AU694701B2 - Peptides with organo-protective activity, their preparation and use - Google Patents

Description

S F Ref: 258860D2

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Appl icant: Predrag Sikiric Jurisceva. HR-41000 Zagreb

CROATIA

Marijan Petek Visnjica 29 ~:HR-41000 Zagreb *o

CROATIA

Sven Seiwerth Palnioticeva 17 HR-41000 Zagreb

CROATIA

ZeIjko Grabarevic Lermanova. 12A HR-41000 Zagreb

CROATIA

Ivo Rotkvic Cvjetno naselje 1/21 HR-41000 Zagreb

CROATIA

Marko Duvnjak R. Luxemburg 4 HR-41000 Zagreb CROATI A Branko Turkovic Bauerova. 19 HR-41000 Zagreb

CROATIA

Stjepan Mise Ruzveltova 37 HR-58000 Split

CROATIA

Ernest Suchanek Aleja V. Popovica 125 HR-41000 Zagreb

CROATIA

CONT 5845 Nmmw 6' 1 Boris Mildner Kopernikova 34 HR-41000 Zagreb

CROATIA

Ivan Udovicic Ennetmoserstr.16 CH-6370 Stans

SWITZERLAND

Actual Inventor(s): Address for Service: Invention Title: Predrag Sikiric, Marijan Petek, Sven Seiwerth, Zeljko Grabarevic, Ivo Rotkvic, Marko Duvnjak, Branko Turkovic, Stjepan Mise, Ernest Suchanek, Boris Mildner, Ivan Udovicic Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Peptides with Organo-Protective Activity, Their Preparation and Use 0 0* 000 04 4 0 e 50 00 0 0 *r 0 *0 .0 O 0 *400 0 40 0 000 0 The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845 Peptides with Organo-Protective Activity, Their Preparation and Use This invention relates to new peptides having high biological activity of the same type as known natural compound BPC, but with shorter amino acid chains.

Background of the invention A biologically active protein with organo-protective activity which was isolated from human or animal body and named BPC (Body Protecting Compound) was disclosed in EP 0 432 400 and also published: P.Sikiric et al, Exp.Clin.

Gastroenterol., 1, 15-26, 1991. This protein has high molecular weight of about 40 000 5000 Daltons, with only partial determined structure. This compound has very broad spectrum of biological activities like: ulcer protective, hepatoprotective, antiviral, antiedematous, general 15 antiinflammation activity, antimalignant activity and others. It should be used in the therapy of cited diseases, .further in the therapy of diseases and disorders of nerve system, disorders of dopaminergic ethiology, surgery, stomatology, in curing of fertility and in veterinary medicine. But this broad spectrum of activity can be probably a consequence of undetermined structure or even of insufficiant purity or homogeneity of isolated compound

BPC.

e Summary of the Invention We have discovered synthetic peptides with only 8 to 15 amino acid residues in chain with the molecular weight between 900 and 1600 Daltons, that have biological activity of the natural body protecting compound BPC, but with increased selectivity.

Our new peptides are more economically produced and are less subject to the effect of side reactions because they contain less amino acid residues than BPC.

According to a first embodiment of the invention, there is provided a biologically active peptide consisting of from 8 to 15 amino acid residues and represented by the formula: Xaa Zaa Pro Pro Pro Xaa Yaa Pro Ala Asp Zaa Ala Xaa Xaa Xaa 1 5 10 in which: Xaa signifies a neutral aliphatic amino acid residue selected from the group consisting of i Ala, bAla, Leu, Ile, Gly, Val, Nle, and Nva, Yaa signifies a basic amino acid residue selected from the group consisting of Lys, Arg, Orn, and His, and Zaa signifies an acidic amino acid residue selected from the group consisting of Glu, Asp, Aad, and Apm.

"According to a second embodiment of the invention, there is provided a biologically active peptide having a molecular weight of 900 to 1600 Daltons and including the sequence: Zaa Pro Pro Pro Xaa Yaa Pro Ala :1 1 5 8 wherein Xaa signifies a neutral aliphatic amino acid residue selected from the group consisting of .i Ala, bAla, Leu, Ile, Gly, Val, Nle, and Nva, Yaa signifies a basic amino acid residue selected from the group consisting of Lys, Arg, orn, and His, and Zaa signifies an acidic amino acid residue selected from the group consisting of Glu, Asp, Aad, and Apm.

According to a third embodiment of the invention, there is provided a therapeutic composition containing a peptide according to the first or second embodiments of the invention in admixture with a pharmaceutically acceptable solid or liquid vehicle.

According to a fourth embodiment of the invention, there is provided a method of normalising organic function in an animal, comprising administering to said animal an effective amount of a peptide according to the first or second embodiments of the invention or a composition according to the third embodiment of the invention.

According to a fifth embodiment of the invention, there is provided a method for the prophylaxis or treatment of a condition or disease in an animal, comprising administering [N:\LIBFFJ00787:RRB to said animal an effective amount of a peptide according to the first or second embodiments of the invention, or a composition according to the third embodiment of the invention.

Description of the Invention In accordance with one aspect of the present invention, there is provided a class of synthetic peptides which exhibit surprisingly high biological activity, especially in the sense of body protection. In any case these synthetical compounds with well defined structure have great advantage in comparison to only partial defined high molecular protein BPC, which is obtainable only by difficult procedure from dubious natural source.

More particularly, this invention relates to a novel class of biologically highly active peptides, comprising from 8 to 15 amino acid residues, which are represented by the following basic structural formula using the three letter amino acid code and the numbers below each amino acid residue refer to the position of the residue in the peptide chain; *ot 44 [N:\LIBFF00787:RRB oo« *o* e e 9, BF]077*R Xaa Zaa Pro Pro Pro Xaa Yaa Pro Ala Asp Zaa Ala Xaa Xaa Xaa 1 5 10 wherein one or more amino acid residues are substituted.

The substituents Xaa,. Yaa and Zaa which may be employed are shown in the following table I.

Table I.

Residue Substituent Xaa a neutral aliphatic amino acid: Ala, bAla, Leu, lie, Gly, Val, Nie, Nva Yaa basic amino acid: Lys, Arg, Orn 2 His Zaa acidic amino acid: Glu, Asp, Aad, Apm Preferred peptides are as follows: Sed. Id. No: 1 15 Leu Glu Pro Pro Pro Gly Lys Pro Ala Asp Asp Ala Leu Gly Val 1 5 10 Seq. Id. No: 2 Gly Glu Pro Pro Pro Gly Lys Pro Ala Asp Asp Ala Gly Leu Val 1 5 10 Sea. Id. No: 3 Leu Glu Pro Pro Pro Leu Lys Pro Ala Asp Asp Ala Leu Gly Val 1 5 10

S

S

rrr 4 In accordance with another respect of the present invention there are provided analogic peptides in amide or carboxy terminated form at C terminus, having the structural formulae hereinabove described, and wherein at least one and at most 7 amino acid residues in region 1-15 are omitted and at least one of the remaining amino acid residues can be substitued according to described schema of substitution in table I.

Preferred peptides are as follows: Seq. Id. No: 4 Leu Glu Pro Pro Pro Leu Lys Pro Ala Asp Ala Leu Gly Val 1 5 10 14 Sea. Id. No: Gly Glu Pro Pro Pro Gly Lys Pro Ala Asp Ala Gly Leu Val 1 5 10 14 Sea. Id. No: 6 SGlu Pro Pro Pro Leu Lys Pro Ala 1 5 8 Sec. Id. No: 7 Asp Pro Pro Pro Ile Arg Pro Ala Asp S1 5 9 .i 9ss _C Sea. Id. No: 8 Glu Pro Pro Pro Leu Lys Pro Ala Asp 1 5 9 In accordance with another embodiment, peptides having the structural formules hereinabove described, and wherein at least one of amino acid residues can be omitted and at least one of the remaining amino acid residues can be substituted as indicated in table I, are transformed in cyclic form by the formation of a new bond CO-NH between the first and the last amino acid residues in the molecule.

Preferred peptides are as follows: Sea. Id. No: 9 Leu Glu Pro Pro Pro Leu Lys Pro Ala Asp Ala Leu Gly Val 2 5 10 13 15 Sea. Id. No: Gly Glu Pro Pro Pro Gly Arg Pro Ala Asp

I

2 5 9 Applicants have found that when employing the peptides as hereinabove described, such peptides display biological 20 activity equal to or greater than the parent protein BPC.

Pharmacological investigation of described peptides has been done on different common models "in vitro" and "in vivo" and following pharmacological properties were found: 4 pi II~- 9L~qlll IPPI P~-~~LLdlC I Gastric ulcer induced by restraint stress Albino Wistar (male) rats (180-200 have been used in the experiments. All the animals have been immobilized in a supine position for 48 hous at room temperature.

Immediately thereafter they were sacrified and measured the lesions. The peptides Sea. Id. No: 4, 6 and 2 have been applied in dosage of 10 gg or l0ng/kg i.p. or i.g. 1 hour before noxious procedure.

Both i.g. and i.p. applications, even in such low doses as 10 ng/kg b.w. were strongly protective.

2. Cysteamine model of ulcer Albino Wistar (female) rats (180-200g), have been used in the experiments. Cysteamine hydrochloride (dissolved in distil. water) in dosis of 400 mg/kg s.c. has been applied. 24 hours later the animals were sacrified. The peptides Seq. Id. No: 4 has been applied in dosage 1.0 Aig, 100 ng and 50 ng/kg i.p. and i.g. A strong and dose dependent protection has been obtained. The same activity has been found for both i.p. and also for intragastrical 20 route of administration.

3. Turpentine induced buccal edemas Animals: Wistar male rats (180-240g), N=10 in each group.

Turpentine was given intramucosally 0.02 ml/rat. Control: 0.9% saline intramucosally (0.02 ml/rat, 2 x) Peptides Seq. Id. No: 2 and 4 have been applied in doses 10 kig and 10 ng/kg, i.p. and 1 hour before turpentine. Buccal c- M 4 edemas were measured 24 hours after edema induction.

Statistical analysis: Mann-Whitney test. The investigated peptides were effective in dosis of 10 Jg/kg b.w.

4. Surqerv: the effect on the skin incissions Male Albino rats (10 for each experiment, 200-250g b.w.) have been used in the experiments. The rats with the skin wounds have been individually maintained in separate cages.

On each animal under light aether anaesthesia after the back skin showing 3 cm incissions were paralely placed through the skin 1.5 cm of either site of the midline. One wound was then coapted with two surgical clips, and the other left untreated. Peptide Sea. Id. No: 4, dissolved in saline, in doses of 1.0 and 1.0 ng/kg or saline ml/kg b.w. in control, have been applied intradermally between the wounds immediatelly after the injury.

A prominent healing effect of investigated peptide has been obvious on both wounds with surgical clips as well as those left without surgical treatment. After 5 days the treateC group showed smaller number of inflammatory cells and 20 significantly better developed reticulin fibres than in the control.

5. Effect on the experimental burns Male albino rats, Wistar strain (N 10, 200-250 g b.w.) have been used in the experiment. Under light aether 25 anaestesia the nasal mucosa has been exposed to a glowing cauter during 5 secons. The peptide Sea. Id. No: 4 has been 41 -BP IIII~ used in dosage of 10 ag/kg i.p. 1 hour before injury induction. Control: 5.0 ml/kg b.w. of saline, i.p. The applied procedure usually produced a prominent swelling of the snout and has been regularly fatal for controls (but not for treated animals!) within nine postwounding days. In contrast to controls, only very modest posttraumatic swelling of the snout has been observed. Subsequently the normal nasal respiration was only slight impa'red and survivial undisturbed.

Effect on fracture healing Male Albino rats of Wistar strain (270-300 g have been used in experiments. Left tibias were fractured manually in the middle part of the bone under aether anaestesia. No immobilisation was used and animals v allowed to move freely in cages. The animals were sacrified on 5 th 8 th 12 th and 30 th day after the injury induction.

Peptide Sea. Id. No: 4 has been given in the dosage of 10.0 pg/kg 1 hour before injury and subsequently once daily (last application 24 h before sacrificion).

Control group received simultaneously an equivolume of saline 5.0 ml/kg A significantly improved healing rate has been noted in all with peptide treated animals at each of the investigated intervals.

Noteworthly, all treated animals showed consistently lower 25 local posttraumatic hematome than control animals and they restored their lesioned function quite sooner.

e6* -1- 9 Antiviral activity Antiviral activity was investigated on new-born mice, BALB- C strain, 24 hours old, both sexes.

ARBO-viruses: TBE (=Tick Borne Encephalitis), Bhania, Dengue 1,2,3,4, Sinbis, West-Nile, Calovo, Hepatitis A, LCM (Limphatic choriomeningitis) and Herpes type I have been applied as viruses suspension in dilution 10- 2 (0.02 mi/mouse), prepared as in generally accepted, and injected i.c. or p.o. (Hepatitis In view of difference in virulence the doses were adjusted in such a way as to be comparable regarding LD 1 0 0 in 0.02 ml i.c. (or p.o., Hepatitis A) inoculum in a 10 2 dilution. In this way, we were able to compare the course of different virus infections, regardless possible inoculation of different viil-s concentrations.

The peptide Sea.Id.No: 4 was used in concentration of 20.0 gg/ml in 0.9% saline solution and applied only once in the dosage of the 2.0 g/kg i.c. or i.p.: t.

a) 2 hours before virus application (-2h) 20 b) simultaneously with infection Control: the same volume of the saline i.c. or i.p. The results are summarized in Table II.: the numbers in the table indicate period (days) till all virus infected mice in peptide or saline (control) treated 25 groups succumbed.

T RE ATM E NT Viruses inf ections saline -2hx. Ox i.p. i.c. i.p. i.c.

synthetic -2hx i.p. i.c.

fragment 4 Ox i.p. TBE a 5 5 Bhania a 5 5 Denguel1 a 5 5 Dengue 2 a 5 5 Dengue 3 a 5 5 Dengue 4 a 5 5 Sinbis a 5 5 WestNile a 5 5 Calovo a 5 5 Hepatitis A+ 5 5 LCI4 5 5 Herpes typel 5 5 None (Healthy) n.d. n.d.

n. d.

n. d.

n. d.

n. d.

n. d.

n. d.

n. d.

n. d.

n. d.

n. d.

n.d.

n. d.

n. d.

n. d.

n. d.

n. d.

n. d.

n. d.

n.d.

n. d.

n. d.

n. d.

n. d.

n. d.

a.

S S a a.

a a a.

9 Ga *aa.

a.

a

S

a.

a.

a n.d. n.d. n.d. n.d. n.d. -n.d.

ni. d. -non determined: animals heve been observed during 40 days after viruses application.

No death has been noted.

a -ARBO-viruses -perora). viruses application x -the animals were treated 2h before viruses application or simultaneously with viruses administration (0) ovs.saline P<0.001;8 mice per each group Antidepression activity For revealing of antidepressant activity the forcec swimming test according Porsolt et al, Eur.J.Pharmacol., 47, 379-391,1978, was used.

Male Wistar rats (180-240 g have been used for this experiment. The investigated peptide Sea.Id.No: 4 was giver at first day after pretest session and at second day one hour before experiment Control group: saline, i.p.

The animals were observed for 5 minutes. The time oj immovability was measured (Ti).

T

i in the control group was about 150 sec., in peptidE treated group was only 60-70 sec. This effect was from lone duration: after 16 days was still present.

Dose reponse: 10 Ag to 10 ng/kg: full effect 10 pg/kg: effect still present 1 pg/kg: effect in disappearance.

a *9 Effect on model of Parkinson disease For investigation the well known models for Parkinson disease have been used (Karakola s. et al, Pharmacol.

Toxicol., 67, 95-100, 1990): reserpine model and MPTP model.

NMRI- Hannover male (for MPTP model) or both sexes (for reserpine model) mice have been used.

a) MPTP was applied in the dosage of mg/kg i.p. once daily for six subsequent days, and then for next four days in a higher dosage of 50 mg/kg b.w. The investigated peptide Seq.Id.No: 4 was applied in dosage of 1.5 pg and 15.0 ng/kg 15 min. before each MPTP administration or 15 min. after each MPTP administration.

b) Reserpine was applied in the dose of 5mg/kg i.p.

The tested peptide was applied in the dosage of 10 pg or ng/kg i.p. 15 minutes before reserpine or 24 hours following reserpine administration in the same dosage.

Controls: an equal volume of the saline, 5 ml/kg b.w. i.p.

44 9.

A significant reduction of hypokinesia, rigidity 20 (catalepsy) and tremor have been observed after pretreatment with peptide.

*a In the MPTP model the pretreatment with this peptide strongly abolished the catalepsy development and diminished a the akinesia and tremor appearance.

4 .1 see Posttreatment (after 15 min) strongly prevented the- further development of MPTP catalepsy and markedly diminished akinesia and tremor. Usualy a high mortality noted after only one application of MPTP has been decreased in peptide pretreated as well in posttreated animals.

The effect of peptide administration in haemorrhaqic Shock Adult female Albino rats of Wistar strain (150-180 g) have been used in all the experiments.

The effects on haemorrhagic shock have been investigated in two series of experiments.

a) The animals were bled (1 ml over 3 minutes, 2 minutes of the rest) till the death. The removed blood volume producing fatal outcome has been assessed in peptide 15 Sea.Id.No: 4 (10.0 pg or 10 ng/kg or saline (5.0 ml/kg pretreated (15 min. before bleeding) animals. A significantly higher fatal blood volume loss in comparison with the control values has been consistently noted in group treated with the higher dose of peptides.

b) The blood pressure has been lowered by controlled blood volume removal and thereafter sustained at the 30-35 mm Hg value5 for 5 minutes period.

Then, the peptide Seg.Id.No: 4 (10.0 ig or 10.0 ng/kg b.w.) or saline (control, 3.0 ml/kg were i.v. applied.

Contrasting with the controls, significantly raised and sustained blood pressure values and complete death abolition have been noted in peptide treated animals.

Therefore, it seems that the investigated peptide is highly effective in ameliorating the consequences of the blood volume loss.

11.) The influence on lethal radiation NMRI Hannover mice, 5 6 weeks old, of both sexes (18 22 g have been used in the experiments. Synthetic peptide Sea.Id.No: 4 has been applied in dosage 20 pg/kg i.p. 1 hour before or after irradiation. The control animals received simultaneously an equivolume of 0.9% saline i.p. (5.0 ml/kg). Naive, healthy animals kept in usual conditions, not subjected to drugs or irradiation treatment served as healthy control.

Irradiation: Teatron 80 (Co 60, 2200 Ci). The unanaesthetized mice in cages, 16 mice per group, were 15 exposed to whole body irradiation with a supralethal dose of 9 Gy in the irradiation area of the 20 x 20 cm, distance *80 cm.

Deaths were recorded twice daily for 30 days.

All control animals died within 7-12 days (LD 10 0 12 20 No death has been noted in healthy (non irradiated) 3 .3 controls.

Relative to the control data, no difference has been observed in groups treated with peptide 1 hour after irradiation. However, a significant survival has been noted when the compound has been applied 1 hour before radiation.

a -Y rC ~4p-s~ PeCI II~ -9~ A 70% increase in LD 100 relative to the control could be noted.

12. Influence on induced malformation NMRI Hannover female mice, two months old, 25 g but no previously exposed to copulation have been used in all experiments. The female mice in the estrus phase have been mated overnight with well experienced healthy male mice.

The mice have been sacrified after 20 days of pregnancy.

Vitamin A has been applied in a dose of 15700 U/kg b.w., at tenth day of the pregnancy (0.05 ml/kg Peptide Sea.Id.No: 4 has been given simultaneously in dosage of the 10 pg or 10 ng/kg b.w.,i.p.

Control: saline 5.0 ml/kg i.p.

Animals non treated with vitamin A receiving at the same time i.p. saline or peptide 4 in the same dosage served as healthy control.

No malformation have been noted in healthy (saline) or peptide treated group.

The results summarized in the Table III. show surprisingly 20 a significant and dose dependent protective effect of investigated peptide against vitamin A induced malformations.

*o ill slPB 16 Table III. Macroscopic examination Treatment Total number of fetuses Number of malformated fetuses control group 55 0 (saline) vitamin A 54 36 vitamin A peptide 4 36 13 ng/kg vitamin A peptide 4 18 1 gg/kg .c 13.) Acute toxicity Acute toxicity of synthetic peptides was determined in female mice (weight ca. 20 Groups of 6 animals were used for each experiment and control.

Peptides Seq.Id.No: 2,3,4 and 6 were applied in the dosage 8, 25 and 50 mg/kg i.v. Control group was treated with saline 5.0ml/kg The atimals have been observed for signs of toxicity for next 15 days.

In the applied dosage no signs of toxicity or death have been observed. An interesting phenomenon of significantly increased motility and livelines in duration of 2 hours after application of peptides was observed.

Following the results of pharmacological investigation these peptides exhibit activity in the sense to protect organism against stress ard diseases, in general to normalize the organic functions.

Their use should be also effective for prevention and therapy of several human or animal diseases and disorders.

More particularly, these compounds will be used for the treatment of: 15 stress induced disorders and diseases, gastrointestinal ulcers of diverse ethiology, inflammation and edemas of diverse ethiology traumas, burns, bone fractures and general in surgery, 20 viral infections, shock, Parkinson's disease, for protection of damages of radiation, for protection of malformations.

25 In general, the described peptides can also be employed in a wide variety of pharmaceutical compositions in combination with a non-toxic pharmaceutical carrier or vehicle such as a filler, non-toxic buffer, or -18physiological saline solution. Such pharmaceutical compositions can be used topically or systemically and can be in any suitable form such a. a liquid, solid, semi-solid, injectable ion, tablet, ointment, lotion, capsule, lingualette or similar.

These peptides will be in general administered in a dosage of from 10- 5 to 10- 2 mg/kg of body weight, when applied systemically. When administered topically, they will be used in higher concentration, e.g. 0.1% to Very favourable is the absence of any signs of toxicity until doses of 50 mg/kg b.w.

and also good activity of compounds by peroral administration (intragastrically).

The peptides described herein can be synthesised using step by step condensation of protected amino acids in homogenous liquid system or preferably using solid phase method. For preparation of cyclic peptides partially protected linear peptides of desired length are prepared as with alkyl ester group on C-terminus, which is converted into azide, following coupled and deprotected. Alternatively partially protected linear peptide with free terminal groups can be cyclized by diphenylphosphorylazide in very 15 diluted solution.

14.) Protective effect on liver and pancreas Protective effect of newly synthesised peptide, coded Seq. Id. No.: 2, 3, 4, 6 having been compared with reference standards (bromocriptin, amantadine, somatostatin) in different experimental models of liver and pancreas injury in the rates: 25h bile duct 20 ligation, 24h bile duct hepatic artery ligation, 48h restraint stress, CC1 4 application.

The used peptide, applied in range between 10 p.g 10 ng/kg either i.g. or i.p.

significantly prevent the liver and pancreas necrosis, as well as fatty changes in animals subjected to 24h bile duct ligation, 24h bile duct hepatic artery ligation, 48h restraint stress, CC1 4 application. Biochemical values of bilirubin, SGOT, SGPT are completely in line with mentioned macro/microscopically data.

[N:\LIfaa]0053:HRNV The effect on kidney lesions a) Unilateral nephrectomy In experiments Wistar albino rats of both sexes, 190-250 g, were used. A unilateral nephrecromy has been done, and as a result, an increase of the remaining kidney weight has been observed in control as well as in groups treated with p.eptides Seq.Id.No.2,3,4,6, given in 10 ig/kg b.w.i.p. lh before surgery. In peptides treated groups a decreases of the remaining kidney raise has consistently been observed.

Biochemical parameters in both groups have been compared. It seems that all of the used ameliorate the function of the remaining kidney.

4* 15 b) Gentamycin application 4In experiments Wistar albino rats, female were used. Tubular damages were induced with gent.amycin 4 application, in the dosage of the 40 mg/kg applied one daily during a period of 30 days. All of the animals were sacrificed 5 days following last drugs S* application. Significant tubular lesions have been observed in control groups, whereas significantly Slesser microscopically lesions were consistently been found in all groups treated with above mentioned peptides.

e c) Mercury chloride In experiments Wistar albino rats were used.

Mercury chloride was applied in the dosage of the 2mg/kg and peptides Seq.Id.No.: 2,3,4,6 in the dose of the 20 pg/kg lh before mercury chloride application. Unlike control data biochemical as well as microscopically results showed a protective effect in groups treated with mentioned peptides.

16.) The influence on testis lesions The influence of peptides Seq.Id.No.: .2,3,4,6 on testis lesions has been investigated by ketoconasol application (24 mg/kg testosterone (30 mg/kg or 6 Gy irradiation. Peptides Seq.Id.No.: 2,3,4,6 were given lh before. Peptides Seq.Id.No.: 2,3,4,6 demonstrat.ed a protective effect.

17.) Fertility/Commercial breeding a) peptides Seq.Id.No. 2,3,4,6 and fertility. The influence on oligoasthenospermis. The research was performed on 10 men, age 30-40 years, with diagnosis of oligoasthenospermis. From these patients an ejaculate was taken after 3 to 4 days 0* 0 of abstinence. After liquefaction (30min) to the of ejaculate is added in layer of 0.5ml of medium. The control medium consisted of HAM-F with 10 of deactivated chordal serum.

Experimental medium contained additionally 2 pg/ml or 4 pg/ml of BPC. After traveling time for 21 minutes at 37"C in atmosphere containing 5% of CO,, in the Horwell Fertility Chamber was deterined the number of progressive movable, movable on place and immovable spermatozoa in 1 ml of ejaculate.

Preliminary results show no effect on mctility of spermatozoa of lower concentration of the mentioned peptides. However, in their higher concentration (4pg) a significantly of motility relative to control was determined.

Peptides Seq.Id.No. 2,3,4,6 and reproduction processes The mentioned peptides were investigated in mice with previous history of three pregnancies. 20 days after cessation of the last lactation, the animals were subjected to copulation. The used peptides were given in a dose of the 10 gg/kg b.w.i.p. once daily during all period of pregnancy (19-21 days) and lactation (next three weeks). Control group received simultaneously an equivolume of saline.

0 The rumber: of offspring per female and the weight of offspring were recorded.

A careful statistical analysis revealed constistently a significantly increased number of offspring per female, as well as surprisingly, no 25 difference between the body weight at the each of the studied time intervals. All of the female were allowed to recover for next 25 days after cessation of the lactation. Then, they were again subjected to copulation for investigaticn of the effect on fifth pregnancy.

With regard to control group, an increased fertility rate with increased capability to lactation was registered in all treated groups treated with mentioned peptides.

It should be noted that mentioned peptides could improved the fertility in quite old mice. As a conclusion, these results are in line with overall beneficial peptides capacity and in vitro data obtained using human sperm. Peptides capacity and in vitro data obtained using human sperm. 1 beneficial peptideoPa c) Commercial breeding 1419 healty pigs (from total 4306 pigs) received immediately after birth 10 gg of the peptides Seq.Id.No.2,3,4,6, other 1440 at the 13th day of life, lh before castration, whereas remaining 1477 received only conventional Fe-therapy. Contusion, culling, mortality, body weight and food S* consumption were assessed aftr 4 weeks. The used 20 peptides, applied once significantly decrease S• culling rate (in the groups treated with peptides immediately after the birth) and contusion rate (in all groups treated with peptides). Same body weight was obtained in peptides treated animals with 25 markedly lower consumption of food. Influence on

**S

r" infection: 40 weaned pigs affected with E.colXi enterocolitis, 28 days old, were investigated. The used peptides were applied in 20 animals (10 pg/kg One month later a significantly lowering of mortality rate was noted in peptides treated animals.

In another experiment mortality rate after the first week was investigated in a total number of 1200 healthy pigs. 400 animals received the mentioned peptides immediately after the birth, in addition to conventional fe-thereapy. A significantly lower mortality was noted in mentioned peptides treated (10 gg/kg b.w.i.m.) treated groups.

18.) Influence on amphetamine behavior The influence of peptides Seq.Id.No,: 2,3,4,6 was investigated in a dosage of the 10 gg/kg or ng/kg 5 minutes before or simultaneously with amphetamine application. In the groups treated with mentioned peptides a significant decrease of the amphetamine induced behavioral disturbances was noted.

19.) Bleeding time The influence of the peptides Seq.Id.No. 2,3,4,6 was investigated by usual bleeding assays (tail 20 cutting or incision) in mice and rats, with or So.

without heparin application (1000 UI/kg s.c. 3h before bleeding). The investigated peptides pg/kg or 10 ng/kg were applied simultaneously with bleeding induction. In all animals treated with studied peptides a significant decrease of the bleeding time was noted.

The influence on consequences of castration A modified procedure according to Allen-Doisy method has been done. Different dosages were applied (10 pg or 10 ng/kg in single application or in once daily regimen, as pre treatment or post treatment. In groups treated with the investigated peptides a prevention or reversion of different castration consequences vaginal epithelium atrophy, oesteoporosis).

*00 9* *9 *e o 9 to 9 *9 9 21.) Hypertension Hypertension was induced by unilateral renal artery occlusion. After 10 days, the animals were treated with peptides Seq.Id.No. 2,3,4,6 (10 pg or 10 ng/kg or saline, using various protocols (single or continuos application) In groups treated with the studied peptides a significant decrease of the raised blood pressure values was observed.

22.) The influence on experimental diabetes mellitus 20 Wistar male albino rats, 175-230 g were used for streptozotocin or alloxan induced diabetes mellitus. Peptides Seq.Id.No. 2,3,4,6, applied in the dosage of the 10 or 100 pg/kg significantly delay the diabetes onset in streptozotocin treated animals. Moreover, they significantly prolonged the survival time.

23.) The influence on thyreoparathyreoidoctomy A total ablation of thyreoparahtyorids glands in Wistar rats has been performed. Thereafter, peptides Seq.Id.No. 2,3,4,6 (10 ng/kg or saline (5 ml/kg using different protocols (single or repeated treatment). In groups treated with the studied peptides the consequences of glands removal have significantly been reduced.

24.) Carotid artery legation Animals were sacrificed 3 hours after both carotid arteries legation. One hour before all of the animals were treated with peptides Seq.Id.No.

2,3,4,6 (10 n/kg or saline (5 ml/kg In investigated peptides treated groups 15 a significantly less brain edema has been found.

25.) Adrenal glands For adrenal glands injury induction the animals received aniline (300 mg/kg s.c. during 7 days).

All of the animals were simultaneously treated with peptides Seq.Id.No.: 2,3,4,6 (10 pg/kg ng/kg or with saline (5 ml/kg The weight of the adrenals and microscopically changes were assessed. Relative to aniline control group, the investigated peptides significantly reduced the aniline-induced increase of the adrenal glands weight in both dosages. Microscopically protection was dose dependent. As it is in y generally accepted, that aniline induced disturbances are related to ACTH hyper secretion, a causal relationship could be established between studied peptides and ACTH. This seems to be also supported by the finding of the same weight in groups treated with the mentioned peptides as in the naive healthy control group.

Additionally, the animals were subjected to 48 h restraint stress procedure, after the pretreatment with investigated peptides (10 pg or 10 ng/kg A decrease of the raised weight of the adrenals in the studied peptides treated animals was consistent with corresponding microscopically observation.

26.) Temperature disturbances An increased of body temperature was provoked by application of yeast solution (4000 mg/kg and a temperature decrease was induced using an immersion in cold water, temperature 4oC, -or reserpine application (5 mg/kg Different regimens of peptides Seq.Id.No. 2,3,4,6 (10 ng/kg investigated on both increased as well as *I ee decreased temperature, have consistently been demonstrated to normalize both temperature disturbances.

27.) Influence on hematopoeasis Albino mice were injected with cytostatics (Endoxan, Vincristin, Adriablastin, Cytosina3a~--YI~ b arabinosid) in LD 50 doses. One hour before cytostatic application, the animals were treated with peptides Seq.Id.No. 2,3,4,6 in a dose of pg, whereas controls received an equivolume of the saline. The animals were sacrificed on 3,5,7 and 11th day of experiment. Parameters in blood (E, Hb, Htc, L, Tb abs. number of neutrophiles), bone marrow, cytology and histology of liver and spleen were assessed.

On the third day of experiment no difference between the groups were observed in L,E,Tb and neutrophiles values, unlike intact haematopoetic cells in bone marrow of the animals treated with peptides, strongly contrasting with aplasia noted in the controls.

The number of neutrophiles and leukocytes significantly increased in animals treated with peptides till fifth day, reaching the normal values till seventh day, unlike controls where 20 normalization should not be observed before llth day.

28.) Pain reduction Pain was induced by an intraperitoneal application (0.2 ml/mouse) 2% MgS04 or 0.6% acetic acid.

Various regimens of peptides Seq.Id.No. 2,3,4,6 relative to the dose or time of application (10 ig or 10 ng/kg simultaneously or 30 min before) significantly reduce the writhing time in comparison with the control groups.

e r I II ebl is 28 29.) Infection induced by Rickettsias 0.2 ml of suspension of Coxielle Burnetti, dissolved 10-9, was injected in the suitable eggs.

In the eggs, the peptides Seq.Id.No. 2,3,4,6, (1 pg, 10 pg, 100 pg and 1 ng) were also injected.

The survival time was significantly longer in the treated groups than in the controls.

Influence on tumor cells a) Two cell lines L-924 and melanoma B-16 were cultivated in vitro under standard conditions. In vitro the influence of peptides Seq.Id.No. 2,3,4,6 on growing of' the mentioned cell cultures was investigated. Two days following the initiation of cell cultures, mentioned peptides were added in 3 15 concentration (0.1 ml). Three days later the numbers of cells per cultures were determined by 4 cell counter. The results showed an inhibitory effect of the mentioned peptides on -16 melan6ma cells.

20 b) Erlich's ascites tumor (EAT) is the tumor which can grow in all strains of mice, either as ascitic or solid, depending upon way of administration of tumor cells. The influence of incubation of EAT cells with peptides Seq.Id.No. 2,3,4,6 on survival time of mice, injected with these cells subcutaneously or intraperitoneally, was investigated. The animals were observed during the period of 45 days. Control of 15 male mice, NMRI strain, received 0.4x106 EAT cells. Prior to the injection tumor cells were incubated for one hour at 4'C in saline. Volume of injection was 0.2 ml.

Median survival time in this group was 36 days, and only 3/15 survived 45 days.

Experimental group of 15 NMRI male mice received the same dose of tumor cells, in the same volume, but these cells were previously incubated in mentioned peptides solution (2 g/ml). Only 2/15 mice died during the observed period (45 days), where all others survived more than 45 days.

Difference between the control and the experimental group was significantly different The same results were observed if the same procedure was carried out and the EAT injected i.p. (instead 15 c) The mice were used for investigation of the influence of peptides Seq.Id.No. 2,3,4,6 (given in a dose of the 10 g/kg on lung metastases induced by melanoma or aplastic mamas cercinoma 20 cells injection. In peptides treated groups a significant decrease of lung metastases number was determined.

Based on the above described data, it could be stated that mentioned peptides prolonged survival time of the animals with tumors, showing an anti tumor activity.

1 U h i 31.) Anti depression activity For revealing of antidepressant activity the forced swimming test according Porsolt et al, Eur. J.

Pharmacol., 47, 379-391, 1978, was used.

Male Wistar rats (180-240 g b.w. have been used for this experiment. The investigated peptide Seq.Id.N. 4 has been applied according to the described protocol. Briefly, control animals received aline, the assessment were for 5 min, and the time of immobility has been calculated. The immobility time in the control was 150 sec, whereas in the peptide treated animals the values of only 60-70 sec have been observed. A dose response curve could be calculated as follows: 10 ug 10 ng/kg 15 full effect, 10 pg/kg b.w. the activity still present, 1 pg/kg the effect not present.

The following results of pharmacological investigation of these peptides exhibit activity in the sense to protect organism against stress and 20 diseases, in general to normalize the organic functions.

Their use should be also effective for prevention and therapy of serveral human or animal diseases and disorders. More particularly, these compounds will be used for the treatment of: stress induced disturbances and illnesses disturbances and lesions of liver and pancreas atrophy of testis and sperms mobility 1 14 i 1 111 ~RIPCII~I~ I~ 31 fertility disturbances commercial breeding improvement rickettsias illnesses thyreoparathyreoidoctomy diabetes mellitus disturbances of adrenal gland disturbances of hematopoetic system disturbances of coagulation disturbances of bleeding 10 post-castration/menopausal disturbances ishemic and toxic lesions psychiatric disturbances hypertension disturbances of body temperature pain tumor depression In general, the described peptides can also be employed in a wide variety of pharmaceutical compositions in combination with a non-toxic p II pharmaceutical -carrier or vehicle such as a filler, non-toxic buffer, or physiological saline solution.

Such pharmaceutical compositions can be .used topical or systematically and can be in any suitable form such as a liquid, solid, semisolid, injectable solution, tablet, ointment, lotion, capsule, lingualette or similar.

These peptides will be in general administered in a dosage of from 10 5 to 10 2 mg/kg of body weight, when applied systematically. When administered topical, they will be used in higher concentration, e.g. 0.1% to Very favorable is the absence of any signs of toxicity till doses of 50 mg/kg b.w. and also good 15 activity of compounds by peroral administration *(intra gastric).

o.

The peptides described herein can be synthesized using step by step condensation of protected amino acides in homogenous liquid system or preferable 'using solid phase method. For preparation of cyclic peptides partially protected linear peptides of desired length are prepared as with alkyl ester group on C-terminus, which is converted into azide, following coupled and de protected. Alternatively 25 partially protected linear peptide with free terminal groups can be cycled by diphenylphosphorylazide in very diluted solution.

The invention will now be described with respect to the following examples, however, the scope of the present invention is not intended to be limited thereby.

i 33 Example 1 Peptide synthesis using Boc strategy Peptide synthesis was performed beginning with 100 mg of Boc-Val-PAM resin to produce C-terminal carboxy peptide (PAM purchased from Applied Biosystems, substitution 0.56 meq/g is an amino-acyl-4-(oxymethyl)-phenylacetic acid derivative of aminopolystyrene). Boc amino acids (Boc tert.-butoxycarbonyl-) were condensed one after other on polymer carrier using diisopropylcarbodiimide (DIPC) as condensation reagent. In each step Boc group was removed with 50 solution of trifluoroacetic acid (TFA) in dichloromethane. Amino group was then deprotonated with "o"o diisopropylethylamine.

The conversion in each step should be higher than 99.5%. If not, the condensation was repeated. After complete synthesis, cleavage was performed using low HF procedure (2 hours at As carbonium ion scavenger was used anisole.

HF was evaporated by a flow of nitrogen. Crude raw peptide was then obtained by pouring of oily residue- into dry ether.

Then the raw peptide was purified by reverse phase HPLC using column 5 x 150 mm filled with silica gel RP-18, gradient elution with solvent system: 0.1% TFA in water/acetonitrile. Detection: ultraviolet absorbtion at 225 nm.

The synthesis of the peptide with Sea.Id.No: 4 is dedicated in Fig. 1.

I

Example 2.

Peptide synthesis using Fmoc strategy Standard Fmoc protected amino acids were used for the synthesis (Fmoc 9-fluorenylmethyloxy-carbonyl-). The side chain functions were protected as 0-tert. butyl esters (Asp, Apm, Glu, Aad) and as Boc derivatives (Lys). First amino acid (Val) was bonded on to polymeric carrier-BHA resin (BHA benzhydrilamino resin) using diisopropylcarbodiimide as coupling reagent. In each step the Fmoc protective group was removed with piperidine. Thereafter the second and so on all next amino acids were introduced using the same way until the synthesis is completed. The cleavage has been done by a mixture of TFA/TFMSA/anisole *2 17 52 (vol/vol).

Peptide was then purified using the HPLC method described in example 1.

The synthesis of the peptide with Seq.Id.No: 2 is illustrated on the Fig. 2.

*9 Example 3.

Peptide synthesis using Ddz strategy All amino acids were used protected at their a-amino function by Ddz a, dimethoxybenzyloxycarbonyl-). The side functions were protected by Z group benzyloxycarbonyl-) for lysine and O-t-Bu group (t-butylester) for aspartic and glutamic acid.

A Merrifield support (crosslinked chloromethylated polystyrene gel) with capacity of 1.4 mmol/g was used for bonding of the first Ddz-amino acid via its cesium salt.

After condensation with dicyclohexylcarbodiimide (DCC) was removed the Ddz protection group in each ste'. -vith 5% TFA in dichloromethane, following by washing and deprotonation with 10% triethylamine in dichloromethane. After deprotonation the next coupling step was mediated, using the same method. These coupling steps were repeated until peptide sequence was completed.

Finally the peptide .as cleaved from polymeric carrier using a mixture HBr/TFA/anisole. After evaporation of volatile part the just cleaved peptide was precipitated from dry ether and dried. The raw peptide was then purified by HPLC method as described in example 1. The synthesis of the peptide with Sea. Id.No.: 6 is illustrated on the Fig,3.

Example 4.

SSynthesis of cyclic peptide Peptide in partially protected form: OBzl NO 2 OBzl Gly-Glu-Pro--Pro-Pro-Gly-Ar:g-Pro-Ala-Asp-OH was previously prepared according to the method desi:.,bed in examples 1 or 2.

The 0.0005 molar solution of this peptide in dimethylformamide was cyclized after addition of I I j 36 diphenylphosphorylazide and triethylanine at 20*C for 12 hours.

Then this mixture was hydrogenated with hydrogen/palladium charcoal catalyst at 25*C for 8 hours.

SThe solvent was carefully evaporated and crude product purified using HPLC method described in example I.

Cyclic eptide with Seg.Id.No: Gly Glu Pro Pro Pro Gly Arg Pro Ala Asp 1 5 10 was obtained in 10% yield.

C.

I5 *SM- U Synthesis of linear and cyclic pelotides usingr the Ddz stratecrV S.The same strategy with the Ddz protecting groups waLs ed 15 for obtaining of linear and cyclic peptides. The side functions were protected with Z~ group (lysine) and with 0benzyl ester groups OBzl (glutamic and aspartic acid).

oPolymeric carrier was HYCRAM Rresin (trademark of ORPEGEN, Heidelberg, Germany), which is 4-bromcrotonyl-B-alanylamidonsthyl-polystyrene. The first amino acid (Ddz-valine) was bonded onto polymer carrier via its cesium salt.

I I I 37 Then the Ddz group was removed using trifluoroacetic acid in dichloromethane and amino group deprotonated with diisopropylethylamine.

In the next step Ddz-glycine was coupled onto valine residue in polymer matrix using diisopropylcarbodiimide (DIPC) in the presence of l-hydroxybenzotriazole.

These steps were repeated until peptide chain was completed. The synthesized peptide in protected form was then cleaved carefully from HYCRAM R carrier with tetrakis- (triphenyl-phosphino)-palladium dissolved in anhydrous tetrahydro-furan in absende of oxygen. An additive like morpholine was used as acceptor molecule for allylic groups.

Polymeric carrier was filtered off and washed with 15 tetrahydrofurane. The solution of peptide was then filtered through a short column of silica gel to remove the palladium catalyst.

The eluate which contains the partially protected peptide Aa. was dried after evaporation of solvent in vacuum.

e i 20 Synthesis of linear peptide with Seq.Id.No: 4 Partially protected peptide 4a. was dissolved in 2,2,2trifluoroethanol and hydrogenated in the presence of palladium on charcoal with hydrogen gas at 30°C. The catalyst was filtered off and solvent evaporated in vacuum.

The crude residue was then purified using HPLC method as described in example 1. After purification the peptide with I 38 Seq.Id.No: 4 was obtained. The product was identical with compound obtained in example 1.

Synthesis of cyclic Deptide with Seq.Id.No: 9 Partially protected peptide 4a was dissolved in dimethylformamide/dichloromethane to form 0.001 molar solution. For cyclisation DIPC and HOBt were added and left to stand at 20*C for 10 hours. Then the solution was concentrated to samll volume, diluted with 2,2,2trifluoroethanol and purified by passing of colunin filled with Sephadex LH-20. Fractions containing the partially protected cyclic peptide 9a were collected and hydrogenated with palladium on charcoal catalyst at 30'C by hydrogen bubling and intensive shaking for 8 hours.

Then the catalyst was removed by filtration and solution 15 dried by solvent evaporation in vacuum. The crude raw peptide was additionally purified by HPLC using the described method from example 1. The obtained pure cyclic S' peptide was identified as peptide with Seq.Id,No:-9.

The synthesis on HYCRAM R polymer carrier using Ddz strategy 20 and cyclisation were illustrated on Fig. 4 and 5. All synthesized peptides were checked on their purity by HPLC method using column with silica gel RP-18 (octadecylsilanized), eluted with a mixture of solvents consisting of water/acetonitrile/trifluoroacetic aced gradiently in usual mode. In all cases the purity was higher than Peptides were characterized with amino acid analysis (values were within 10% of theory), sequence analysis, I I 1 39 molecular weight determined by mass FAB spectrometry, ultraviolet and infrared spectra (Fig.6 and 7).

While only certain embodiments of our invention have been described in specific detail it will be apparent to those skilled in this art that many other specific embodiments may be practiced and many changes may be made, all within the spirit of the invention and the scope of the appended claims.

S.

9*9

Claims (14)

1. A biologically active peptide consisting of from 8 to 15 amino acid residues and represented by the formula: Xaa Zaa Pro Pro Pro Xaa Yaa Pro Ala Asp Zaa Ala Xaa Xaa Xaa 1 5 10 in which: Xaa signifies a neutral aliphatic amino acid residue selected from the group consisting of Ala, bAla, Leu, Ile, Gly, Val, Nle, and Nva, Yaa signifies a basic amino acid residue selected from the group consisting of Lys, Arg, Orn, and His, and Zaa signifies an acidic amino acid residue selected from the group consisting of Glu, Asp, Aad, and Apm.

2. The peptide of claim 1, wherein at least one amino acid residue and at most :seven amino acid residues in region 1 to 15 are omitted.

3. The peptide of claim 1 or 2, wherein at least one of amino acid residues Xaa or Zaa are omitted. 1 5 4. The peptide of any one of claims 1 to 3, wherein at least one and at most seven amino acid residues in region 1 to 15 are omitted with the exception of: Gly-Glu- SPro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Gly-Leu-Val. The peptide of any one of claims 1 to 4, wherein said peptide is cyclized by an amide bond between the first and last amino acid residue. 20 6. A biologically active peptide having a molecular weight of 900 to 1600 Daltons and including the sequence: Zaa Pro Pro Pro Xaa Yaa Pro Ala S1 5 8 wherein Xaa signifies a neutral aliphatic amino acid residue selected from the group consisting of Ala, bAla, Leu, lie, Gly, Val, Nle, and Nva, Yaa signifies a basic amino acid residue selected from the group consisting of Lys, Arg, orn, and His, and Zaa signifies an acidic amino acid residue selected from the group consisting of Glu, Asp, Aad, and Apm.

7. A biologically active peptide substantially as hereinbefore described with reference to any one of the Examples.

8. A therapeutic composition containing a peptide according to any one of claims 1 to 7 in admixture with a pharmaceutically acceptable solid or liquid vehicle. [N:\LIBFF]00787:RRB

9. A method of norrmalising organic function in an animal, comprising administering to said animal an effective amount of a peptide according to any one of claims 1 to 7 or a composition according to claim 8. A method for the prophylaxis or treatment of a condition or disease in an animal, comprising administering to said animal an effective amount of a peptide according to any one of claims 1 to 7 or a composition according to claim 8.

11. The method according to claim 10 wherein said condition or disease is a stress related condition or disease.

12. The method according to claim 10 wherein said condition or disease is any one of inflammation, oedema, trauma, burns, a viral infection, a disease of the immune system, CNS disorder, a gastrointestinal ulcer of diverse ethiology, a bone fracture, surgery related shock and/or trauma, a cerebral disorder, depression, Parkinson's disease, radiation damage or malformation induced by a chemical agent.

13. The method according to claim 10 wherein said condition or disease is any 15 one of a disturbance and/or lesion of the liver or pancreas or both, atrophy of testis or sperm mobility or both, a fertility disturbance, a commercial breeding improvement, a rickettsia illness, thyreoparathyreoidoctomy, diabetes mellitus, a disturbance of the adrenal gland, a disturbance of the hematopoetic system, a disturbance of coagulation, a disturbance of bleeding, a post-castration or menopausal disturbance, an ischemic or toxic lesion or both, a psychiatric disturbance, hypertension, a disturbance of body temperature, pain or tumour.

14. The method according to any one of claims 9 to 13 wherein said animal is a mammal. The method according to claim 14 wherein said mammal is human.

16. A biologically active peptide consisting of from 8 to 15 amino acid residues, substantially as hereinbefore described with reference to any one of the Examples.

17. A therapeutic composition containing a peptide according to claim 16, in admixture with a pharmaceutically acceptable solid or liquid vehicle.

18. A method of normalising organic function in an animal, comprising administering to said animal an effective amount of a peptide according to claim 16 or a composition of claim 17. [N:\LIBFF]00787:RRB t- 42

19. A method for the prophylaxis or treatment of a condition. or disease in an animal, comprising administering to said animal an effective amnou~nt of a peptide according to claim 16 or a composition of claim 17. Dated 29 May, 1998 Predrag Sikiric Marijan Petek Sven Seiwerth Zeljko Grabarevic Ivo Flotkvic Marko Duvnjak Branko Turkovic Stepan Mise Ernest Suchanek Boris Mildner Ivan Udovicic Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [N:.LIBFF]00787;RRB Abstract Peptides with Organo-Protective Activity, Their Preparation and Use A biologically highly active peptide which comprises from 8 to 15 amino acid residues and is represented by the general formula: Xaa Zaa Pro Pro Pro Xaa Yaa Pro Ala Asp Zaa Ala Xaa Xaa Xaa 1 5 10 in which: Xaa signifies a neutral aliphatic amino acid residue; Yaa signifies a basic amino acid residue; Zaa signifies an acidic amino acid residue; wherein at least one of residues Xaa or Zaa is optionally omitted. [N:\LlBaa]00353:MCN