WO1992005189A1

WO1992005189A1 - Analogues of cyclolinopeptide a and the use thereof

Info

Publication number: WO1992005189A1
Application number: PCT/SE1991/000628
Authority: WO
Inventors: Zbigniew Wieczorek; Z. Ignacy Siemion; Jerzy Trojnar
Original assignee: Ferring Ab
Priority date: 1990-09-25
Filing date: 1991-09-18
Publication date: 1992-04-02
Also published as: SE467410B; CA2090859A1; FI931259A; EP0553133A1; FI931259A0; SE9003040D0; SE9003040L; AU8653191A; HU9300847D0

Abstract

New analogues of Cyclolinopeptide A having a cyclic formula derived from the sequence of Cyclolinopeptide A (I) which sequence is extended between any two amino acid residues with the cystine derivative residue (II) in which X is selected from -H and -OH, and Y is selected from (a) and -H, wherein the amino acid residues are of L- or D-configuration, are disclosed. The above analogues can be used as medicaments, especially as immunosuppressive agents. The use of the above analogues for the preparation of a medicament for immunosuppressive treatment and pharmaceutical preparations comprising said analogues are also disclosed. A mammal, including man, in need of immunosuppressive treatment can be treated with a medicament comprising a pharmacologically effective amount of the above analogues.

Description

ANALOGUES OF CYCLOLINOPEPTIDE A AND THE USE THEREOF

The present invention relates to new analogues of Cyclolinopeptide A, in particular analogues having a cyclic formula derived from the sequence of Cyclolinopeptide A, which sequence is extended between any two amino acid residues with a defined cystine derivative residue. Additionally, the invention 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 medicament for immunosuppressive treatment, to pharmaceutical preparations 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, W.M. Eds., North Holland, Amsterdam, pp. 158-1723. The chemical structure of Cyclolinopeptide A is

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 knowledge, this was the first biological activity that have been found for this natural product. 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 requires 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=O NH

| |

X-C-H H-C-Y

| |

CH₂ CH₂

| |

S

S in which X is selected from -H and -OH, and

Y is selected from -C=O and -H,

|

NH₂ 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 medicament for immunosuppressive treatment. 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 invention 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 immunosuppressive therepy.

The pharmaceutical preparation comprising an analogue of the invention can be formulated into oral preparations or preparations for infusion using pharmaceutically acceptable carrier(s), excipient(s) and/or diluent(s) suitable for such preparations, but keeping in mind that said analogue is insoluble 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 Cyclolinopeptide A. Accordingly, the analogues of the invention are to be used in medicaments for immunosuppressive treatment of mammals, including man, for all indications where immunosuppressive treatment is warranted. Examples of when immunosuppressive treatment is warranted is for the prevention of allograft rejection after organ transplantation and bone marrow transplantation, prophylactic or therepeutic treatment of graf t-versus-host-disease (GHHD), and autoimmune diseases, such as systemic lupus erythematosus (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-NH₂.

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 invention 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. Chem. Soc. 82 (1960) 4579-4581; Zhuze, A.L., Jost, K. Kasafirek, E. & Rudinger, J . : Coll. Czechoslovak Chem Commun. 29 (1964) 2648-2662, modified by Larsson L.-E., Lindeberg, G., Melm, P. & Pliska, V.: J . Med. Chem. 21 (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 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 König, W. & Geiger, R.: Chem. Ber. 103 (1970) 788.

GENERAL DESCRIPTION OF SYNTHESIS

All the peptides in the examples below were synthesized on an Applied Biosystems 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 International, Louisville, KY, USA). The final product of the synthesis 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-sulf ide 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 cm³ 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% trifluoroacetic acid water solution. The samples collected from the column were analyzed by analytical HPLC (Varian 5500, Sunnyvale, CA, USA) equipped with a Bondapak C₁₈ column

(Millipore, Milford, Mass., USA). Fractions containing pure substance were pooled, the solvent was evaporated and the product freeze-dried from 1 % acetic acid in water. The final HPLC analysis was performed on ready product and the structure of the peptide in question was confirmed by ammo 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 α-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-Cys-NH₂

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. [M+H]⁺ = 1246.

EXAMPLE I I

1-Mpa-LA-D-Cys-NH₂

The peptide was prepared according to the general description of synthesis. 3-Mercaptopropionic acid [S-(p-methoxy-)benzyl] was used for position I and Boc-D-cysteine

[-(p-methoxy-)-benzyl] for position 2.

Purity (HPLC):95.7%

The structure was confirmed by amino acid analysis and by FAB-MS. [M+H]⁺ = 1246.

EXAMPLE III 1-Hmp-LA-Cys-NH₂

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. [M+H]⁺ = 1262. EXAMPLE IV

1-Mpa-D-LA-D-Cys-NH₂

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

3-Mercaptopropionic acid [S-(p-methoxy-)bezyl] was used for pσstion 1. Purity (HPLC):98.0%

The structure was confirmed by ammo acid analysis and by FAB-MS: [M+H]⁺ = 1246. LIST OF COMPOUNDS USED AS ENZYME INHIBITORS AND AS IMMUNOSUPPRESSIVE AGENT

CS-A = Cyclosporin A (Reference)

CLA = Cyclolinopeptide A (Reference)

1-Mpa-LA-Cys-NH₂ Leu-Val-Pro-Pro-Phe

Ile-Ile-Leu-Mpa Cys-NH₂

I I

(Compound of Example 1)

1-Mpa-LA-D-Cys-NH₂ Leu-Val-Pro-Pro-Phe

Ile-Ile-Leu-Mpa D-Cys-NH₂

(Compound of Example 2)

1-Hmp-LA-Cys-NH₂ Leu-Val-Pro-Pro-Phe

Ile-Ile-Leu Hmp Cys-NH₂

L J

(Compound of Example 3) 1 -Mpa-D-LA-D-Cys-NH₂ D-Leu-D-Val-D-Pro-D-Pro-D-Phe

\

D- I le-D- I le-D-Leu-Mpa D-Cys-NH

(Compound of Example 4) wherein Mpa is 3-mercapropropionyl residue (-S-CH₂-CH₂-CO-) and Hmp is 2-hydroxy-3-mercaptopropionyl residue

OH

I

(-S-CH₂-CH-CO-)

INHIBITION OF THE ENZYME PPIase

It is known that the peptidyl-prolyl cis-trans isomerase (PPIase) catalyses the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and Nobuhiro Takahashi, et al (Nature vol. 337, Letters to Nature, pp 473-475, 1989) suggested that the peptidyl-prolyl cis-trans isomerizing activity of PPIase may be involved in events, such as 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 cyclophilm, 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 PPIase

The pig kidney cortex was homogenized in equal volume of saccharose (0.2 M) and centrifuged 60 min. at 10,000 x g. The supernatant was adjusted to pH 5.5 by addition of 1 M acetate buffer, pH 4.0. The precipitate was discarded and the supernatant was brought to pH 7.0. The extract was brought to 40% saturation of ammonium sulphate. To the supernatant obtained after 15 min. centrifugation at 20,000 x g solid sodium sulphate was added to get a final saturation of 60%. The precipitate obtained by centrifugation 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 KCl (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 chymotrypsin. Substrate (50 μM), previously dissolved m DMSO, was equilibrated at

10°C with the appropriate amount of PPIase in the thermostated 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 isomerization 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 incubating 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 activity 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_t was plotted against time.

The first order rate constant k (s^-1) was calculated from the resulting straight lines.

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

Materials and methods

Animals: CBA/ I iw mice 8 - 10 weeks old

Antigen: SRBC (sheep red blood cells)

Reagents: 1-Mpa-LA-Cys-NH₂, 1-Mpa-D-LA-D-Cys-NH₂,

1 -Mpa-LA-D-Cys-NH₂ , and CLA we re prepared by

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 intraperitoneally (ip.) or intravenously (iv.):

The reagents dissolved in one of solvents were diluted to desired concentration in PBS or in intralipid, and 0.2 ml 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 mtraperitoneally with 0.2 ml of 10% suspension 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 magnitude of the humoral immune response was expressed as the number of PFC per 10⁶ splenocytes. The results obtained are given 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% NH₄Cl buffered with 0.017 M Tris buffer to remove 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 10⁶

cells/ml) 0.1 ml of the reagent was added followed by addition of 0.1 ml of 0.005% suspension of SRBC in the cell culture medium, and incubated for 4 days at 37°C, 5% of CO₂ 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 was 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 hypersensitivity (DTH)

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

Immunosuppressive activity of CLA analogues in

Graf t-versus-host reaction (GvH)

Animals: Hybride F₁ (C₃H/Iiw x B₆/Iiw) and female B₆ mice,

8-10 weeks old.

Preparation of parental cells:

Female B₆ 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. GvH test:

GvH reaction was performed according to Twist and Barnes (Twist V.S., Barnes R.D.: Transplantation, 1973, 15, 1982). Hybride mice, F₁ (C₃H x B₆) were injected subcutaneously in the left hind footpad with 5 x 10⁶ parental lymphoid cells (B). After 7 days, draining lymph nodes 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.

Table A The number of PFC in the spleen cell of mice treated intraperitoneally with two doses (-3 hrs and + 24 after SRBC) of 1-Mpa-LA-D Cys-NH₂, 1-Mpa-LA-Cys-NH₂ and CLA dissolved in

Intralipid® (int.) (A fatty emulsion for intravenous

nutrition from Kabi, Stockholm, Sweden)

Preparation and introduction of the substance, see explanation after Table C.

The results are expressed as a mean ± SE of 6 mice. Table B

The number 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.)

The results are expressed as a mean ± SE of 6 mice . Table C

The number of PFC in the spleen of mice treated intravenously (iv.) with two doses of 1-Mpa-LA-Cys-NH₂, CLA and CS-A dissolved 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

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 di luted directly in int ral ipid to desired concentration. In the control 0.2 ml of PBS or intralipid was introduced.

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.)

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

Bg - background 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.

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

CS-A - dissolved in PBS.

Table F

The effect of CLA, CS-A and analogues of the invention dissolved 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 10⁵ SRBC. After 4 days the reaction was elicited by an intradermal introduction of 10⁸ 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. Table G Immunosuppressive activity of 1-Mpa-LA-Cys-NH₂ and

I

1 -Hmp-LA-Cys-NH₂ in GvH react ion ( c e l lular immunity ) .

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

respectively.

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 "Ol" - olive oil 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^-3s^-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—without inhibitor k=52 x 10^-3s^-1 O— O—with 1-Mpa-LA-Cys-NH₂ 2μg 41 ●—●—LA - inactive 10μg 35 ♡—♡—with CLA 20μg 24.1 Δ—Δ—without PPIase 7.5

Claims

1. An anlogue of Cyclolinopeptide A, characterised by 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 residue

C=O NH

| |

X-C-H H-C-Y

| |

CH₂ CH₂

I |

S —-------------S in which X is selected from -H and -OH, and

Y is selected from -C=O and -H

NH₂ wherein the amino acid residues are of L- or D-configuration.

2. An analogue according to claim 1, wherein said cystine derivative residue is located between the amino acid residues Leu and Phe in the formula of Cyclolinopeptide A.

3. An analogue according to claim 2, wherein X is -H and Y is -C=O , and

I

NH₂ all the amino acid residues are of L-configuration.

4. An analogue according to claim 2, wherein X is -H and Y is -C=O , and

I

NH₂ the cystine derivative residue is of D-configuration and the rest of the amino acid residues are of L-configuration.

5. An analogue according to claim 2, wherein X is -OH and Y is -C=O , and

I

NH₂ all the amino acid residues are of L-configuration.

6. An analogue according to claim 2, wherein X is -H and Y is -C=O , and

I

NH₂ all the amino acid residues are of D-configuration.

7. An analogue according to anyone of claims 1-6 for use as a medicament.

8. An analogue according to anyone of claims 1-6 for use as an immunosuppressive agent.

9. Use of an analogue according to anyone of claims 1-6 for the preparation of a medicament for immunosuppressive

treatment.

10. A pharmaceutical preparation comprising, as an active ingredient, an analogue according to anyone of claims 1-6, together with pharmaceutically acceptable carrier(s),

excipient(s) and/or diluent(s).

11. 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 according to anyone of claims 1-6.