The present invention relates to new analogues of Cyclolino¬ peptide A, in particular analogues having a cyclic formula derived from the sequence of Cyclolinopeptide A, which se¬ quence is extended between any two amino acid residues with a defined cystine derivative residue. Additionally, the inven¬ tion relates to the analogues of the invention for use as a medicament, in particular for use as an immunosuppressive agent. Furthermore, the invention relates to the use of the analogues of the invention for the preparation of a medica¬ ment for immunosuppressive treatment, to pharmaceu ical pre¬ parations comprising an analogue of the invention, and a method of treating a mammal, including man, in need of immunosuppressive treatment.

BACKGROUND

Cyclolinopeptide A was first isolated from linseed cake by Kaufmann H.P. and Tobschirbel A., in 1959 (Chem. Ber., 92.

2805 (1959)) and its chemcial structure was suggested in 1966 by Prox.A. and Weygand, F. C(1967) in "Peptides: Proceedings of the 8th European Peptide Symposium", Beyerman, H.C., van de Linde, A. and van den Brink, .M. Eds., North Holland, Amsterdam, pp. 158-1723. The chemical structure of Cyclolino¬ peptide A is

Pro-Phe-Phe-Lejj

Kessler, H., et al (Ang. Chemie Int. Ed. .25, 997-999 (1986)) showed that natural Cyclolinopeptide A inhibits the uptake of cholate into hepatocytes, and stated that, to their know- ledge, this was the first biological activity that have been found for this natural product.

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However, it was surprisingly found that Cyclolinopeptide A can be used as an immunosuppressive agent, and this has been disclosed in our previous Swedish patent application number 8804640-4 filed December 23, 1988 (corresponding PCT application PCT/SE89/00732) .

Immunosuppressive treatment of a patient in need thereof re¬ quires large amounts of the immunosuppressive agent used. When Cyclolinopeptide A is synthesized according to the method used in our previous patent application, 60-70% of the linear peptide is lost during the cyclization. It is evident that large scale production of Cyclolinopeptide A is very expensive, and industrial production of Cyclolinopeptide A is thus deferred.

Surprisingly, we found that the immunosuppressive activity of Cyclolinopeptide A is retained when the sequence thereof is extended by a cystine derivative residue. The new analogues of Cyclolinopeptide A enables large scale production thereof, since in the cyclization step practically no linear peptide is lost .

DESCRIPTION OF THE INVENTION

In one aspect of the invention there is provided new analogues of Cyclolinopeptide A having a cyclic formula derived from the sequence of Cyclolinopeptide A

which sequence is extended between any two amino acid residues with the following cystine derivative residues

C=0 NH

X-C-H H-C-Y

CH ₂ CH ₂

in which X is selected from -H and -OH, and Y is selected from -C=0 and -H,

NH-

wherein the amino acid residues are of L- or D-configuration .

In a preferred embodiment of this aspect of the invention the above cystine derivative residue is located between the amino acid residues Leu and Phe in the formula of Cyclolinopeptide A. In fact the location of said cystine derivative residue in the sequence of Cyclolinopeptide A was randomly chosen. Thus it can be expected that the immunosuppressive activity of

Cyclolinopeptide A is retained irrespective of between which two amino acid residues in the sequence thereof the cystine derivative residue is introduced.

In an additional aspect of the invention there is provided an analogue of the invention for use as a medicament.

In another aspect of the invention there is provided the use of an analogue of the invention for the preparation of a me- dicament for immunosuppressive treatment.

SUBSTITUTE SHEET

In still another aspect of the invention there is provided a method of treating a mammal, including man, in need of immunosuppressive treatment, comprising administering to said mammal a pharmacologically effective amount of an analogue of the invention.

In yet another aspect of the invention there is provided a pharmaceutical preparation comprising an analogue of the in- vention together with pharmaceutically acceptable carrier(s), excipient(s) and/or diluent(s) .

The amount of an analogue of the invention to be administered to a mammal in need of immunosuppressive treatment has to be decided by a physician who is experienced in immunosuppres¬ sive therepy.

The pharmaceutical preparation comprising an analogue of the invention can be formulated into oral preparations or prepa- rations for infusion using pharmaceutically acceptable carrier(s), excipient(β) and/or diluent(s) suitable for such preparations, but keeping in mind that said analogue is in¬ soluble in water. The pharmaceutical preparation can e.g. comprise an analogue of the invention in a pharmacologically effective amount suspended in an olive oil.

The present invention reveals that analogues of the invention have immunosuppressive activity, which is comparable to that of Ciclosporin (also called Cyclosporin A) and Cyclolino- peptide A. Accordingly, the analogues of the invention are to be used in medicaments for immunosuppressive treatment of mammals, including man, for all indications where immuno¬ suppressive treatment is warranted. Examples of when immuno¬ suppressive treatment is warranted is for the prevention of allograft rejection after organ transplantation and bone marrow transplantation, prophylactic or therepeutic treatment of graft-versus-host-disease (GVHD), and autoimmune diseases,

such as systemic lupus erythe atosus (SLE), rheumatoid arthritis (RA) and diabetes mellitus.

SHORT DESCRIPTION OF THE DRAWINGS

Fig. 1 shows a graph of the inhibition of PPIase activity by Cyclosporin A.

Fig. 2 shows a graph of the inhibition of PPIase activity by CLA, LA, and 1-Mpa-LA-Cys-NH ₂ .

SYNTHESIS OF CLA ANALOGUES OF THE INVENTION

The CLA analogues of the invention can be produced by any known method in the art of peptide chemistry, whereby a linear peptide is formed, which is then subjected to an oxidation step in order to cyclize two cysteine related residues, whereby a sulphur bridge is formed between said residues, thus forming a cystine derivative residue.

The linear linopeptide (LA) analogues according to the inven¬ tion can thus be synthesized in conventional manner e.g. by step wise coupling of one amino acid residue to the next in liquid phase, e.g. according to the method of Law, H.B. & Du Vigneaud, V. :J. Am. Chβ . Soc . 82. (1960) 4579-4581; Zhuze, A.L., Jost, K. Kasafirek, E. & Rudinger, 3 . : Coll. Czecho¬ slovak Che Commun. 1 (1964) 2648-2662, modified by Larsson L.-E., Lindeberg, G., Melm, P. & Pliβka, V. : 3 . Med. Chem. 2Λ_ (1978) 352-356. The coupling of amino acid residues to one another, whereby so called peptide bonds are produced may also be performed starting with a solid phase (usually a resin) to which the C-terminal of the first ammo acid is coupled, whereupon the C-terminal of the next amino acid is coupled to the N-terminal of the first amino acid, etc., finally releasing the built-up peptide from the solid phase. In the examples below this so called solid phase technique

SUBSTITUTE SHEET

has been utilized in accordance with the method of Merrifield, R.B. ; J. Am. Chem. Soc . .85 (1963) 2149; Merrifield, R.B.: Biochem. 3_ (1964) 1385 and Kdnig, W. & Geiger, R. : Chem. Ber . 103 (1970) 788.

GENERAL DESCRIPTION OF SYNTHESIS

All the pept'ides in the examples below were synthesized on an Applied Bioεystems 430A Peptide Synthesizer using a double coupling program with termination step after the second coupling. The resm used was of 4-methyl-benzhydrylamine type with a theoretical loading of 0.66 meq/g (Peptides Interna¬ tional, Louisville, KY, USA). The final product of the syn¬ thesis was dried in vacuo over night. The peptide was then cleaved from the resin by treatment with liquid hydrogen fluoride in the presence of anisole and ethyl-methyl-sulfide as scavangers (HF:anisole:EMS - 10:05:05). After removal of hydrogen fluoride by evaporation the residue was suspended in ethyl acetate (100 ml) and filtered. The solid was washed on filter with additional ethyl acetate (3 x 100 ml) and the cleaved peptide extracted with acetic acid (100 ml). The extract was promptly diluted to a volume of 2 cm^ with 20% acetic acid in methanol and treated with 0.1 M solution of iodine in methanol until the faint brown colour persisted. Then the Dowex 1 x 8 ion exchanger in acetate form was added (3 g) (Bio-Rad, Richmond, CA, USA) and the mixture was filtered. The filtrate was evaporated and the residue freeze- -dried from 1% acetic acid in water. The product was then purified by reversed phase liquid chromatography on a column filled with Vydac 20-25 μ (Separation Group, CA, USA) in a suitable system containing acetonitrile in 0.1% trifluoro- acetic acid water solution. The samples collected from the column were analyzed by analytical HPLC (Varian 5500, Sunny¬ vale, CA, USA) equipped with a Bondapak C _j s column (Millipore, Milford, Mass., USA). Fractions containing pure substance were pooled, the solvent was evaporated and the

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product freeze-dried from I X acetic acid in water. The final HPLC analysis was performed on ready product and the struc¬ ture of the peptide in question was confirmed by am o acid analysis and FAB-MS (Fast atom bombardment spectrometry). The FAB-MS analyses were performed by M-Scan Ltd. Sunninghill, Ascot, Berkshire, England.

All amino acids used during the synthesis were protected with tert-butoxycarbonyl group at (X-amino function. The thiol function of Mpa, Hmp and Cys was protected with 4-methoxy- bensyl group. The amino acid derivatives were delivered by Bachem AG, Switzerland.

In the Examples below the abbreviation LA is used for the linear peptide Leu-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe

EXAMPLE I

1-Mpa-LA-Cyε-NH ₂

^' 1

The peptide was prepared according to the general description of synthesis. 3-Mercaptopropionic acid CS-(p-methoxy-)benzyl ] was used for position 1.

Purity (HPLC) :99.8%

The sructure was confirmed by amino acid analysis and by FAB- MS. CM+H3 ⁺ = 1246.

EXAMPLE I I

1-Mpa-LA-D-Cys-NH ₂

SUBSTITUTE SHEET

The peptide was prepared according to the general description of synthesis. 3-Mercaptopropionic acid CS-(p-methoxy-)benzyl3 was used for position I and Boc-D-cysteine C-(p-methoxy-)-benzylϋ for position 2.

Purity (HPLC) : 95.7%

The structure was confirmed by amino acid analysis and by FAB-MS. CM+H3+ = 1246.

EXAMPLE III

1-Hmp-LA-Cys-NH ₂

The peptide was prepared according to the general description of synthesis. 2-Hydroxy-3-mercaptopropionic acid was used for position I .

Purity (HPLC) :97.5%

The structure was confirmed by amino acid analysis and by FAB-MS. CM+H3 ⁺ = 1262.

EXAMPLE IV

1-Mpa-D-LA-D-Cyε-NH ₂

The peptide was prepared according to the general description of synthesis using amino acids of D-configuration.

3-Mercaptopropionic acid CS-(p-methoxy-)bezylD was used for pσstion 1.

Purity (HPLC) :98.0%

The structure was confirmed by am o acid analysis and by FAB-MS: CM+H3 ⁺ = 1246.

SUBSTITUTE SHEET

LIST OF COMPOUNDS USED AS ENZYME INHIBITORS AND AS IMMUNO¬ SUPPRESSIVE AGENT

CS-A = Cyclospoπn A (Reference)

CLA = Cyclolinopeptide A (Reference)

1-Mpa-LA-Cys-NH ₂ Leu-Val-Pro-Pro-Phe

Phe

Ile-Ile-Leu-Mpa Cys-NH ₂

I I (Compound of Example 1)

1-Mpa-LA-D-Cys-NH ₂

(Compound of Example 2)

1 -Hmp-LA-Cys-NH ₂

(Compound of Example 3 ⁾

SUBSTITUTE SHEET

1 -Mpa-D-LA-D-Cys-NH D-Leu-D-Val-D-Pro-D-Pro-D-Phe

\

D-Phe

D- I le-D- I le-D-Leu-Mpa D-Cyε-NH

I I

(Compound of Example 4)

wherein Mpa is 3-mercapropropionyl residue ⁽ -S-CH ₂ -CH ₂ -CO-) and Hmp is 2-hydroxy-3-mercaptopropionyl residue

OH I (-S-CH ₂ -CH-CO-)

INHIBITION OF THE ENZYME PPIase

It is known that the peptidyl-prolyl cis-tranε isomerase (PPIase) catalyses the cis-tranε isomerization of proline i idic peptide bonds in oligopeptideε and Nobuhiro Takahashi, et al (Nature vol. 337, Letters to Nature, pp 473-475, 1989) suggested that the peptidyl-prolyl cis-tranε iεomerizing ac¬ tivity of PPIaεe may be involved in events, εuch aε those occuring early in T-cell activation, that are suppressed by Cyclosporin A. Gunter Fischer, et al (Nature Vol. 337,

Letters to Nature, pp 476-478, 1989) reported that PPIase is probably identical to cyclophil , a recently discovered mammalian protein which binds tightly to Cyclosporin A. We have purified PPIase and tested analogues of the invention as inhibitors of PPIase.

PURIFICATION OF PPIaBe

The pig kidney cortex was homogenized in equal volume of saccharose (0.2 M) and centrifuged 60 in. at 10,000 x g. The

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supernatant was adjusted to pH 5.5 by addition of 1 M acetate buffer, pH 4.0. The precipitate was discarded and the super¬ natant was brought to pH 7.0.

The extract was brought to 40% saturation of ammonium sul¬ phate. To the supernatant obtained after 15 min. centrifuga- tion at 20,000 x g solid sodium sulphate was added to get a final saturation of 60%. The precipitate obtained by centri- fugation was dissolved in a small volume of 10 mM Tris buffer, pH 7.6 and dialysed 24 h. against the same buffer. The dialysed solution was loaded onto a DEAE-Sephadex A-50 column, that had been previously equilibrated with the same buffer and eluted without changing the buffer. The eluate rich in PPIase activity passed through the column in one active peak.

The active fractions were applied on a CM-Sephadex C-50 column and eluted with a linear gradient of KC1 (0-1.2 M) as one active peak.

The enzyme was stored as suspension in 60% saturated ammonium sulphate. After 5 months the enzyme shows full enzymatic activity.

DETERMINATION OF PPIase ACTIVITY

The cis-trans isomerization of the Ala-Pro peptide bond of the peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide was measured in a coupled assay with chy otrypsin. Substrate (50 μM) , previously dissolved m DMSO, was equilibrated at

10°C with the appropriate amount of PPIase in the thβrmostat- ed spectrophotometer cell. The reaction was started by adding chymotrypsin (final concentration 21 μM) . The trans-peptide was cleaved within the deadtime. The rate of cis-trans isome- rization was followed by the decrease in transmittance at 390 nm. The spectrophotometer Specord UV-Vis (Jena, GDR) equipped with a cell stirrer was used.

The effect of the inhibitors on PPIase was examined by mixing the studied substance with PPIase for 1 min. before incubat¬ ing the substrate. The substances tested were dissolved in dioxane containing 0.5 - 1% of Triton X-100 or in DMSO . There was no difference observed in the effect on the PPIase acti¬ vity depending on the kind of solvent.

The results are given in Fig. 1 and Fig. 2, where the log of the difference between transmittance at steady state A _x and transmittance at a given time A^ was plotted against time.

The first order rate constant k (s-*) was calculated from the resulting straight lines.

IMMUNOSUPPRESSIVE ACTIVITY OF CLA, CS-A AND THE ANALOGUES OF THE INVENTION.

Materials and methods

Animals: CBA/ Iiw mice 8 - 10 weeks old Antigen: SRBC (sheep red blood cells)

Reagents: 1-Mpa-LA-Cys-NH ₂ , 1-Mpa-D-LA-D-Cys-NH ₂ ,

I 1 I I

1-Mpa-LA-D-Cys-NH , and CLA were prepared by

1 I

FERRING AB, Sweden, and Cyclosporin A (CS-A), Sandimmun

Sandoz, Basel, Switzerland Solvents: Mixture of Cremophor EL (SIGMA) and 94% ethanol (6.5 : 3.5), ethanol 96%, olive oil, intralipid and PBS (phosphate buffer solution)

Treatment of mice with reagent intraperitoneallv (jp.) or intravenously (iv.):

The reagents dissolved in one of solvents were diluted to desired concentration in PBS or in intralipid, and 0.2 ml

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thereof was introduced ip. in two doses. First 3 hrs before the antigen, second 24 hrs later. The activity of analogues studied was compared to the activity of CLA and CS-A.

Treatment of mice with reagents per os ⁽ pp.) . directly into the stomach:

The reagents dissolved in one of the solvents diluted to desired concentration in olive oil, PBS or in intralipid was introduced in the volume of 0.2 ml in to the animal. First dose 3 hrs before the antigen, second dose 24 or 48 hrs later .

The activity of the derivatives studied was compared to the activity of CLA and CS-A.

Effect of CLA analogues on humoral immune response in mice immunized with SRBC

Mice were injected traperitoneally with 0.2 ml of 10% sus- pension of SRBC in PBS, 3 hrs before the antigen the first dose of the reagent was introduced, ip., po . or iv. After 4 days the number of plaque forming cells (PFC) in the spleen was determined according to the Mishell-Dutton (Mishell R. I., Dutton R. W. : J. Exp. Med . , 1967, 126, 423) . The magni- tude of the humoral immune response was expressed as the num¬ ber of PFC per 10^ splenocyteε. The results obtained are gi¬ ven in tables A, B and C.

Effect of CLA analogues on humoral immune response to SRBC in vitro

Priming of mice and isolation of spleen cells: Mice were primed intravenously with 0.2 ml of 1% suspension of SRBC in PBS. Four days later the animals were killed and their spleens were minced, pressed through a plastic screen into 0-83% NH ₄ C1 buffered with 0.017 M Triε buffer to remove

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erythrocytes . Then the cells were washed three time with PBS and finally resuspended in RPMI medium supplemented with 10% of fetal calf serum.

Culture conditions and determination of PFC number:

To one milliliter of spleen cell suspension (5 x 10^ cells/ml) 0.1 ml of the reagent was added followed by addi¬ tion of 0.1 ml of 0.005% suspension of SRBC in the cell cul¬ ture medium, and incubated for 4 days at 37°C, 5% of C0 and 100% of humidity. The number of PFC was measured using the procedure of Mishell-Dutton. As a control PBS or appropriate concentration of the solvent waε used. The experiments were performed using NUNC 24-well Tissue Culture Plate. The results are shown in Tables D and E.

Effect of CLA analogues on delayed type hypersensitivitv (DTH)

The results and description of the test are given in Table F.

Immunosuppressive activity of CLA analogues in Graft-versus-host reaction (GvH)

Animals: Hybride F^ (C3H/I1W x B _β /Iiw) and female B _β mice, 8-10 weeks old.

Preparation of parental cells:

Female B _β mice were killed, their popliteal nodes were pressed through a plastic screen into PBS. Then the cells were washed three times with PBS and resuspended in RPMI medium .

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GvH test:

GvH reaction was performed according to Twist and Barnes (Twist V.S., Barnes R.D. : Transplantation, 1973, 15, 1982) . Hybride mice, F _j (C3H x Bg) were injected subcutaneously in the left hind footpad with 5 x 10" parental lymphoid cells (B) . After 7 days, draining lymph nodeε were isolated and weighed. As a control, the weight of popliteal node isolated from the right leg was measured.

The intensity of GvH reaction was expressed as a ratio of weights of popliteal lymph nodes isolated from the left and right legs (index of GvH reaction) . The results are given in Table G.

SUBSTITUTE SHEET

Table A

The number of PFC in the spleen cell of mice treated intra- peritoneally with two doses (-3 hrs and + 24 after SRBC) of 1-Mpa-LA-D Cys-NH ₂ , 1-Mpa-LA-Cys-NH ₂ and CLA dissolved in i i i i

Intralipid® (int.) (A fatty emulsion for intravenous nutrition from Kabi , Stockholm, Sweden)

Reagent P μg/mouse PFC/10 ⁶ - SE Student test

Control int. 3047 358

1-Mpa-LA-Cys-NH ₂ 0.001 0.001

CLA 0.05 0.001

Preparation and introduction of the substance, see explana¬ tion after Table C.

The results are expressed as a mean — SE of 6 mice.

SUBSTITUTE SHEET

Table B

The umber of PFC in the spleen of mice treated with two doses (-3 hr and 24 hr after SRBC) of CLA, CS-A and analogues of the invention dissolved in olive oil, introduced per os (po. )

Reagent P μg/mouse PFC/10 ⁶ i SE Student test

Control olive oil 2183 166

The results are expressed as a mean i SE of 6 mice

SUBSTITUTE SHEET

Table C

The number of PFC in the spleen of mice treated intravenously (iv.) with two doses of 1-Mpa-LA-Cys-NH ₂ , CLA and CS-A dis¬ solved in intralipid (int.)

The preparates in the volume of 0.2 ml were introduced twice, first dose 3 hrs before sensitization with antigen second 24 hrs later. Preparation of CLA and 1-Mpa-LA-Cys-NH2 : 2 mg of

1 I the substance was dissolved in 0.15 ml of hot ethanol and 0.35 ml of intralipid, then diluted to desired concentration in intralipid.

CS-A was diluted directly in intralipid to desired concentra- tion. In the control 0.2 ml of PBS or intralipid was intro¬ duced.■ The results are expressed as a mean -± SE of 6 mice.

Table D

The number of PFC in the spleen cell cultures treated with CLA, CS-A and analogues of the invention dissolved in intralipid (int.)

Bg 65 17

The results are expressed as a mean - SE of 6 wells Bg - background

SUBSTITUTE SHEET

Table E

Imunosuppressive activity of CS-A and analogues of the invention measured by the number of PFC. In in vivo studies, mice were treated with two doses of the preparations dissolved in cremophor or in olive oil. First dose 3 hours before immunization, second 24 hours later.

Reagent Route of μg/mouse introduc¬ PFC/10 ⁶ i SE

Control "C" i.p. 1330 109

Control PBS i.p. 1056 95

CS-A 10 μg i.p. 124 36 0.001

Control "C" - cremophor in appropriate concentration needed to prepare 10 μg of the peptide. CS-A - dissolved in PBS.

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Continued

Control "01" p.o 2428 329

1-Mpa-LA-Cys-NH ₂ 10 μg p.o 1214 91 0.02

100 μg p.o 406 73 0.001

CS-A 10 μg p.o 1888 272 NS 100 μg p.o. 834 142 0.01

Control "01" .o 1693 133

1-Mpa-LA-Cys-NH ₂ 100 μg p.o 444 66 0.001

1-Hmp-LA-Cys-NH ₂ 100 μg p.o 672 74 0.001

Control PBS p.o 1788 219

CS-A 100 μg p.o 561 88 0.001

Control "C" in vitro 3690 321

1-Mpa-LA-Cys-NH ₂ 1 μg/ml in vitro 65 11 0.001

CS-A lμg/ml in vitro 20 12 0.001

Control "01" - olive oil

SUBSTITUTE SHEET

Table F

The effect of CLA, CS-A and analogues of the invention dis¬ solved in olive oil on delayed type hypersensitivity (DTH) in mice. Preparates were introduced per os (po.) twice, first dose 3 hours before sensitization, second 48 hours later.

One unit = 0.1 mm

DTH was induced in mice according to Lagrange et al (Lagrange P.H., Mackaness G.B., Miller T.E., Pardon P: J. Immunol.,

1975, 114, 447) . Mice were sensitized intravenously with 10 ⁵ SRBC. After 4 days the reaction was elicited by an intra- dermal introduction of 10 ⁸ SRBC into left, hind foot pad. The magnitude of the reaction was measured as the increase of the foot pad thickness at 24 hrs following administration of the challenging dose. The results are expressed as a mean — SE of 10 mice.

SUBSTITUTE SHEET

Table G

Immunoβuppressive activity of 1-Mpa-LA-Cys-NH ₂ and

I I

1 -Hmp-LA-Cys-NH ₂ in GvH react ion ( c e l lular immunity ⁾ .

I I

Preparations dissolved in cremophor were given i.p. -4 and 48 hrs before and after introduction of parental cells, respectively .

Reagent Route of GvH i SE μg/mouse introduc- (index) tion

Control "C" i.p. 3.17 0.36

1-Mpa-LA-Cys-NH ₂ 10 μg i.p 1.96 0.27 0.05

I I

1-Hmp-LA-Cys-NH ₂ 10 μg i.p. 1.54 0.22 0.01

Control PBS i.p. 3.09 0.42

CS-A 10 μg i.p. 1.41 0.16 0.001

Control PBS - phosphate buffer solution

Control "C" - cremophor at a concentration needed to dissolve 10 μg of the peptides or 1 μg <in in vitro)

Control "01" - olive oil

SUBSTITUTE SHEET

Description of the drawings:

Fig.1 Inhibition of PPIase activity by Cyclosporin A, Catalysis of proline isomerization by PPIase:

- X-X—without CS-A k=26 X 10 ^"3 s~ ¹ O-O—0.25 μg CS-A 12.9 D-D— 0.50 μg CS-A 10.9 Δ-Δ— 1.5 μg CS-A 7.0

- •- —•—without PPIase 7.0

Fig.2 Inhibition of PPIase activity

Catalysis of proline isomerization by PPIase: X—X— ithout inhibitor k=52 x 1θ ^"3 s ^"1 O—O—with 1-Mpa-LA-Cys-NH ₂ 2μg 41 •—•—LA - inactive 10 g 35 O—D—with CLA 20ug 24.1 Δ—Δ—without PPIase 7.5

SUBSTITUTE SHEET