WO2000030674A1

WO2000030674A1 - Neuropeptide y y4 agents in the treatment of reproductive disorders

Info

Publication number: WO2000030674A1
Application number: PCT/GB1999/003963
Authority: WO
Inventors: Pierre Broqua; Karen Akinsanya; Amanda Hayward
Original assignee: Ferring Bv
Priority date: 1998-11-26
Filing date: 1999-11-26
Publication date: 2000-06-02

Abstract

A pharmaceutical composition for the treatment of human reproductive disorders which comprises an effective amount of an NPY-Y4 rerceptor ligand.

Description

NEUROPEPTIDE Y Y4 AGENTS IN THE TREATMENT OF REPRODUCΗVE DISORDERS

FIELD OF INVENTION

The present invention relates to pharmaceutical agents that can be used in the treatment of disorders of the human reproductive system.

BACKGROUND TO THE INVENTION

Studies on the physiology of the hypothalamic-pituitary-gonadal (H-P-G) axis have established the importance of gonadotropin releasing hormone (GnRH, otherwise known as luteinizing hormone releasing hormone, LHRH), follicle stimulating hormone (FSH), luteinizing hormone (LH) and steroids (such as oestradiol and testosterone) in the regulation of the function of the reproductive system. More recently, it has become clear that other control mechanisms exist, either acting upstream of the H-P-G axis or directly on relevant target organs. These mechanisms might be involved in the timing of the onset of puberty, in the maintenance of fertility and, in the female, in the timing of the onset of menopause. Clearly, agents that can modulate these mechanisms are of potential interest in the treatment of disorders such as precocious or delayed puberty, reduced fertility and early-onset menopause.

One particularly important hormone appears to be leptin (ob protein). Originally identified as a regulator of body fat content, leptin has been shown to have a significant role in the onset of puberty. Mice lacking the ability to produce leptin fail to reach sexual maturity, and the same appears to be true of humans. Low leptin levels have been suggested as a causative factor in the amenorrheic status of female athletes. In some respects, however, the leptin system is not an ideal target for therapeutic intervention in human reproductive disorders. Because leptin appears to control several other physiological processes, leptin-modifying agents might be expected to have effects beyond the reproductive system, which effects might be detrimental to the health of the patient. If the downstream signals that link leptin to the H-P-G axis could be identified, the potential for identifying a selective therapeutic agent would be improved.

There is now clear evidence that leptin modulates the release of neuropeptide Y (NPY), and it is known that the administration of NPY to animals has an effect on their reproductive status. However, since NPY appears to have a number of physiological actions, this finding does not necessarily advance the search for a selective agent. The requirement is for an agent that acts only to mimic (or counteract) the effects of NPY on the H-P-G axis.

NPY is one of a family of neuropeptides. Other members of this family include peptide YY (PYY) and pancreatic polypeptide (PP). The sequences of these three peptides in humans (h) and of PP in rats (r) are listed below.

5 10 15 20 hNPY Tyr Pro Ser Lys Pro AspAsn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr hPYY Tyr Pro lie Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr hPP Ala Pro Leu Glu Pro Val Tyr Pro Gly AspAsn Ala Thr Pro Glu Gin Met Ala Gin Tyr rPP Ala Pro Leu Glu Pro Met Tyr Pro Gly Asp Tyr Ala Thr His Glu Gin Arg Ala Gin Tyr

25 30 35

HNPY Tyr Ser Ala Leu Arg His Tyr He Asn Leu Leu Thr Arg Pro Arg Tyr NH₂ SEQUENCE

ID1 HPYY Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gin Arg Tyr NH₂ SEQ UENCE

ID 2 HPP Ala Ala Asp Leu Arg Arg Tyr lie Asn Met Leu Thr Arg Pro Arg Tyr NH₂ SEQ UENCE

ID 3 RPP Glu Thr Gin Leu Arg Arg Tyr He Asn Thr Leu Thr Arg Pro Arg Tyr NH₂ SEQ UENCE

ID 4

At the cellular level, NPY exerts its effects by interacting with a membrane-bound receptor that relays a signal to cytoplasmic second messengers. A number of NPY receptors have been identified. Together, they form a family within the superfamily of heptahelical receptors, also called the seven-transmembrane domain receptors. At least five such receptors (designated NPY-Y1 to NPY-Y5) have been characterised at the DNA level and the proteins have been expressed in transformed cells. A number of NPY analogues have been described, and their affinities for the various NPY receptors have been reported. Crucially, the particular receptor that is responsible for the effects of NPY on the H-P-G axis has eluded positive identification. SUMMARY OF THE INVENTION

We present here our finding that the receptor by which NPY acts to control the H-P-G axis is the NPY-Y4 receptor. We describe how this finding can be applied to the development of therapeutic agents for the treatment of reproductive disorders. In one embodiment, the invention comprises a pharmaceutical composition for the treatment of reproductive disorders, which composition is characterised in that it comprises a compound with high affinity for the NPY-Y4 receptor. The compound may be either a peptide or a non-peptide, and may act as an agonist or antagonist at the NPY-Y4 receptor. In another embodiment, the invention comprises a method of treatment of various reproductive disorders wherein a pharmaceutical composition comprising an NPY-Y4 receptor ligand is administered to an individual in need of such treatment.

DESCRIPTION OF THE INVENTION

The present invention is based on our novel finding that the effects of NPY on the reproductive system of mammals are mediated by the NPY-Y4 receptor. The experimental evidence is described in detail in the Examples (below). Briefly, it has been found that the administration of selective NPY-Y4 agonists (compounds that activate the NPY-Y4 receptor) to experimental animals causes an increase in the circulating levels of LH. These compounds also improve the fertility of animals with compromised reproductive function. Based on these findings, we disclose herein the use of NPY-Y4 ligands in the treatment of reproductive disorders.

In the following, the term "NPY-Y4 ligand" includes peptide and non-peptide agonists and antagonists at the Y4 subtype of neuropeptide Y receptor.

NPY-Y4 ligands are known in the art. NPY is an agonist at this receptor, but is not selective for this subtype. PP is a very selective NPY-Y4 agonist. A synthetic analogue of NPY, known variously as GR231118 and 12229U91 , was originally described as an NPY-Y1 antagonist, but has more recently been shown to be an NPY-Y4 agonist. Non-peptide NPY receptor ligands have also been reported, although the receptor subtype specificity is not generally discussed in these reports.

In a first aspect, the invention disclosed herein comprises a pharmaceutical composition for the treatment of reproductive disorders, which composition is characterised in that at least one of the active agents is an NPY-Y4 ligand. Such compositions will also include one or more excipients, such as diluents, preservatives and the like. These excipients are well known in the art. The composition is formulated as appropriate for the intended route of administration. In general, peptides are not well suited to oral administration. Accordingly, when the active agent is a peptidic NPY-Y4 ligand, it is preferred that the composition according to the present invention should be formulated for injection (for example, intravenous, subcutaneous or intramuscular injection) or for transmucosal delivery (for example, by intranasal, vaginal or rectal administration). One particularly preferred composition is a sterile solution of the NPY-Y4 ligand in isotonic saline, optionally buffered to pH 4-7 with citrate and/or phosphate. Such a composition is suitable for injection or for intranasal administration. Another particularly preferred composition is this solution microencapsulated in a biodegradable polymer such as copoly(lactide-glycolide). Such a formulation is appropriate for intramuscular injection when a slow release of the active agent is required. Non-peptide ligands may also be formulated in these ways, but may preferably be formulated as tablets, capsules and the like, for oral administration.

In a preferred embodiment, the NPY-Y4 ligand is an NPY-Y4 agonist and the composition is for the treatment of a decreased reproductive function. Examples of conditions resulting in decreased reproductive function include delayed puberty and amenorrhea (for example related to intensive exercise).

In another preferred embodiment, the NPY-Y4 ligand is an NPY-Y4 antagonist and the composition is for the treatment of supranormal function of the reproductive organs. Examples of such conditions include precocious puberty, polycystic ovary syndrome, endometriosis and benign prostatic hyperplasia.

In a second aspect, the invention disclosed herein comprises a method for the treatment of reproductive disorders wherein an effective amount of a pharmaceutical composition comprising at least one NPY-Y4 ligand is administered to a person in need of such treatment. The administration may be by injection (for example, intravenous, subcutaneous or intramuscular injection) or by any other appropriate route (for example, by oral, intranasal, vaginal or rectal administration). For long-term therapy, a particularly preferred method of administration is by intramuscular injection of a controlled-release formulation of the composition. The precise quantity of the composition to be administered will be determined by the supervising physician, but will generally be an amount corresponding to between 5μg and 50mg of the active agent per day for an average adult, given as a single dose or divided into multiple doses.

In a preferred embodiment, the method is a method of treatment of impaired reproductive function, such as delayed puberty or amenorrhea, by the administration of a composition comprising an NPY-Y4 agonist. This embodiment also includes the administration of such compositions in the context of assisted reproduction regimes.

In another preferred embodiment, the method is a method of treatment of supranormal function of the reproductive axis and organs, such as precocious puberty, polycystic ovary syndrome, endometriosis and benign prostatic hyperplasia, by the administration of a composition comprising an NPY-Y4 antagonist. EXAMPLES

Animals

All experiments were conducted in male Sprague-Dawley rats (200-220g) obtained from Iffa Credo (L'Arbresle, France), fed with standard laboratory chow ad libitum and kept on a 12h light-dark in temperature and humidity controlled room.

Surgery

Implantation of intracerebroventricular cannula: Rats were weighed and anaesthetised with ketamin/xylazin (3 and 7mg/kg, i.p., respectively). Canulae, aimed at the right lateral ventricle, were placed 1 mm posterior and 2mm lateral to bregma and extended 2mm below the outer surface of the skull. The injection canula extended beyond the guide canula 4mm ventrally to the skull surface. Rats were allowed to recover for 7 days.

Implantation of a jugular catheter. Under the same anaesthetic, the ventral side of the throat was shaved and an incision was made down the centre of the throat. The right jugular vein was exposed and canulated using a polythene tubing (OD 1.0mm) connected to a medical grade silicone tubing (OD 0.94mm) (siiicone side in the jugular vein). The tubing was secured and the polythene side was externalised through an incision made on the dorsal side of the neck. The catheter was rinsed with 300μL of a ringer solution containing 0.1% heparin. Rats were allowed to recover for at least 24 hours in individual cages with food and water available ad libitum.

Experimental procedure

All experiments were carried out between 14h and 18h. The different NPY-Y4 agonists, dissolved in sterile distilled water, were injected (i.c.v.) in a volume of 5μL. Blood samples (200-250μL) were removed immediately before and 15, 30, 60, 90 and 120min after central injections. Each blood sample was replaced with an equivalent volume of a Ringer solution containing 0.1% heparin. Plasma was extracted and stored at -20°C until determinations of LH and FSH by RIA. At the end of the study, the rats were anaesthetised and an i.c.v. injection of 5μL methylene blue dye was made. Animals were then killed by decapitation and the brain inspected for uniform and complete spread of the dye in the lateral ventricles. Data from any subject with inadequate spread of the marker were discarded. EXAMPLE 1

Stimulation of the gonadotropic axis by GR231118 (12229U91) in rats

GR231118 is a mixed Y4 agonist/Y1 antagonist (Schober et al., 1998; Parker ef a/., 1998). Intracerebroventricular injection of GR231118 (1.2-12nmol/rat) dose-dependently increased plasma LH levels (Table 1).

Table 1: Effects of GR231118 on plasma LH levels in the intact rat

p< 0.05, Kruskal-Wallis Anova on ranks followed by Dunn's multiple comparison procedure.

EXAMPLE 2

Stimulation of the gonadotropic axis by rat Pancreatic Polypeptide in rats

rPP is a Y4 agonist with very weak or no affinity for other NPY-receptor subtypes (Gerald ef al., 1996). Intracerebroventricular injection of rPP (3-30nmol/rat) dose-dependently increased plasma LH levels (Table 2).

Table 2: Effects of rPP on plasma LH levels in the intact rat

p< 0.05, Kruskal-Wallis Anova on ranks followed by Dunn's multiple comparison procedure. EXAMPLE 3

Reversion by GR231118 (12229U91) of fasting-induced inhibition of the gonadotropic

Based on the observation that fasting induces inhibition of the gonadotropic axis in rats, we have infused centrally NPY-Y4 agonists in fasted male rats and observed that fasting induced-decrease in seminal vesicle and testis weights were normalised in fed rats receiving central infusion of NPY-Y4 agonists. LH and testosterone pulsatility in fasted rats were also restored.

Central infusion of GR23118 in the lateral ventricles of fasting rats (42nmol/day) prevents the decrease in testicular and seminal vesicle weights (Table 3).

Table 3: Effects of GR231118 on testicular and seminal vesicle weights in fasted rats

*: p < 0.01 vs fed, vehicle injected rats

°: p < 0.01 ; °°: p < 0.001 vs fasted, vehicle injected rats

Anova followed by Newman-Queuls multiple comparison procedure

EXAMPLE 4

Time-Course of Effect of Various Compounds on Plasma LH Levels.

In a series of experiments analogous to those described in Examples 1 and 2, the time- course of the LH response to various peptides was determined. The results are summarised in Table 4, which also provides receptor binding data. Table 4: Affinity to Y1 and Y4 receptor subtypes and LH response of NPY, rPP, 1229U91 and related compounds.

Peptide Dosage (icv) Y1 Y4 ΔLH

(nmol) (Ki nM) (Ki nM) (ng/mL) pNPY 10 0.49 ± 0.2 117 0.55 ± 0.08 (10) (15 min) rPP 10 >1000 0.026 ± 0.013 0.40 ± 0.11 (14) (15 min)

Sequence ID5 12.7 0.11 ± 0.06 0.031 ± 0.013 6.53 ± 0.89 (9) (120 min)

Sequence ID6 10 0.28 ± 0.10 0.066 ± 0.010 1.79 ± 0.22 (9) (30 min)

Sequence ID7 10 0.18 ± 0.05 0.16 ± 0.04 1.26 ± 0.13 (8) (30 min)

Sequence ID8 10 0.12 ± 0.03 0.11 ± 0.03 1.23 ± 0.11 (6) (30 min)

The number of rats and time of maximal effect are indicated in parentheses.

In binding studies, n=3 for determinations with error. Peptides are as follows:

ID 5: (lle-Glu-Pro-Dpr-Tyr-Arg-Leu-Arg-Tyr-NH₂)₂ cyclic (2,4'), (2',4)-diamide {1229U91}

ID 6: (lle-Glu-Pro-Orn-Tyr-Arg-Leu-Arg-Tyr-NH₂)₂ cyclic (2,4'), (2',4)-diamide

ID 7, 8: (lle-Asn-Pro)₂-Pim-(Tyr-Arg-Leu-Arg-Tyr-NH₂)2 (two diastereomers) (17a, 17b)

These peptides are described in Daniels et al. (International Patent Application, Publication No. WO94/00486) as seq. ID numbers 4, 2, 17(a) and 17(b) respectively. Abbreviations : Dpr : L-diaminopropionic acid, Pirn : 2,6-diaminopimelic acid

These results demonstrate that NPY-Y4 agonists are capable of increasing the level of LH in the circulation and of promoting the growth of the reproductive organs. Therefore it can be predicted that such compounds will be useful in the restoration of fertility in humans. It also follows that NPY-Y4 antagonists will decrease the levels of circulating LH, hence decreasing the activity of the target organs of this hormone. Hence, such antagonists are predicted to be useful in pathologies arising from a supranormal function of these organs. Such pathologies include (but are not limited to) precocious puberty, endometriosis, benign prostatic hyperplasia, polycystic ovary syndrome and hormone-dependent neoplasias (including breast and prostate cancer).

References:

Schober DA, Van Abbema AM, Smiley DL, Bruns RF and Gehlert DR, 1998, The neuropeptide Y Y1 antagonist 1229U91 , a potent agonist for the human pancreatic polypeptide preferring (NPY Y4) receptor. Peptides 19 (3): 537-542 Parker EM, Babij CK, Balasubramaniam A, Burrier RE, Guzzi M, Hamud F, Mukhopadhyay G, Rudinski MS, Tao Z, Tice M, Xia L, Mullins D and Salisbury BG, 1998, GR231118 (12229U91 ) and other analogues of the C-terminus of neuropeptide Y are potent neuropeptide Y Y1 receptor antagonists and neuropeptide Y Y4 receptor agonists. Eur. J. Pharmacol. 349: 97-105

Gerald C, Walker MW, Criscione L, Gustafson EL, Batzl-Hartmann C, Smith KE, Vaysse P, Durkin MM, Laz TM, Linemeyer DL, Schaffhauser AO, Whitebread S, Hofbauer KG, Taber Rl, Branchek TA and Weinshank RL, 1996, A receptor subtype involved in neuropeptide Y- induced food intake. Nature 382:168-171.

In sequence ID Number 6, Orn is ornithine.

Sequence ID Numbers 7 and 8 are fully disclosed in PCT Patent Publication No. WO94/00486 (Daniels et. al.). These are two diastereomers of Example 14 of WO94/00486 (Sequence 17 of that application):

Ile-Asn-Pro-NH -CH-CO--Tyr-Arg-Leu-Arg-Tyr-NH₂

I

(CH₂)₃

I

Ile-Asn-Pro-NH -CH-CO~Tyr-Arg-Leu-Arg-Tyr-NH₂

Claims

1. A pharmaceutical composition for the treatment of human reproductive disorders which comprises an effective amount of an NPY-Y4 receptor ligand.

2. A pharmaceutical composition for the treatment of reduced reproductive function, which composition is characterised by the inclusion of an NPY-Y4 receptor agonist as an active agent.

3. A pharmaceutical composition for the treatment of delayed puberty, which composition is characterised by the inclusion of an NPY-Y4 receptor agonist as an active agent.

4. A pharmaceutical composition according to any of Claims 1 - 3, wherein the active agent is selected from human, rat or bovine pancreatic polypeptide or GR231118.

5. A pharmaceutical composition for the treatment of supranormal function of the reproductive organs, which composition is characterised by the inclusion of an NPY-Y4 receptor antagonist as an active agent.

6. A pharmaceutical composition for the treatment of precocious puberty, endometriosis, polycystic ovary syndrome or benign prostatic hyperplasia, which composition is characterised by the inclusion of an NPY-Y4 receptor antagonist as an active agent.

7. A method for the treatment of human reproductive disorders which comprises the administration to a person in need of such treatment of a composition containing an effective amount of an NPY-Y4 receptor ligand.

8. A method for the treatment of impaired reproductive function which comprises the administration to a person in need of such treatment of a composition containing an effective amount of an NPY-Y4 receptor agonist.

9. A method for the treatment of delayed puberty or amenorrhea which comprises the administration to a person in need of such treatment of a composition containing an effective amount of an NPY-Y4 receptor agonist.

10. A method for the treatment of supranormal function of the reproductive organs which comprises the administration to a person in need of such treatment of a composition containing an effective amount of an NPY-Y4 receptor antagonist.

1. A method for the treatment of precocious puberty, endometriosis, polycystic ovary syndrome or benign prostatic hyperplasia, which comprises the administration to a person in need of such treatment of a composition containing an effective amount of an NPY-Y4 receptor antagonist.