker group ( -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 steps 2 and 3 as necessary

PG-Bbb-Aaa-L-Res - - - PG-Nnn-..-Bbb-Aaa-L-Res 5. Cleave/deprotect

PG-Nnn-..-Bbb-Aaa-L-Res ~ H-Nnn-..-Bbb-Aaa-OH (or -NH,)

In step one, a protected amino acid is reacted with a functionalised resin. The protecting group (PG) is most commonly tert-butyloxycarbonyl (Boc) or 9- fluorenylmethyloxycarbony! (Fmoc). The functional group on the resin (FG) is commonly a chioroalky! group, a hydroxyl group or an amine group. When FG is a chloroalkyi or hydroxy! group, the linker group (L) is an oxygen atom (-O-). When

FG is an amine group, L is -NH-. in step two, the protecting group (PG) is removed from the o-amino group. When

PG is Boc, this can be accomplished by treating the resin with acids such as trifluoroacetic acid or hydrogen chioride in dichloromethane. When PG is Fmoc, the deprotection can be accomplished by treating the resin with bases such as piperidine.

In step three, the peptide chain is extended by one amino acid residue. A protected amino acid is coupled to the amine group liberated in step two. Many reagents are known in the art for achieving this conversion. One combination is dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBt). Generally, a base will also be necessary. Suitable bases include triethylamine and N.N- diisopropylethylamine. The solvent will generally be dichloromethane, dimethylformamide, or a mixture of these.

SUBSTITUTE SHEET (RULE 26)

If the side chains of the amino acids (Aaa - Nnn) contain reactive groups (for . example amino groups. carboxylic acid groups, hydroxyl groups) then these will need protecting. The protecting groups chosen for the side chains are generally those that are stable under the conditions required to remove the protecting group (PG) from the o-amino group. If PG is Fmoc, then the side chain protecting groups can conveniently be based on tert-butyl chemistry. On the other hand, if PG is Boc, then the side chain protecting groups can be based on fluorenylmethyl chemistry. Other protecting groups known in the art can also be used.

In step four, the deprotection/chain extension cycle is repeated untii the desired peptide sequence has been constructed.

In step five, the completed peptide is liberated from the resin. Protecting groups are removed from the side chains either before or after the cleavage. When L is -NH-, the peptide liberated is in the form of the C-terminal amide. When L is -O-, the peptide liberated is often the C-terminal free acid and a second step is required to form the C-terminal amide.

The peptides may also be prepared by solution-phase synthesis, and this may be more convenient when large quantities of material are needed.

The polymers required for the formulation are generally well known in the art. As stated previously, the formulation may take the form of a simple dispersion of the peptide in a matrix of the polymer, or the peptide may be microencapsulated with the polymer. Dispersions can be prepared by mixing the peptide (as a solid) and the polymer to homogeneity, then compressing the mixture to form a solid mass. It may be necessary to add a binding agent to the mixture in order to ACHIEVE 4 SUEY — cohesive composition. The mass can then be ground up to give particles suitable for suspension in a biologically compatible liquid (such as water or isctonic saline) and injection.

Microencapsulated formulations can be prepared either from the solid peptide (as a powder) or from a solution, and particularly an aqueous solution, of the peptide. The polymer is first 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. A second organic solvent is then added. This second solvent is 6

SUBSTITUTE SHEET (RULE 26)

"3 WO 00/32218 PCT/GBI9/04045 } chosen to reduce the solubility of the polymer in the organic phase. The polymer comes out of solution to form a coating around the particies of solid peptide (or around the droplets of dispersed aqueous solution). The resultant microcapsules are then hardened by washing to remove traces of the organic solvents. They are then ready to be suspended in an appropriate liquid for administration.

The above general description is further elaborated below in a number of examples.

These are intended to illustrate certain aspects of the invention. They are not intended to be limiting in any way.

EXAMPLES

Example 1 - Synthesis of GnRH-lI 1A. Preparation of resin-bound protected peptide. 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 methods starting from Boc-

Gly-esterified Merrifield resin (60 g, 1 mmol/g). The synthesis was performed in a manual synthesizer, with a total solvent and reagent volume of 300 mL for each operation. The standard deprotection/wash/coupling protocol is summarised in

Table 1.

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Table 1 | } ™

Step Reagent Time (min) Number of

Deprotection of Boc HCI/DCM* 60 | 1 * Gaseous hydrogen chloride was bubbled through a suspension of the resin in DCM > Completeness of reaction was determined by a negative ninhydrin

Benzotriazolyl esters were used as the activated esters throughout the synthesis.

These were prepared from the corresponding protected amino acids by reaction with 1-hydroxybenzotriazole (1 eq.) and dicyclohexyicarbodiimide (1 eq.). The quantities used (in relation to the resin substitution capacity) are listed in Table 2.

Table 2

Cycle Amino acid derivative Molar

Fa lll A r 8 I + Jecwmpon 20

EE = E ET 8

SUBSTITUTE SHEET (RULE 26)

x WO 00/32218 PCT/GR99/04045 , Following the final coupling. the resin was washed with dichlorcmethane (3 x 3 L) and dried under reduced pressure at +40°C to constant weight.

Amino acid analysis: Consistent with proposed sequence 1B. Cleavage and deprotection pyroGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH, (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 to a final pressure of 4 atm. After 72h the excess ammonia was vented and the resin was extracted with acetic acid (3x100mL) and ethanol (3x100mL). The combined extracts were degassed with nitrogen, 10% palladium-on-carbon was added, and the mixture was stirred under an atmosphere of hydrogen. When the reaction was complete (as 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 peptide

Copoly(D,L-lactic acid, glycolic acid) with a lactic acid/glycolic acid ratio of 50/50 is used. To a solution of this polymer (3.7g) in dichloromethane (100mL) in a reaction vessel equipped with a stirrer is added GnRH-1l acetate (0.15g, prepared by dissolving the peptide of example 1 in acetic acid and lyophilising the resultant solution). The mixture is stirred at 500revolutions/minute, then silicone oil (Dow Corning 360 Medical

Fluid®, 45g) is added over 10 minutes. The mixture is then introduced as a thin jet into caprylic-capric acid-triglyceride (Miglyol® 812, 3.3L) with continuous stirring at 1000revolutions/minute. When addition is complete, stirring is continued for 1 hour, then the microcapsules are collected by filtration, washed twice with isopropanol, and finally dried.

Example 3 - Analysis of the effects of GnRH-Il and analogues on Osteogenic cell populations in vitro. (a) Human osteoblasts were isolated from cancerous bone from orthopaedic surgery (Nilsson et al., 1995) according to standard procedures known in the art. The bone 9

SUBSTITUTE SHEET (RULE 26)

explants were minced into small bene chips and then washed extensively in N

Dulbecco's modified Eagle's medium (DMEM)/F12 (1:1 Gibco, Paisley, U.K).

These osteoblast like cells, Murine osteoblastic MC3T3-E1 cells and human clonal osteosarcoma cell lines MG-63 (non-mineralising) and Sa0S-2 (mineralising osteosarcoma) were cultured in DMEM:F12, 1:1 with the addition of 10% fetal calf serum (FCS, Gibco), fungizone (500mg/l), gentamycin sulphate (50mg/l). L- glutamine (2mM) and i-ascorbic acid (100mg/l) in a humidified CO, chamber at 37°C. (b) Human bone marrow stromal ceils were isolated from bone fragments rinsed in phosphate-buffered saline. Bone marrow cells were collected and spun through a column of Ficoll Hypaque (Kimble et al J. Clin. Invest. 93 1859-1967, 1994) Cells at the interface were pelleted, counted and seeded into 75cm? flasks. The cells were incubated in a humidified CO, chamber at 37°C and the medium changed weekly. At confluence, the cells were harvested using trypsin EDTA and re-seeded in a-minimum essential medium (ax-MEM) supplemented with 10% fetal calf serum (FCS, Gibco), penicillin (100U/ml), streptomycin (100mg/ml), fungizone and L- glutamine (2mM). (c) All cells were serum-starved for 48h before addition of GnRH-I and GnRH-II. Cells were placed in DMEM without phenol red (in order to avoid oestrogen-like effects of phenol red) containing 10% charcoal-stripped serum for 48 hours in 12 well plates.

Dose dependent effects of GnRH-I and GnRH-Il and analogues of the peptides were studied following the addition of peptides at final concentrations ranging from 10° to 10°M. 1mM dibutyryl cAMP was used as a control. The cells were oo incubated for 24, 48 and 96h with the peptide being replaced every 24 Rours. (d) TO assess the effects of the peptides on cell proliferation, [PH}thymidine was added at 1mCi/ml for an additional 24hours and [°H]thymidine incorporation was determined. Radioisotope incorporation was determined using a scintillation counter and the results were calculated as cpm/mg of total protein. (e) Expression of osteoblastic differentiation markers was also determined (Tintut Y et al. J Biol Chem 273 7547-53, 1998). Total RNA was isolated at several stages

SUBSTITUTE SHEET (RULE 26)

Claims (7)

yi * CLAIMS

1. A pharmaceutical formulation for the controllec release cf a therapeutic peptide or a sait theraaf. which peptide has the sequence : pyraGiu-His-Trp-Ser-Xaa'-Gly-Xaa*-Xaa®-Pro-Gly-NH. wherein ~~ Xaa'is His or Tyr, Xaalis Trp or Lau, and Xaa®is Tyr or Arg, provided that when Xaa' is Tyr and Xaa? is Leu, then Xaa' is not Arg, and which formulation further comprises a pharmaceutically acceptable biodegradable polymer. .

2. The pharmaceutical campasition according to Claim 1, wherein the peptide is pyroGiu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH,

3. The formulation according to Claim 1, wherein the polymer is a polymer of a ) hydroxy derivative of a carboxylic acid, or a copolymer of such derivatives.

4. The formulation according to Claim 3, wherein the poiymer is a polymer of glycolic acid, a polymer of lactic acid, or a copalymer of lactic and glycalic acids.

5. The formulation accarding te Claim 1 wherein the peptide is microencapsulated by the polymer.

6. Use of a controlled release formulation of a peptide according to any of the preceding Claims, for the treatment of a human medical condition, which method comprises the administration to an individual in need of such treatment.

7. A formulation according to any of claims 1 to 5S for treatment of or for protection against disorder of bane growth or disorder of prostate growth, - FURTHER AMENDED SHEET