WO1999054358A1

WO1999054358A1 - Melanocortin receptor ligands

Info

Publication number: WO1999054358A1
Application number: PCT/GB1999/001195
Authority: WO
Inventors: Roger Antonius Henricus Adan; Willem Hendrik Gispen
Original assignee: Quadrant Holdings Cambridge Ltd
Current assignee: Quadrant Holdings Cambridge Ltd
Priority date: 1998-04-17
Filing date: 1999-04-19
Publication date: 1999-10-28
Anticipated expiration: 2000-10-17
Also published as: GB9808229D0; AU3615599A

Abstract

Peptides (many of which are novel) interact selectively with melanocortin receptors, particularly the MC3, MC4 and MC5 receptors. The peptides have been characterised in vivo, and shown to have an effect upon behaviour or metabolism.

Description

1

MELANOCORTIN RECEPTOR LIGANDS Field of the Invention

The present invention relates to peptides which interact selectively with melanocortin (MC) receptors and which have been characterised in vivo. Background of the Invention

The melanocortins were originally described as mediators of pigmentation. Forexample, US-A-5674839 and US-A-5714576 disclose cyclic and linear analogues of α-MSH for stimulating melanocytes in vertebrates to treat hypopigmentation disorders. One such known peptide is Ac-Ser Tyr Ser Nle c[Asp His D-Phe Arg Trp Lys] Gly Pro Val-NH₂.

Melanocortins are now known to modulate neurophysiological and neuropathological phenomena such as conditioned avoidance, central control of autonomic systems, different types of behaviour (grooming, female sexual behaviour, anxiety, aggression), drug addiction and nerve regeneration (De Wied and Jolles, Physiol. Rev. (1982) 60:976-1059). The demonstration of binding sites for melanocortins in the brain (Tatro, Brain Res. (1990) 536:124-132) and the cloning of brain-specific MC receptors (Mountjoy er a/, Mol. Endocrinol. (1994) 8:1298-1308; Chhajiani et al, Biochem. Biophys. Res. Commun. (1993) 195:866-873; and Rehfuss-Roselli et al, Proc. Natl. Acad. Sci. USA (1993) 90: 8856-8860) further underscored the significance of the brain melanocortin system. A number of distinct receptor sub-types have now been shown to be expressed in the brain.

Although the precise function of each sub-type has yet to be determined, it is clear that the receptors are important in many regulatory mechanisms. For example, the MC3 receptor has been implicated in increasing blood pressure and heart rate, whereas the MC4 receptor is believed to decrease blood pressure and heart rate. In addition, the MC4 receptor is believed to be a mediator of melanocortin - induced grooming. The receptors may also play a role in nerve regeneration, as treatment with melanocortins has been shown to enhance maturation of the nervous system. In particular, the MC4 and MC5 receptor may be important in this process. The deletion of the MC4 receptor gene from mice and the identification of certain endogenous antagonists have shown that the MC4 receptor may be implicated in the brain melanocortin system regulation of weight homeostasis (Shutter et al, Genes Dev. (1997) 11: 593-602; Huszarer a/, Cell (1997) 88: 131-141). However, the exact involvement of the melanocortin system in neurophysiology and weight homeostasis is not yet fully understood. Likewise, the mechanisms underlying the effects of melanocortins on the diverse brain functions described above are unknown. Nevertheless, a number of ligands for MC receptors are known. α-Melanocyte- stimulating hormone (α-MSH) is a linear tridecapeptide which is involved in the regulation of skin pigmentation and has various actions on the brain which affect behaviour. In the brain, α-MSH has been shown to interact with several melanocortin receptors, such as MC3, MC4 and MC5. In addition, some α-MSH analogues, such as cyclic lactam analogues, have been shown to be agonists of MC receptors activating both MC1 receptor activity and other MC receptors. Although some in vivo data is available for MC receptor ligands, in vivo activity is poorly characterised. Peptide activity in vivo has been assessed, for example, in rats by means of a grooming assay. The stretching and yawning syndrome (Ferrari, Nature (1958) 925-926) and grooming behaviour in the rat (Gispen et al, Life Sci. (1975) 17: 645-652) were among the first described behaviours which are under the control of the melanocortins. Grooming behaviour consists of activities directed to the animal body surface like face washing, body grooming, licking, scratching and genital grooming. Grooming behaviour is thought to be under control of the MC4 receptor (Adan et al, Mol. Pharmacol. (1994) 46: 1182-1190).

Despite some in vitro binding analysis, there is still a need for the provision of peptides which have a family for specific MC receptors, particularly MC3, MC4 and MC5 receptors, and whose activity has been characterised in vivo. Summary of the Invention

According to the present invention, peptides have been identified which, surprisingly, exhibit differential binding to the MC3, MC4 and MC5 receptors, and which exhibit biological activity in vivo. The peptides are modifications of the α-MSH peptide and show distinct neurophysiological effects that are manifested as behavioural patterns.

In a further aspect of the invention, the peptides are useful to mediate physiological effects controlled or affected by the MC3, MC4 and MC5 receptors, including metabolic and behavioural changes. The invention, thus, extends to include pharmaceutical preparations containing the peptides, and methods of treatment which involve the use of the peptides.

Peptides according to the present invention are: (i) Ac-Nle c[Asp His D-Tyr Arg Trp Lys]-NH₂; (ii) Ac-Ser Tyr Ser c[Cys Gly His D-Phe Arg Trp Cys] Lys Pro Val-NH₂;

(iii) Ac-Tyr Val Nle Gly His Phe Arg Trp Asp Arg Phe Gly-NH₂; and

(iv) Ac-c[Cys Gly His D-Nal Arg Trp Cys]-NH₂.

In addition, the peptide: (v) Ac-Ser Tyr Ser Nle c[Asp His D-Phe Arg Trp Lys] Gly Pro Val-NH₂ may be used to treat various disorders in which the melanocortin receptors are implicated.

"Ac" represents acylation, and "cQ" represents a cyclic structure. "Nle" represents the oxidatively stable isostere for methionine.

A preferred peptide is (iii) which has good selectively for the MC3 receptor as opposed to the MC4 receptor. Description of the Invention

The peptides may have many uses in therapy. In particular, the peptides may be used in the manufacture of compositions for therapy (including prophylaxis where relevant) of neurological disorders including memory deficit and nerve damage, behavioural disorders including compulsive behaviour, stress, anorexia and addiction, cardiovascular disorders including cerebrovascular disorders such as stroke, metabolic disorders including obesity, sexual dysfunction including erectile dysfunction, or inflammatory events such as pain and fever.

The peptides may also be used to prevent or reduce nerve damage, e.g. as caused by other drugs or chemotherapy. They may therefore be co-administered with, say, cytotoxic agents.

The peptides of the present invention may be administered by any suitable means, many of which will be apparent to the skilled person. For example, the peptides may be administered orally, buccally ortransdermally, or by the intravenous, intramuscular, pulmonary, mucosal, rectal or subcutaneous route. The formulation of the peptides into suitable compositions for delivery, will be apparent to the skilled person. For example, it will be readily apparent which pharmaceutically acceptable excipients or diluents are appropriate.

The amount of peptide to be delivered may be determined by the route of administration, the extent of damage to be treated, and the relative activity of the peptide. Suitable amounts, and other relevant factors, can be determined by a skilled person.

The present invention will now be discussed with respect to the following, illustrative Examples. Example 1 Synthesis of Peptides (Table 1)

The peptides shown in Table 1 were synthesised using F-Moc synthesis (Fields er a/, Pept. Res. (1991) 4:95) on solid phase.

Peptides identified as MT-II, D-Tyr-MT-ll, RM12004 and SHU 9119 (SEQ ID Nos 3, 4, 6 and 8 respectively) are cyclised via the side chains of Asp and Lys Cyclisation was carried out in solution using Py-BOP. A 30 mmol scale was used with double coupling steps of 30 min and 4 equivalents of amino acid. Py-BOP (26 mg, 50 mmol) was dissolved in 20 ml of peptide grade DMF. Diisopropylethylamine (DIEA, 17 ml, 100 mmol) was added, followed by 20 mg (12 mmol) of deprotected peptide; the reaction was followed by HPLC analysis and found to be complete after 1 hour. The reaction was stopped after 2 hours, and the product purified using preparative HPLC.

Peptides identified as RMI 2001 and RMI 2005 (SEQ ID Nos. 5 and 9, respectively) are cyclised via disulphides. For example, the deprotected peptide of SEQ IS No 5 was dissolved in 40 ml of 0.5% NH₄HCO₃(pH 8). After 24 hours, no free sulphydryl could be detected by Elmanns reagent, and the solution was acidified with acetic acid. The product was lyophilised and purified using preparative HPLC. Example 2 Receptor binding activity of peptides

Transfected HEK 293 cells (transfected with the rat MC3 or rat MC4 receptor) were grown in poly-L-lysine (Sigma) coated 24 well Costar plates. Two days after transfection, the cells were incubated with 100,000 cpm of ¹²⁵I-NDP-MSH (final concentration 0.1-0.2 nM) and various concentrations of peptides in Ham's F10 medium (Gibco) pH 7.4 containing 2.5 mM CaCI₂, 0.25% BSA, 10 mM Hepes and 50 mg/ml (150 KlU/ml) aprotinin. After incubation for 30 min at room temperature, the cells were washed twice with ice-cold TBS containing 2.5 mM CaCI₂ and lysed in 1 M NaOH. Radioactivity of the lysates was counted in a Packard Cobra γ-counter.

Table 2 (below) shows the corrected IC₅₀ value (K,) of these peptides on the rat MC receptors MC3-R and MC4-R. The peptides NDP-MSH, RMI-2001 , Nle-γ-MSH, RMI-2004, RMI-2005, SHU9119 and MT-II all had an affinity on the rat MC3-R in the low nanomolar range, whereas α-MSH had an affinity which was 2-10 fold lower. [D-Tyr]-MTII had an affinity in the submicromolar range. α-MSH had a similar affinity on the rat MC4-R as compared to the rat MC3-R.

NDP-MSH and MT-II had affinities in the low nanomolar range on the rat MC4-R, similar to the affinity for MC3-R. However, in contrast to their affinity for the rat MC3 receptor, RMI-2001 , RMI-2005 and SHU9119 had affinities in the subnanomolar range for the rat MC4-R, and [D-Tyr]-MTII had an affinity in the low nanomolar range on the rat MC4-R. Thus, RMI-2001 , [D-Tyr]-MTII, RMI-2005 and SHU9119 had an affinity on the rat MC4-R which is significantly higher as compared to the rat MC3-R. In contrast, Nle-γ-MSH and RMI-2004 had an affinity which is lower on the rat MC4-R as compared to the rat MC3-R. Example 3 in vitro activity assay of receptor binding

Functional activity was assayed by the reporter activity of a LacZ gene, which is expressed under the control of a cAMP regulated promotor in a pCREIacZ construct to detect changes in intracellular cAMP as a result of receptor activation (Chen et al, Anal. Biochem. (1995) 226: 349-354 ). 293 HEK cells expressing either the rat MC3 receptor (Rehfuss-Roselli et al, supra) or the rat MC4 receptor (Alvaro et al, Mol. Pharmacol. (1996) 50:583-591) were grown in modified Eagle's medium (DMEM) supplemented with 10% FCS. MC-receptor expressing cells were transfected with 10 mg of the pCREIacZ construct using the calcium phosphate precipitation method. 20 hours after transfection, the cells were split into 96-wells plates. The next day, cells were treated for 6 hours with melanocortin receptor ligands in DMEM supplemented with 0.5% BSA and 25 mM HEPES (pH7.4). The agonist activity was measured by stimulating the cells with varying concentration of α-MSH, NDP-MSH, MT-II and RMI-2001, Nle-γ-MSH, [D-Tyr]-MTII and RMI-2004. After treatment, cells were lysed in PBS with 0.1% Triton X-100, frozen, thawed and assayed for β-galactosidase activity. The results are also shown in Table 2. In addition, antagonist activity was demonstrated with a fixed concentration of agonist and increasing concentrations of antagonists SHU9119 and RMI-2005. The antagonists were also screened for (partial) agonistic activity.

NDP-MSH, MT-II and RMI-2001 were the most potent ligands on the rat MC4-R, having EC50 values of less than that 0.1 nM. α-MSH and RMI-2004 had comparable activities on the rat MC4-R which were in the (sub)nanomolar range [D-Tyr]-MTII had a subnanomolar EC50 value and Nle-γ-MSH had an EC50 value of 11 nM. On the rat MC3-R, only NDP-MSH and MT-II had a subnanomolar EC50 value. RMI-2001, Nle-γ-MSH and RMI-2004 had EC50 values in the low nanomolar range, whereas [D-Tyr]-MTII had an EC50 value of 20 nM. Thus, MT-II, RMI-2001 and [D-Tyr]-MTII showed the highest difference between rat MC3-R and MC4-R, the activity being higher on the rat MC4 receptor. Only Nle-γ-MSH was more potent on the rat MC3-R than on the rat MC4-R.

Partial agonistic activity on the rat MC3 receptor was observed for SHU9119 (maximal at 10 nM being 40% of maximal activity) and RMI-2005 (maximal at 40 nM being 25 % of maximal activity). RMI-2005, but not SHU9119, had also partial agonistic activity on the rat MC4-R, being 20 % of maximal receptor activation at 20 nM. Example 4 in vivo activity of peptides administered cerebrally

Male Wistar rats weighing 120- 130 g were used. Rats were housed in single cages in a light-dark cycle of 12 h. Cannulas made from polypropylene tubes were implanted into the foramen intraventriculare under anaesthesia (Brakkee et al, Lab. Animal Sci. (1979) 29: 78-81). Rats were allowed to recover for 3 days and used for experiments during the next 10 days. Peptides dissolved in 3 ml of saline (154 mM NaCI) were injected intracerebroventricularly (i.c.v.) by means of a Hamilton syringe. Grooming tests were performed according to (Gispen et al, supra). Briefly, rats were transported to an observation room at least 1 hour before start of the test. Grooming was induced either by agonist injection or by exposure to a novel environment which consisted of a Plexiglass box (30 cm/15 cm/15 cm) covered with a metal cover in which naive rats were kept during observation. Rats were placed into the observation boxes immediately afterthe injection. Observation started 15 min after the injection and continued for 50 min. Grooming was scored each 15 sec over 50 min, thus the maximum grooming score for a rat is 200. Rat activities such as vibration, face washing, genital grooming, body licking, scratching were considered as grooming. Each experimental group consisted of at least 6 rats. Figures 1a and 1b are graphs of grooming score against agonist, and show the results of the grooming experiments. α-MSH, NDP-α-MSH, RMI-2001, [D-Tyr]-MTII, RMI-2004 and MT-II induced grooming after i.c.v. injections, whereas Nle-γ-MSH did not. Also RMI-2005 and SHU9119 did not induce grooming behaviour even at the highest tested dose of 1 nmol. RMI-2001 , NDP-α-MSH and MT-II had similar potency with submaximal doses between 2 and 10 pmols. The lowest dose of these peptides that significantly increased grooming as compared to saline treated rats was 1.5 pmol for NDP-α-MSH and RMI-2001 and 4.5 pmol for MT-II. Thus, the three most potent MC4 receptor agonists were also the most potent peptides in inducing excessive grooming behaviour. Furthermore, the pharmacological profile of melanocortin-induced grooming fits best with that of the rat MC4-R and not with that of the rat MC3-R. For instance Nle-γ-MSH is more potent and [D-Tyr]-MTII is less potent than α-MSH on the rat MC3-R, but [D-Tyr]MT-ll is more potent than α-MSH on induction of grooming behaviour, whereas Nle-γ-MSH did not induce grooming. Example 5 in vivo activity of peptides administered intraveneously

For intravenous injections via the tail, peptides were dissolved in 100 ml saline. Rats were injected intravenously with 100 mg α-MSH, NDP-MSH, MT-II, D-Tyr, RMI-2001 or RMI-2004, and grooming behaviour was observed. MT-II potently activated grooming behaviour, whereas NDP-MSH, D-Tyr, MY-II and RMI-2001 significantly stimulated grooming behaviour although to a lesser extent. RMI-2004 and α-MSH did not stimulate grooming behaviour. Example 6 Peptide binding to mouse MC5 receptors

A number of ligands were screened for binding to the mouse MC5 receptor. IC50 values were determined according to the method given in Example 2. The results are given in Table 3.

Table 3

Peptide Receptor IC50

RMI-2001 Rat MC5 2.7 e-07

RMI-2004 Rat MC5 1.5 e-09

RMI-2005 Rat MC5 3.6 e-09 (agonist)

Example 7 Peptide binding to the human receptors A number of ligands were screened for binding to the human receptors, including the human MC5 receptor. IC50 values were determined according to the method given in Example 2. The results are given in Table 4. 8 Table 4

Peptide Receptor IC50 value

RMI-2001 Human MC3 7.5 e-09 Human MC4 5.6 e-09

RMI-2004 Human MC3 1.5 e-09 Human MC4 9.7 e-09

D-Tyr-MTII Human MC3 4.4 e-07 Human MC4 2.8 e-08 Human MC5 1.1 e-09

RMI-2005 Human MC3 1.4 e-08 Human MC4 1.3 e-09 Human MC5

1.9 e-07 (agonist)

Table 1 o vø α-MSH SEQIDNol Ac- Ser Tyr Ser Met Glu His Phe Arg Trp Gly Lys Pro Val -NH₂ <

NDP-MSH SEQID 02 Ac- Ser Tyr Ser Nle Glu His D-Phe Arg Trp Gly Lys Pro Val -NH₂ 05

MT-II SEQIDNo3 Ac- Nle c[Asp His D-Phe Arg Trp Lys] -NH₂

D-Tyr-MTII SEQID o4 Ac- Nle c[Asp His D-Tyr Arg Trp Lys] -NH₂

RMI-2001 SEQIDN05 Ac- Ser Tyr Ser c[Cys Gly His D-Phe Arg Trp Cys] Lys Pro Val -NH₂

RMI-2004 SEQID 06 Ac- Ser Tyr Ser Nle c[Asp His D-Phe Arg Trp Lys] Gly Pro Val -NH₂

Nle-γ-MSH SE^QIDNo7 Ac- Tyr Val Nle Gly His Phe Arg Trp Asp Arg Phe Gly -NH₂

SHU9119 SEQID 08 Ac- Nle c[Asp His D-Nal Arg Trp Lys] -NH₂

RMI-2005 SEQIDNo9 Ac- c[Cys Gly His D-Nal Arg Trp Cys] -NH₂

CD

O

H o W vo vo v© en

Table 2 vo

>o tn

4-. W CΛ

00

MC3R MC4R

LIGAND Ki EC50 Ki EC50 α-MSH 9,40 ±3,17 11,6 ±3,2 9,17 ±4,23 1,34 ±0,50

NDP-MSH 1,19 ±0,51 0,309 ±0,170 3,14 ±1,18 0,0927 ±0,0471

MT-II 4,77 ±2,13 0,780 ±0,173 1,74 ±0,77 0,0111 ±0,0043

D-Tyr-MTII 204 ±87,2 20,3 ±7,1 3,84 ±0,83 0,466 ±0,189

RMI-2001 4,96 ±1,06 2,22 ±0,50 0,260 ±0,098 0,0300 ±0,0131

RMI-2004 1,23 ±0,13 3,86 ±0,92 4,51 ±0,99 0,326 ±0,155

Nle-r-MSH 1,44 ±0,26 1,26 ±0.10 77,5 ±37,7 11,0 ±3,92

SHU9119 0,879 ±0,170 0,238 ±0,060

RMI-2005 4.56 ±1.30 0.485 ±0.169

π H

O 93 vo vo

VO

Claims

11CLAIMS

1. Use of any of the peptides

(i) Ac-Nle c[Asp His D-Tyr Arg Trp Lys]-NH₂;

(ii) Ac-Ser Tyr Ser c[Cys Gly His D-Phe Arg Trp Cys] Lys Pro Val-NH₂; (iii) Ac-Tyr Val Nle Gly His Phe Arg Trp Asp Arg Phe Gly-NH₂;

(iv) Ac-c[Cys Gly His D-Nal Arg Trp Cys]-NH₂; and

(v) Ac-Ser Tyr Ser Nle cfAsp His D-Phe Arg Trp Lys] Gly Pro Val-NH₂, for the manufacture of a composition for the therapy of a disorder in which melanocortin receptors are implicated.

2. Use according to claim 1 , wherein the disorder is neurological.

3. Use according to claim 1 , wherein the disorder is behavioural.

4. Use according to claim 1 , wherein the disorder is cardiovascular.

5. Use according to claim 1 , wherein the disorder is metabolic.

6. Use according to claim 1 , wherein the disorder is sexual dysfunction.

7. Use according to claim 1 , wherein the disorder is nerve damage caused by other therapy.

8. The peptide (i), (ii), (iii) or (iv) defined in claim 1.

9. The peptide (iii) defined in claim 1.

10. A peptide according to claim 8 or claim 9, for use in therapy.