WO2002004005A2

WO2002004005A2 - Short peptides comprising histidine or alanine and their therapeutic use

Info

Publication number: WO2002004005A2
Application number: PCT/GB2001/003125
Authority: WO
Inventors: Joseph Chayen
Original assignee: Ks Biomedix Limited
Priority date: 2000-07-11
Filing date: 2001-07-11
Publication date: 2002-01-17
Also published as: WO2002004005A3; GB0017028D0; AU2001270794A1

Abstract

The present invention relates to the use of peptides, in particular alanyl and histidyl dipeptides, as therapeutic agents that act through stimulating the production of nitric oxide.

Description

PEPTIDES AND THEIR THERAPEUTIC USE

This invention relates to peptides, in particular dipeptides, containing alanine and or histidine for use as therapeutic agents. 5

It is known that muscle contains significant levels of histidyl dipeptides. Examples of these dipeptides include carnosine (β-alanyl-L-histidine), its Nι_ derivative anserine, and homocarnosine (y-aminobutyryl-L-histidine). The most common of these dipeptides is carnosine, which has been investigated

10. extensively in a variety of physiological processes, but, at present, its precise physiological function remains unknown. Carnosine is recognised as a potent buffer at physiological pH and is also known to restore functional capacity to fatigued muscles. Carnosine has also been reported to have several other roles including as a neurotransmitter, as an antioxidant, scavenging free radicals,

15 stabilisng membranes, and chelating metal ions (Quinn et al)

A description of the use of carnosine in the treatment of heart failure is contained in US5585396. In this patent there is a detailed description of the administration of carnosine to enhance cardiac contractile function and to 0 stimulate heart rate. There is also mention of the use of carnosine to accelerate wound healing and an indication that carnosine may increase collagen deposition.

The gaseous small molecule nitric oxide (NO) has been discovered to be an 5 important biological mediator with physiological and pathophysiological roles in nearly every organ system. In addition, nitric oxide plays a role in immunity and inflammation. Research into the pathology of various diseases has indicated that there is a link between the development of certain conditions and low nitric oxide production. There is a wide range of diseases that show this potential involvement, including GI disorders (Perner et al), skin complaints (Bruch-Gerharz et at), insulin resistance, cardiovascular complications (Sartori et al ), and . renal insufficiency (Noris et al). The range of diseases cited illustrates the broad involvement of this molecule in vivo.

It has now been found that administration of certain peptides can be used to increase nitric oxide production. In particular, histidyl dipeptides may be administered to increase nitric oxide production.

Accordingly, in one aspect the present invention a pharmaceutical composition for stimulating the production of nitric oxide comprising one or more peptides selected f om dipeptides, tripeptides, tetrapeptides, and/or pentapeptides, wherein each peptide comprises at least one alanine or histidine amino acid residue, optionally together with one or more pharmaceutically acceptable carriers, diluents or excipients.

In another aspect, the present invention provides the use of one or more peptides selected from dipeptides, tripeptides, tetrapeptides, and/or pentapeptides, wherein each peptide comprises at least one alanine or histidine amino acid residue, in the manufacture of a medicament to stimulate the production of nitric oxide.

The peptides used can be naturally occurring peptides such as carnosine. Alternatively, they could be synthetic peptides (i.e. non-naturally occurring)> The function of the peptides is to deliver the necessary substrate for NO synthesis.

The medicament or pharmaceutical composition of the invention may be adapted for administration by any appropriate route, for example by the oral ( cluding buccal or sublingual), rectal, nasal, topical (including buccal, sublrngual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carriers) or excipient(s).

Pharmaceutical formulations adapted for oral aό^ninistration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in- water liquid emulsions or water-in-oil liquid emulsions.

Pharmaceutical formulations adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6), 318 (1986).

Pharmaceutical formulations adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For applications to the eye or other external tissues, for example the mouth and skin, the formulations are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in- water cream base or a water-in-oil base.

Pharmaceutical formulations adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

Pharmaceutical formulations adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or enemas.

Pharmaceutical formulations adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is adrninistered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for aclrninistration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient. Pharmaceutical formulations adapted for adm sfration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators.

Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical formulations adapted for parenteral adminisfration include aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyopbilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient. The dosage preferably ranges from 0.1 to lOOmg/kg depending on the route of adminisfration. .

It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents. In one preferred embodiment the medicament of the present invention is administered by the oral, transdermal or intramuscular route.

Alternatively, the peptides of the present invention can be incorporated into a foodstuff. Therefore, in another aspect the present invention provides a foodstuff supplemented with one or more peptides selected from dipeptides, tripeptides, tetrapeptides, and/or pentapeptides, wherein each peptide comprises at least one alanine or histidine amino acid residue.

The foodstuff of the invention may be a dry product (with approximately 5 to 12% moisture), a semi-moist product (with approximately 12 to 70% moisture) or a wet product (with approximately 70 to 90% moisture).

The foodstuff according to the present invention encompasses any product that an animal, including humans, consumes in its diet. The invention covers standard food products as well as snacks (for example, snack bars, biscuits and sweet products).

The foodstuff is preferably packaged. In this way, the consumer is able to identify, from the packaging, the ingredients in the foodstuff. The packaging may be metal (usually in the form of a tin or flexifoil), plastic (usually in the form of a pouch or bottle), paper, glass or card. The amount of moisture in any product may influence the type of packaging, which can be used or is required.

The foodstuff can be provided as a food supplement. The food supplement can be a powder, sauce, topping, packet or tablet that can be administered with or without an additional foodstuff. Where the food supplement is administered with an additional foodstuff, the food supplement can be administered sequentially simultaneously or separately. The food supplement may be mixed with the foodstuff, sprinkled or poured over the foodstuff or served separately. Alternatively, the food supplement can be added to a liquid provided for drinking such as water or milk.

From the data enclosed herein, it can be seen that histidine and alanine are good substrates for stimulating the production of nitric oxide. Therefore it is well know to a person skilled in the art that small peptides containing these amino acids, delivered in an appropriate manner can be used, to stimulate the production of nitric oxide. The peptides of the present invention can contain only one type of amino acids, e.g. a pentamer of histidine residues, or a mixture of histidine and alanine residues, or a combination of one to four other naturally occurring amino acids with alanine and/or histidine.

In one preferred embodiment of the present invention, the peptide is a dipeptide, preferably an alanyl dipeptide or a histidyl dipeptide. The histidyl dipeptide is most preferably selected from carnosine, anserine or homocarnosine.

Without being bound by theory, it is believed that the peptides, e.g. histidyl dipeptides, and carnosine in particular, act as substrates for nitric oxide synthase. The nature of the mammalian nitric oxide synthase enzymes has been reviewed by Stuehr et al, Adv. Enzymol., (1992);65:287-346. The production of NO is believed to result from the reaction of arginine and NADPH. However, it has now been found that the histidyl dipeptides are also effective in stimulating the production of nitric oxide, and therefore administration of histidyl dipeptides may enable the nitric oxide to exert a beneficial effect in numerous processes. For example, nitric oxide stimulates cytosolic guanylate cyclase activity, resulting in the production of neuro transmitters in the brain. It also stimulates the production of cyclooxygenase II and acts as a vasodilator in blood vessels.

The present invention may also be Useful in the treatment of the following disorders, all of which have been associated with nitric oxide imbalance: Acute myocardial ischemia due to impaired coronary blood flow regulation (Feliciono et al); Renal hemodynamic and nonhemodynamic disorders (Noris et al); Insulin-resistance (Sartori et al); Lower urinary tract disorders (Mumtaz et al); Brain damage including Alzheimer's disease (Ronson et al, Licinio et al, McCarty); Chronic inflammation, including bowel disorders and asthma (Goodwin et al, Bhagat et al, Perner et al); and Cancer (Wink et al).

This aspect of the invention is illustrated in the following Example. Example

The measurement of nitric oxide synthase activity may be carried out substantially as described in Mehdizadeh et al., J. Histochem. Cytochem. 43:12351995), but with the substitution of carnosine or a derivative for arginine. Briefly, samples of liver are cut into 14 μM sections in a cryostat (Bright Instruments: Huntingford, U.K.;) fitted with an automatic drive to ensure a constant thickness of the sections (Chayen J. and Bitensky L, Practical Histochemistry. 2nd Edition. (1991) Wiley, New York, London). The measurement of guanylate cyclase activity is carried out in media containing 10 mM guanosine triphosphate lithium salt; 32 mg/ml lead ammonium citrate/acetate (LACA) dissolved in a minimum amount of dilute ammonia; 0.2 mM sodium acetate; 20 mM manganese chloride; 10 mM sodium fluoride; 0.4 mM L-p-bromotetramisole; 2.5 mM sodium azide; and 35 g/100 ml Polypep 5115. The sections are reacted in an atmosphere of 95% oxygen and 5% C0₂ in the presence and absence of 5 mM carnosine and 1 mM NADPH.

At the end of the reaction, the samples are rinsed in water, air-dried and mounted in absolute glycerine. The coloured reaction product present is measured with a Vickers M85A scanning and integrating microdensitometer (Biorad Instruments, York, U.K.) with a x40 0.6 NA objective and light measurementat 585 nm wavelength. The reaction may be carried out at different time intervals.

Table 1 shows the results from experiments carried out using carnosine or various amino acids in place of arginine. MIE x 100 refers to the mean integrated extinction multiplied by 100.

Table 1

The results show that carnosine has greater activity than arginine in stimulating the production of soluble guanylate cyclase.

References

1. Feliciono et al, Clinical Cardiology, (1999); vol 22, No 12:775-786.

2. Noris et α/., Proc. Assoc. Am. Physicians, (1999); vol 111, No. 6: 602- 610.

3. Sartori et al, J. of Hypertension, (1999); vol 17, No. 11:1517-1525.

4. Mumtaz et al, Endothelium-New York, (1999); vol 7, No 1 : 1-9.

5. Ronson et al, Cardiovascular Research, (1999); vol 44, No 1: 47-59.

6. lAcvcάo et al, Molecular Medicine Today, (1999); vol 5, No 5: 225-232. 7. Goodwin et al, FASEB Journal, (1999); vol 13, No 10:1121-1136.

8. Bhagat et al, Current Opinion in Nephrology and Hypertension, (1999); vol 18, No 1:89-96.

9. Perner et al, Alimentary Pharmacology & Therapeutics, (1999); vol 13, No 2: 135-144. 10. McCarty, Medical Hypotheses, (1998); vol 51, No 6: 465-476.

11. Wink et al, Biochemistry-Moscow, (1998); vol 63, No 7:802-809.

Claims

1. A pharmaceutical composition for stimulating the production of nitric oxide comprising one or more peptides selected from dipeptides, tripeptides, tetrapeptides, and/or pentapeptides, wherein each peptide comprises at least one alanine or histidine amino acid residue, optionally together with one or more pharmaceutically acceptable carriers, diluents or excipients.

2. A pharmaceutical composition as claimed in claim 1 wherein the composition comprises a dipeptide.

3. A pharmaceutical composition as claimed in claim 2 wherein the dipeptide is an alanyl dipeptide.

4. A pharmaceutical composition as claimed in claim 2 wherein the dipeptide is a histidyl dipeptide.

5. A pharmaceutical composition as claimed in claim 4, wherein the histidyl dipeptide is any one of carnosine, anserine or homocarnosine.

6. A pharmaceutical composition as claimed in any one of claims 1 to 6 for use in the treatment of inflammation, insulin resistance, and/or renal insufficiency

The use of one or more peptides selected from dipeptides, tripeptides, tetrapeptides, and/or pentapeptides, wherein each peptide comprises at least one alanine or histidine amino acid residue, in the manufacture of a medicament to stimulate the production of nitric oxide.

8. The use as claimed in claim 7 wherein the peptide is a dipeptide.

9. The use as claimed in claim 8, wherein the dipeptide is an alanyl dipeptide.

10. The use as claimed in claim 8, wherein the dipeptide is a histidyl dipeptide.

11. The use as claimed in claim 10, wherein the histidyl dipeptide is any one of carnosine, anserine or homocarnosine.

12. The use as claimed in any one of claims 7 to 11, wherein the medicament is adapted to be administered by the oral, transdermal or intramuscular route.

13. The use as claimed in any one of claims 7 to 12, wherein the medicament is for the treatment of inflammation, insulin resistance or renal insufficiency.

14. A method for stimulating the production of nitric oxide in vivo, comprising administering to a patient a therapeutically effective amount of one or more peptides selected from dipeptides, tripeptides, tetrapeptides, and or pentapeptides, wherein each peptide comprises at least one alanine or histidine amino acid residue.

15. The method as claimed in claim 14, wherein the peptide is a dipeptide.

16. The method as claimed in claim 15, wherein the dipeptide is an alanyl dipeptide.

17. The method as claimed in claim 15, wherein the dipeptide is a histidyl dipeptide.

18. The method as claimed in claim 17, wherein the histidyl dipeptide is any one of carnosine, anserine or homocarnosine.

19. The method as claimed in any one of claims 14 to 18, wherein the patient is suffering from inflammation, insulin resistance or renal insufficiency.

20. A foodstuff supplemented with one or more peptides selected from dipeptides, tripeptides, tetrapeptides, and/or pentapeptides, wherein each peptide comprises at least one alanine or histidine amino acid residue.

21. The foodstuff as claimed in claim 20, wherein the peptide is a dipeptide.

22. The foodstuff as claimed in claim 21, wherein the dipeptide is an alanyl dipeptide.

23. The foodstuff as claimed in claim 21, wherein the dipeptide is a histidyl dipeptide.

24. The foodstuff as claimed in claim 23, wherein the histidyl dipeptide is any one of carnosine, anserine or homocarnosine.

25. A kit comprising a pharmaceutical composition as claimed in any one of claims 1 to 6.