WO2005079831A1

WO2005079831A1 - Use of hcnp peptides for the modulation of cardio-vascular physiology and diuresis

Info

Publication number: WO2005079831A1
Application number: PCT/EP2004/002395
Authority: WO
Inventors: Yannick Goumon; Marie-Hélène METZ-BOUTIGUE; Dominique Aunis
Original assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm)
Priority date: 2004-02-13
Filing date: 2004-02-13
Publication date: 2005-09-01
Also published as: WO2005079832A1

Abstract

The present invention relates to a method of use of HCNP peptides or their derivatives or functional equivalents as active ingredients in medicaments for the modulation of cardio-vascular physiology and diuresis.

Description

Use of HCNP peptides for the modulation of cardio-vascular physiology and diuresis

The present invention relates to a method of use of HCNP peptides or their derivatives or functional equivalents as active ingredients in medicaments for the modulation of cardiac physiology and diuresis.

Phosphatidylethanolamine-binding protein (PEBP) is a 21 kDa protein which has been found in numerous tissues of many species including rat, human, plants, bacteria and yeast, indicating its ubiquitous occurrence. PEBP has previously been described in a large variety of tissues including brain and adrenal gland. Several reports have also described the presence of PEBP immunoreactivity in biological fluids including rat haploid testicular germ cell secretions, testicular interstitial fluid, the culture media of transfected Rat-1 fibroblasts, and more recently in the conditioned medium from adult rat hippocampal progenitors. PEBP and its N-terminal-derived peptide named hippocampal cholinergic neurostimulating peptide (HCNP), corresponding to the first eleven N- terminal amino acid residues (PEBPl-11), were also found in synaptic vesicles after subcellular fractionation. In addition, HCNP derived peptides were detected by immunohistochemistry in nerve cell terminals of neurons in rat brain and small intestine. Furthermore, HCNP was shown to be released from rat hippocampal. More recently, HCNP has also been detected in the cerebrospinal fluid of Alzheimer patients. EP0 390 602 discloses neurotrophic peptides derived from brain hipocampal tissue in mammals able to enhance synthesis of acetylcholine in cholinergic neurons, as well as pharmaceutical compositions comprising these neurotrophic peptides for the treatment of neurological degenerative disorders and dementia. This patent discloses in particular a neurotrophic peptide of sequence Ala Ala Asp He Ser Gin Trp Ala Gly Pro Leu which is rat HCNP peptide as well as HCNP derivatives having a smaller molecular weight. EP0 511 816 discloses the human-derived neurotrophic peptides of HCNP or derivatives thereof. These peptides are also useful for the treatment of neurological degenerative disorders and dementia. The inventors found that HCNP and HCNP derivatives or functional equivalents modulate the cardiac physiology and diuresis. HCNP and PEBP are natural, stable and are easy to synthetize. As these peptides are present in all organisms, there is no possible antigenicity between species. Accordingly, one aspect of the invention is to provide a therapeutic method of use of a polypeptide containing at least the amino acid sequence of formula SEQ ID N°l or derivative or functional equivalent of this sequence for the modulation of cardiac physiology. Another aspect of the invention is to provide a therapeutic method of use of a polypeptide containing at least the amino acid sequence of formula SEQ LD N°l or derivatives or functional equivalents of this sequence for the modulation of diuresis. Another aspect of the invention is the use of a peptide of sequence SEQ LD N°l or derivatives and functional equivalents for the modulation of cardiac physiology. Another aspect of the invention is the use of a peptide of sequence SEQ LD N°l or derivatives and functional equivalents for the modulation of diuresis. Another aspect of the invention is a medicament comprising at least one peptide, derivative and functional equivalent of the invention for the modulation of cardiac physiology. Another aspect of the invention is a medicament comprising at least one peptide, derivative and functional equivalent of the invention for the modulation of diuresis. Other aspects will become apparent on reading the description and examples, which are not restricted and with reference to the following drawing:

The subject matter of the invention is the use of polypeptide comprising at least a peptide of sequence XaaιXaa₂AspXaa₄SerXaa₆TrpXaa₈GlyProLeu (SEQ ID Nj 1) in which Xaa_t is Pro or Ala, Xaa₂ is Nal or Ala, Xaa₄ is Leu or He, Xaae is Lys or Glu and Xaa₈ is Ser or Ala, derivatives or functional equivalents of this peptide for the preparation of a medicament for the modulation of cardiac physiology and diuresis. According to the present invention, the term "derivatives" as used herein means NH2 and/or COOH terminal modified sequences of the peptide of the invention. Modifications encompass, but are not limited to, modifications resulting from posttranslational processus, or chemical modifications by known techniques in the art (glycosylation, phosphorylation, sullfatation, addition of methyl group, acetyl group, amide group, lipid fixation, fatty acid fixation, nucleotides fixation, cyclization...) According to the present invention, the term "functional equivalent" as used herein means sequences which function in a similar way but may have deletions, additions or substitutions that do not substantially change the activity or function of the sequences. Here the functions are to act as a modulator of either cardiac physiology or diuresis. Substitutions well known of the skilled man in the art, encompass substitution of an amino acid by another one having same characteristics (charge, size hydrophilicity, etc.... For example substitution between Asp and Glu, between Lys and Arg, between Gly, Ser, Thr, Cys, Tyr, Asn, Gin, His and Trp, between Ala, Val,

Leu, He, Pro, Phe and Met, between Phe and Tyr. The functional equivalent of polypeptides according to the invention may be constituted of amino acids residues of configuration D or L. These functional equivalent encompass also retropeptides. The peptides according to the invention may be natural or prepared by methods including (but not restricted to) solid or solution phase chemical synthesis of polypeptide or/and by recombinant DNA/genetic engineering procedures using well known methods. Preferably, this polypeptide is the Phosphatidylethanolamine Binding

Protein (PEBP), which is a precursor of HCNP. The PEBP protein can be obtained by means described in the article "Purification and characterization of a basic 23kDa cytosolic protein from bovine origin" (I. Bernier & P. Jolles, (1984) Biochimica and

Biophysica Acta, 790, ppl74-181). More preferably, the polypeptide PEBP of the invention is the human PEBP or the murine PEBP (including mouse or rat). In another embodiment, a peptide of sequence SEQ ID N°l or a derivative or a functional equivalent thereof is used for the manufacture of a medicament for the modulation of cardiac physiology and diuresis. Preferably, the peptide of sequence SEQ ID N°l is the Hippocampal Cholinergic Neurostimulating Peptide (HCNP). More preferably, the peptide used according to the invention is the HCNP in which amino acids Xaa_l5 Xaaj, Xaa₄, Xaa₆ and Xaa₈ are defined as in SEQ LD N°2. Preferably, the amino acid sequence of the peptide according to the invention is the amino acid sequence of human HCNP. In another embodiment, the peptide used according to the invention is the

HCNP in which amino acids Xaa_l5 Xaa₂, Xaa₄, Xaag and Xaa₈ are defined as in SEQ LD N°3. Preferably, the amino acid sequence of the peptide according to the invention is the amino acid sequence of murine HCNP. β-adrenergic stimulation, like the stimulation with catecholamines, is indeed known to display a positive inotropic effect on the mechanical cardiac performance. The peptides according to the invention are able to display a dramatic negative inotropic effect and are able to inhibit isoproterenol (ISO which is a β-adrenergic agonist inducing a positive inotropic effect) stimulation of the heart (as shown in FIG. 1). Since catecholamines are in particular secreted in response to a stress, the peptides of the invention are also useful in case of pathologies related to stress. The peptides of the invention are useful for the preparation of a medicament intended to modulate cardiac physiology and diuresis. The term "modulation of cardiac physiology" as used herein encompasses the decrease of cardiac performances also called inotropic negative effect linked to myocardial infarction or heart failure. The peptides of the invention are useful for the preparation of a medicament intended to modulate diuresis. Peptides of the invention can be incorporated in a composition containing a pharmaceutically acceptable medium for the preparation of the medicament for the modulation of cardiac physiology or diuresis. Preferably, the concentration of the peptide, derivative or functional equivalent according to the invention is from 10^"11 to 10^"4moles/L in the peripheral blood. The peptides, derivatives and functional equivalent thereof according to the invention are used on a pharmaceutically acceptable salt form. These pharmaceutically acceptable salts include conventional non-toxic salts of these peptides and quaternary salts thereof. These salts may be formed from inorganic or organic acids or bases. Examples of such acid addition salts are salts of acetic acid, butyric acid, citric acid, lactic acid, tartaric acid, oxalic acid, maleic acid, succinic acid, fumaric acid, hydrochloric acid, hydrobromic acid and sulfuric acid. Salts of bases are ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with amino acids such as arginine, lysine, etc. Such salts can be readily produced by known methods. For example, in the case of citrates, the citrates can be prepared by dissolving the peptide according to the invention in water and adding a necessary amount of citric acid to the solution. Such compositions may further contain protease inhibitors as, for example, PMSF or PHP. In a preferred embodiment, peptides of the invention are on a citrate salt form. Indeed, in case of intraveinous route, this formulation as a citrate salt avoids coagulation. Peptides of the invention may be used alone, in combination of two or more peptides and/or in combination with others conventional cardiomodulateurs. In a preferred embodiment, the composition further comprises other cardiomodulators. Such modulators are for example beta-blockers, such as carvedilol, metropolol-XL, diuretics as spironolactone, angiotensin-converting enzyme inhibitors and nesiretide (commercially available). Animal to which the peptides of the present invention are applicable is not limited. The peptides of the present invention can be applied to human beings as well as to other various species of mammals such as mouse, rat, dog, calf, horse, goat, sheep, rabbit, hog, etc. The peptides of the invention can be administered to these animals and human by ordinary routes, for example, orally, intramuscularly, intraveinously, subcutaneously, intraperitoneally and pernasally. Dose and time of administration vary depending upon animal species, administration routes, condition of disease, body weight, etc... In human, the peptides can be administered to adult in a daily dose once or in several portions. Examples of pharmaceutical compositions include powders, granules, tablets, capsules, suppositories, injections, nasal preparations, etc . The pharmaceutical compositions can be prepared in a conventional manner, using conventional carriers for preparations. That is, in the case of preparing oral preparations, excipients or carriers are added to the active ingredient and if necessary, binders, disintegrators, lubricants and coloring agents are further added thereto and then prepared into tablets, granules, powders, capsules, etc... In the case of preparing injections, pH regulators, buffers stabilizers, solubilizing agents, etc., are added depending upon necessity and prepared into injections in a conventional manner.

The examples, which follow, are intended to illustrate the invention without, however, being limiting in nature. Example 1: Synthesis of HCNP peptide Peptides were synthesized on an Applied Biosystems 432A peptide synthesizer (Synergy, Foster City, USA), using the stepwise solid-phase synthetic approach with 9-fluoromethoxycarbonyl (Fmoc chemistry). Then, the synthetic peptides were purified by liquid high performance chromatography (Akta Purifier, Amersham Biosciences, Orsay, France) on a C18 column (Vydac, Hesperia, USA;

218 TP 5μ 10 _ 250 mm). After lyophilization, synthetic peptides were analyzed by sequencing (473A sequencer, Applied Biosystems, Boston, MA) and MALDI-TOF mass spectrometry with a Bruker BIFLEX MALDI-TOF mass spectrometer (Wissembourg, France). N-terminal acetylated peptides were synthesized using Acetylated synthons. Example 2: Effects of HCNP on isolated and perfused working heart physiology The isolated avascular frog hearts, were used in order to examine the role of HCNP on heart physiology. Frog hearts were isolated from specimens of both sexes of Rana esculenta (weighting 22.0 ±1.2g; mean value ± SE) and connected to a perfusion apparatus as described in Sys, S. U., et al, (1997) J Exp Biol 200, p3109-3118. Experiments were done at room temperature (18-21°C) from autumn to spring. A Grass S44 stimulator was used (single pulses of 20 V, 0.1s) to electrically pace the heart preparations; the stimulation rate was identical to the control (unpaced) rate. The saline buffer composition was: 115mM NaCl, 2.5mM KC1, l.OmM CaC12, 5.6mM anhydrous glucose, 2.15mM Na2HPO4, 0.85mM NaH2PO4, pH 7.2 equilibrated with air. Mean input pressure and minimal output pressure (diastolic afterload) were regulated by moving the reservoirs up or down with reference to the level of the atrium and the aortic trunk, respectively. The hearts were stabilized at basal conditions (see below) for 15-20min before being treated with drugs. Pressure was measured through T-tubes placed immediately before the input cannula and after the output cannula, using two MP- 20D pressure transducers (Micron Instruments, Simi Valley, CA, USA) and connected to a Unirecord 7050 (Ugo Basile, Comerio, Italy). Pressure measurements were expressed in kPa and corrected for cannula resistance. The heart rate was calculated from pressure recording curves. The cardiac output was collected over 1 min and weighted. Values were corrected for temperature and fluid density by calculation and expressed as volume measurements. The afterload (mean aortic pressure) was calculated as 2/3 diastolic pressure plus 1/3 maximum pressure.

Cardiac output and stroke volume (SV = cardiac output/ heart rate) were normalized per kg of wet body weight. Stroke volume at constant pre- and afterload in paced hearts was used as measure of ventricular performance. Changes in stroke volume under these conditions were considered inotropic effects. Ventricular stroke work (SW), an index of systolic functionality, was calculated as mJ/g (afterload-preload) x stroke volume/ventricle weight. The duration of the systolic phase and the height of peak pressure were determined from recording traces. Cardiac output, heart rate and aortic pressure were measured simultaneously during the experiments. Hearts that did not stabilize within lOmin from the onset of perfusion were discarded. The basal condition parameters of cardiac performance were measured after a 20min perfusion according to Sys, S. U., et al, (1997) / Exp RtoZ 200, p3109-3118.

After the 20min-control period, the treated hearts were perfused for 20min without or with HCNP-enriched saline. Other experiments were repeated in presence or absence of isoproterenol in the saline buffer. Each heart was tested for one concentration of HCNP. These values were not significantly different when the second perfusion step lasted as much as 40min. Results are shown on FTGl: inotropic effect of HCNP peptide on frog heart. Concentration-dependent responses of the peptide alone (left panel) and in the presence of isoproterenol (ISO) (right panel) are shown. Percent changes (Δ%) were evaluated as means ± S.E. of the experiments:

- concentration-dependent curve n=6 or 7 hearts;

- ISO n=3.

- Significance of difference from control values; *= p<0.05 ,**=p<0.01. Experiments performed on perfused isolated frog heart indicated that HCNP exerts a significant inotropic negative action from 10^"11 to 10^"7M (Fig. 1). Control experiments with either bovine serum albumin (1 to 15 nM) or the frog CGA (chromogranin A) amino-terminal fragment (CGA₄.₁₆) synthesized with the same method as for the HCNP (10 to 200 nM) did not affect the hemodynamic parameters (SV and SW). Since HCNP can be released together with catecholamines, its effect has been examined during the stimulation of cardiac β-adrenergic receptors with isoproterenol, the β-adrenergic agonist known to produce a positive inotropic effect Gattuso, A., et al., (1999) Am J Physiol 276, H633-641 et Sys, S. U., et al, (1997) J Exp Biol 200, 3109-3118. As shown in FIG. 1 (right panel), 10^"10M of HCNP was found to counteract the positive inotropic effect of isoproterenol (10^"8M). Example 4: Formulation of a medicament containing peptides of the invention The peptides are formulated as a citrate salt form for intravenous injections as it was reported for others commercialized vasoactive peptides (nesiritide). Briefly, the peptide according to the invention is diluted in 100/ l of physiological serum to obtain a final concentration of 1.5 10^"5M of peptide. This composition is then placed in sealed sterile vials before use. For a rat, an intraveinous injection is made every 3 hours during heart crisis.

Claims

CLAIMS I . Method of use of a polypeptide containing at least the sequence SEQ LD N°l or a derivative or a functional equivalent for the manufacture of a medicament for the modulation of cardio-vascular physiology. 2. Method of use of a polypeptide containing at least the sequence

SEQ LD N°l or a derivative or a functional equivalent for the manufacture of a medicament for the modulation of diuresis. 3. Method of use of a polypeptide according to claim 1 or 2, in which the polypeptide is Phosphatidylethanolamine Binding Protein (PEBP). 4. Method of use of a polypeptide according to claim 3, in which

PEBP is of human or bovine origin. 5. Method of use of a polypeptide according to claim 3, in which PEBP is of murine origin. 6. Method of use of a peptide of sequence SEQ ID N°l or a derivative or a functional equivalent for the manufacture of a medicament for the modulation of cardio-vascular physiology. 7. Method of use of a peptide of sequence SEQ ID N°l or a derivative or a functional equivalent for the manufacture of a medicament for the modulation of diuresis. 8. Method of use of a peptide according to claim 6 or 7, which the peptide is Hippocampal Cholinergic Neurostimulating Peptide (HCNP) or a derivative or a functional equivalent. 9. Method of use of a peptide according to anyone of claims 6 to 8, in which amino acids Xaa_1; Xaa₂, Xaa₄, Xaa₆ and Xaa₈ are defined as in SEQ ID N°2. 10. Method of use of a peptide according to claim 9, of human or bovine origin. II. Method of use of a peptide according to anyone of claims 6 to 8, in which amino acids Xaa_1; Xaa₂, Xaa₄, Xaa₆ and Xaa₈ are defined as in SEQ LD N°3. 12. Method of use of a peptide according to claim 11, of murine origin. 13. Method of use of a peptide according to claims 1 to 12 in combination with other cardiomodulators for the manufacture of a medicament for the modulation of cardio-vascular physiology.

14. Method of use of a peptide according to claims 1 to 12 in combination with other cardiomodulators for the manufacture of a medicament for the modulation of diuresis. 15. Method of use of a composition containing at least one peptide according to claims 1 to 12 in a pharmaceutically acceptable medium for the preparation of a medicament for the modulation of cardio-vascular physiology. 16. Method of use of a composition containing at least one peptide according to claims 1 to 12 in a pharmaceutically acceptable medium for the preparation of a medicament for the modulation of diuresis. 17. Method of use according to claim 15 or 16, in which the concentration of the peptide is from 10^"n to 10^"4 moles/L in the peripheral blood. 18. Method of use according to anyone of claims 15 to 17, further comprising other cardio-vascular modulators. 19. Method of use according to anyone of claims 15 to 17, further comprising other diuretics.