CA2090859A1 - Analogues of cyclolinopeptide a and the use thereof - Google Patents

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

' W092/05189 1 PCT/SE91/~628 2~0~:~9 t ANALOGUES OF CYCLOLINOPEPTIDE A AND THE USE TH~QE

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 a6 a medicament, in particular for use a9 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 pharmaceutical 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 w~s first ~solated from linseed cake by Kaufmann H.P. and Tobschirbel A., in 1959 ~Chem. Ber., 92, 2805 ~1959)) and ~t8 chemclal Ltructure was suggested ln 1966 by Prox,A. and Weygand, F. t~1967) in "Peptides: Proceedings of the 8th European P-ptide Symposium", Beyerman, H.C., van de Linde, A. and van den Brink, W.M. Ed~., North Holland, Am~t-rdam, pp. 158-172~. The chemlcal structure of Cyclol~no-p-ptid- A i~ :~

Pro-Phe-Phe-L ~
¦ le Pro-Val-Leu-11e ~

K---ler, H., et al ~An9. Chemie Int. Ed. 25, 997-999 ~1986)) how-d that natural Cyclolinopeptide A inhibits the uptake of cholate into hepatocyte-, and stated that, to thelr know-ledge, this was the first biological activity that have been found for this natural product.
SUBSTITUTE SHEET

W092/05~89 2 PCT/SE91/~K~
t~
2~8~9 However, it wa~ surprisingly found that Cyclolinopeptlde A
can be used a~ an immunosuppressive agent, and this has been dificlosed in our previou~ Swedlsh patent application number ô904640-4 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 prevlou~ patent application, 60-70X of the linear peptide 18 lost durlng the cyclization I~ 18 evident that large scale production of Cyclolinopeptide A is very expensive, and lndustrial production of Cyclolinopeptlde A is thus deferred Surprisingly, we found that the immunosuppressive actlvity of Cyclolinopeptide A is retained when the sequence thereof is extended by a cystlne derlvat~ve residue The new analogue6 of Cyclolinopeptide A enables lar~e scale production thereof, slnce ln the cycllzatlon ~tep practically no linear pept~de is lost DESC~IPTION OF THE INVENIION
In one aspect ot the lnventlon there is provided new analogu-s of Cyclolinopeptide A havln~ a cyclic formula d-riv-d from the eguence of Cyclolinopeptide A

Val-Pro-Pro-Phe he eu-lle-lle-Leu .

... . . . , .. ~....... .
' ' ~

WO92/05189 ~ PCT/SE91/00628 ,~. 2~9V535~

which sequance i~ extended between any two amino acid residues with the followlng cy~tine derivative residues C=O N~
I
X-C-H H-C-Y
I
; CH2 CH2 ~0 S S -, :

in which X is ~elected from -H and -OH and Y is selected from -C=O and -H

wherein the amino acid residues are of L- or D-configuration.
.~
In a preferred embodlment of thls aspect of the inventlon the above cystine derivative residue is located between the amlno ac~d residues Leu and Phe ~n the formula of Cyclollnopept de A. In fact the locatLon of said cystine derivative residue ir.
the sequence Or Cyclol~nopeptlde A was randomly chosen. Thus lt can bo expocted that the immunosuppressive activity of ~yclolinop-ptido A ~ r-ta~ned ~rrespect~ve of between whlch two amino acid residue~ in the sequence thereof the cyst-ne derivatlve ree~due ~ ntroduced.

In an additional aspect of the invention there is provided an analogue of the ~nvent~on for u~e a~ a medicament.

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

SUE~STITUTE SHEET

... , . . . ~ . . : ~ , `~: . , : ` , : . : :. -. ~ . . .

8 ~ 9 In still another aspect of the invention there i~ provided a method of treating a mammal, including man, in need of immunosuppressive treatment, comprislng administering to sald mammal a pharmacologically effective amount of an analogue of the invention In yet another aspect of the invention there i8 provided a pharmaceutical preparation comprising an analogue of the in-vention together w~th phnrmaceutically acceptable carrier(s), excipient(s) and/or diluent( 8 ) . h The amount of an analo~ue of the ~nvention to ~e administered to a mammal in need of immunosuppressive treatment has to be decided by a phys~cian who 18 experlenced in immunosuppres-~ive therepy The pharmaceutlcal preparation compri~ing an analogue of the invention can be formulated into oral preparations or prepa-rations for infuslon using pharmaceutically acceptable carrier(s), excipient~s) and/or diluent(s) suitable for such preparations, but k--pin~ in mlnd that said analogue i8 ln-oluble in water The pharmaceutical pr-paration can e g compri-e an analogue of the lnventlon ln a pharmacolo~lcally tt-ctiv- amount urp-nd-d in an olive oil The pr-~en- ~nvent~on r-v-al- hat analogues of the lnventlon ha~- immuno-upPre-sive activity, which i8 comparable to that of Ciclosporin ~al-o call-d Cyclo-porin A) and Cyclollno-p-ptid- A Accordin~ly, the analo~ues o~ the inv-ntion are to be u~-d in medlc-m-nt- for immuno-uppre--ive treatment of mammal-, including man, for all indications where immuno-suppre--ive treatment 1~ warrant-d Examples of when lmmuno-uppr-~-ive treatment i- warranted is for the prevention of allogratt reJectlon aft-r or9an transplantation and bone marrow transplantation, prophylactic or therepeutic treatment ot graft-versus-ho~t-dlsease ~GVHD), and autoimmune dlsease6, ~. . -: : . -.. ~ .

~, ,?;
2~8~9 such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and diabetes mellitus.

SHO~ DES~R~'rION OF THE DRAWINGS
Fig. 1 shows a graph of the inhibition of PPIase actl~ity by Cyclosporin A.

Fig. 2 shows a graph of the inhibition of PPlase actlvity 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 peptlde chemistry, whereby a linear peptide i8 formed, which is then subjected to an oxidatlon step ln order to cycllze two cysteine related residues, whereby ~ sulphur bridge is formed between said residues, thus formlng a cystlne derivative residue.

The linear linopeptide ~LA) analogues according to the inven-tlon can thus be synth-s~z-d ln conventional manner e.g. by ~tep wi~e coupling of one amino acid residue to the next in liquid pha~e, e.g. accord1n~ to the method of Law, H.B. 8 Du Vigneaud, V.:J. Am. Chem. Soc. 82 ~1960) 4579-4581; Zhuze, A.L., Jo~t, K. Kasafir-k, E. ~ Rudin~er, J.: Coll. Czecho-lovak Chem Commun. 29 ~1964) 264B-2662, modified by Larsson r .-E., Lindeberg, G., Melln, P. ~ Pliska, V.: J. Med. Ch-m.
21 ~1978) 352-356. The coupling of amino acid residues to one another, whereby ~o call-d peptide bonds are produced may also be performed starting with a solid phase ~usually a re~in) to which the C-terminal of the first amino acid ~8 coupled, whereupon the C-terminal of the next amino acid is coupled to the N-terminal of the flrst amino acid, etc., finally releasing the built-up peptide from the solid phase.
In the example6 below thls 80 called rolid phase techn~que ~ . :

~ 5~ 6 PCT/SE91/~628 has been utilized in accordance with the method of Merrifield, R.~.; J. Am. Chem. Soc. 05 ~1963) 2149;
Merrifield, R.B.: Biochem. 3 (1964) 1335 and Konig, W. 8 Geiger, R.: Chem. Ber. 103 ~1970) 758.
GENER~L DESCRIPTION OF SYNTHESIS

All the peptides in the examples below were synthesized on an Applied Bio~ystems 430A Pept~de Synthesizer using a double coupling program with termination step after the second coupling; The resln used was of 4-methyl-benzhydrylamine type with a theoretical loading of 0.66 meq/g ~Peptides Interna-tional, Louisville, KY, USA). The flnal product of the syn-thesis was dried in vacuo over night. The peptide was then cleaved from the re~in by treatment with liquid hydrogen fluoride in the presence o anisole and ethyl-methyl-sulfide as scavangers ~HF:anisole:EMS - 10:05:05). After removal of hydrogen fluoride by evaporation the residue wa6 ~uspended ln ethyl acetate ~100 ml) and flltered. The solid was washed on ~ilter with addltional ethyl acetate ~3 x 100 ml) and the cleaved pept~de extracted with acet~c acid ~100 ml). The extract was promptly diluted to a volume of 2 cm3 with 20X
acetic acid ln methanol and trested w~th 0.1 M solut~on of iodine ln methanol until the fa~nt brown colour persisted.
Th-n the Dow-x 1 x 8 ~on exchanger ~n acetate form was added ~3 ~) ~3io-Rad, Richmond, CA, USA~ and the mixture was filter-d. She filtrate was evapora-ed and the re-idue freeze--dri-d from 1~ acetic acid in water. The product was then purifi-d by rever~-d pha-e liqu~d chromatography on a column ~ d with Vydac 20-25 ~ tSeparation Group, CA, USA~ in a ~uitable system containinp acetoni-r~le in O.lX trifluoro-acetic acid water olution. The samples collected from the column were analyzed by analytical HPLC ~Varian 5500, Sunny-vale, CA, USA) equipped with a ~ondapak C1g column ~Millipore, Milford, Ma~s., VSA~. Fractions containing pure ~ubstance were pooled, the ~olvent was evaporated and the SUBSTITUTE SHEET

.. . .
. . .

- .
,. . ` , ~ . ..

WO92/05189 7 PCT/SE9l/00628 7 } ~ ! .
product freeze-drled from lX acetic acid in water. The final HPLC analysi~ was performed on ready product and the struc-ture of the peptlde in question was confirmed by amlno acid ~nalysis and FAB-MS ~Fast atom bombardment spectrometry). The FA~-MS analyses were performed by M-Scan Ltd. Sunnln~hill, Ascot, Berkshire, England.

All amino acids used during the synthesi~ were protected with tert-butoxycarbonyl group at -amino function. The thiol function of Mpa, Hmp and Cys wa~ protected with 4-methoxy-bensyl group. The amino acid derivative~ were dellvered by Bachem AG, Switzerland.

~ .
In the Examples below the abbreviation LA is used for the linear peptide Leu-lle-lle-Leu-Val-Pro-Pro-Phe-Phe EXAMPLE I

1-Mpa-LA-Cy6-NH2 The peptide was prepared accordlng to the gener~l de~crlptlon of synthesi6. 3-Mercaptopropionic acid tS-~p-methoxy-)benzyl~ -was used for po~ltion 1.

Pur~ty ~HPLC):99. ax The sructure was conf~rmed by am~no acid analy~is and by FAB-MS. tM~H~ = 1246.

EXAMe~E 11 1-Mpa-LA-D-Cys-NH2 SUBSTITUTE SHEET

2~9~38~9 The peptide wa~ prepared according to the general de~cript~on of ~ynthesis. 3-Mercaptopropionic acid ~S-~p-methoxy-)benzyl]
was used for positlon I and Boc-D-cysteine ~-(p-mothoxy-)-benzyl] for po6ition 2.
s Purity ~HPLC):95.7X

The structure was confirmed by amlno acid analy6i6 and by FA~-MS. CM+H]+ = 1246.
EXAMPLE III

l-Hmp-LA-Cys-NH2 The peptide was prepared accord~ng to the general descrlptlon ot ~ynthesis. 2-Hydroxy-3-mercaptopropionic acid was used for position I.

Purity ~HPLC):97.5X

The structure wa6 conflrmed by amino acid analysls and by FAL-MS. ~M~H~+ = 1262.

l-Mpa-D-LA-D-cys-NH2 The p-ptide was pr-par-d accordlns to the goneral descript~on Or synthesis using amino acids of D-configuration.

3-Mercaptopropionic acld tS-~p-methoxy-)bezyl~ was used for postion 1.

Purity ~HPLC~:96.0X

The structure wa6 conflrmed by amlno acid analysi~ and by FAL-MS: tM~H~ = 1246.
~U~STITUTE SltEET

. , . ~ ., ~

. ,. . .. ~.. . ` .

.

- ..

2~08~9 ~
LIST OF COMPOUNDS USED AS ENZYME INHIBITORS AND AS IMM~NO-SUPPRESSIVE AGENT

CS-A = Cyclo~porln A ~Referenc8) CLA = cyclolinopeptide A (Reference) 1-Mpa-LA-Cy~-NH2 Leu-Val-Pro-Pro-Ph ~
¦ ¦ = Phe / .: ,' Ile-Ile-Leu-Mpa ~ s-NH2 ~5~Compound of Example 1) 1-Mpa-LA-D-Cys-NH2 Leu-Val-Pro-Pro-Phe I ¦ = Phe . -I: Le-Ile-Leu-Mpa D-Cys-NH2 ~Compound o~ Ex~mple 2~

1-Hmp-LA-Cys-NH2 L-u-Val-Pro-Pro-Phe ¦ ¦ _ Phe 1. .e-lle-Leu-Hmp Cys-NH2 ~Compound of Example 3~

SUBSTITUTE SHEET

2~9~8~9~-~

l-Mpa-D-LA-D-Cys-NH2 D-Lju-D-Val-D-Pro-D-Pro-D-Phi . , D-Ile-D-Ile-D-Leu-Mpa D-Cy~-NH ~ -(Compound of Example 4) wherein Mpa i~ 3-mercapropropionyl residue (-S-CH2-CH2-CO-) and Hmp is 2-hydroxy-3-mercaptopropionyl residue OH
I

(-5-CH2-CH-CO-) INH181TION OF THE ENZYME PPlase It i~ known that the peptidyl-prolyl cis-tran~ isomerase ~PPIase~ catalyses the cls-trans ~somerization of prollne 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 isomeriz~ng ac-t~v~ty of PPIa~e may be ~nvolved ln events, such a~ those occuring oarly in T-cell activation, that are suppressed by Cyclorpor~n A. Gun--r F~scher, et al ~Nature Vol. 337, L-tters to Nature, pp 476-478, 1989) reported that PPIase ~8 probably identical to cyclophilln, a recently dlscoverod mammalian protoin which binds tightly to Cyclosporin A. We have purified PPS-~- and tosted analogues of the ~nvent~on a8 inhibitor~ of PPIase.

PURIFICATION OF PPla~e The pig kidney cortox was homogenized in equal volume of 3accharose (0.2 M) and centrifuged 60 min. at 10,000 x 9. The SUBSTITUTE SHEET

. ~ ~
. . ..

.. . ~ , . ` , ,. .~ ,' .

W092~05189 11 PCT/SE91/~628 ~, ~ 2 ~ 9 supernatant wa~ ad~ueted to pH 5.5 by addition of ~ M acetate buffer, pH 4Ø The precipitate was discarded and the ~uper-natant wa~ brought to pH 7Ø

The extract was brought to 40X 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 ~inal ~aturation of 60X. The precipitate obtained by centri-fugation was dissolved in a small volume of 10 mM Tri~
buffer, pH 7.6 and dialysed 24 h. again6t the same buffer.
The dialysed solution was loaded onto a DEAE-Sephadex A-50 column, that had been prevlously eguilibrated with the same buffer and eluted without changing the buf~er. The eluate rich in PP~a~e act~lty pa~ed through the column ln one lS active peak.

The active fract~ons were appl~ed on a CM-Sephadex C-50 column and eluted with a linear gradient of KCl (0-1.2 M) as one actlve peak.
The enzyme was etored as euspens~on in 60X saturated ammon~um sulphate. After 5 month8 the enzyme shows full enzymatic ~ct~ity.

DETERMINAT~ON OF PPlase ACSIVITY

The ci~-trans iromerlzation of the Ala-Pro peptide bond of the peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanlllde waa m-a~ur-d in a coupled assay with chymotryp~in. Substrate ~50 ~M~, prev~ously dic~olved in DMSO, wae equilibrated at 10C w~th the appropr~ate amount of PPlaee in the thermostat-ed ~pectrophotometer cell. The reaction wa8 started by adding chymotryp~in (f~nal concentration 21 ~M). The trans-peptide was cleaved within the d-adtime. The rate of cis-trans isome-rizat~on was followed by the decrease in tran~m~ttance at 390 nm. The ~pectrophotometer Specord UV-Vic ~Jena, GDR~ :~
eguipped with a cell stirrer was used.

;, . . ,. ;. . , . ' ....................... , . ,. :, . :.
- ... . i ., . : . ~ .. .. . .

:, ~. , : ' W092/05189 S2 PCT/SE91/~628 '~ 0 ~

The effect of the lnhibitors on PPIase was examined by mixing the studied sub~tance with PPIase for 1 min. before incubat-ing the ~ubstrate The substances tested were dissolved in dioxane containing 0.5 - lX of Triton X-100 or in DMSO. There was no difference ob~erved in the effect on the PPIase acti-vity depending on the kind of solvent.

The result~ are given in Fig. 1 and Fig. 2, where the log of the difference between transmittance at ~teady state Ax and transmittance at a given time At was plotted against time.
The first order rate constant k ( 5-l ) was calculated from the resulting straight lines.

IMMUNOSUPPRESSIVE ACTIVlTY OF CLA, CS-A AND THE ANALOGUES
OF THE ~ NVENTION.

Material~ and methods Animals: CBA/Iiw mice ~ - 10 wee~ old Antlgen: SR8C ~sheep red blood cells) Reagents: 1-Mpa-LA-Cys-NH2, 1-Mpa~ -LA-D-CT8-NH2~

1-Mpa-LA-D-Cyr-NH2, and CLA were prepared by FERRING AB, Sweden, and Cyclosporin A ~CS-A), Sand~mmun Sandoz, Ba-el, Switzerland Solv-nts: Mixture of Cr-mophor EL ~SIGMA) and 94X ethanol ~6.5 : 3,5~, eth~nol 96X, olive oil, intralipid and PBS ~pho~phate buffer ~olution~

T~QatmQnt çf mice with reaoent intraPeritoneallv ~i~.) or ntravenouslv ~iv.):
The reagents dissolved ~n one of solvents were diluted to desired concentration in PBS or in intralipid, and 0.2 ml SUBSTITUTE SHEET

~. ~
t ' , ' ' ' '; ' ': ' .: ",'. ' ~ .' ~' `' W092/05189 13 PCT/SE91/~628 ~r ~39~ 9 thereof wa~ introduced lp in two dose~ First 3 hr~ before the antigen, second 24 hrs later The activity of analogues studied wa~ compared to the activity of CLA and CS-A

S Treatment of mice with ~ea~ents Der 09 (PO.). directlv into the ~tom~ch The reagents dis~olved 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 4~ hr~ ~ -later The activity of the derivatives studied was compared to the activity of CLA and CS-A
.:
Effect of CLA analoaues on humoral immune resDonse in mlce immunized with SRBC

Mice were injected lntraperitoneally wlth 0 2 ml of lOX sus-pension of SRBC in PBS, 3 hrs before the antigen the first dose of the reagen- WaB introduced, ip , po or iv After 4 days the number of plaque forming cells (PFC) in the spleen was detsrmined accordlng to the Mishell-Dutton <Mishell R
1 , Dutton R W J Exp Med , 1967, 126, 423) The magnl-tude of th- humoral lmmune resPOnSe wa8 expressed as the num-b-r ot PFC p-r 106 ~plenocytes The results obtained are al-ven in table~ A, B and C

Ett-ct of CLA analoau-s on humoral immune re~Donse to SRBC ln vitro Priming ot mice and l-olat~on of spleen cell~ Mice were primed intrav-nously with 0 2 ml of lX 8uspension of SRBC in PBS Four days later the an~mals were killed and thelr spleens were minced, pres~ed through a pla8tic screen into 0-~3X NH4Cl buffered with 0 017 M Tris buffer to remove SU~STITUTE SHEET

. ., ., . .. . . - .. ,; . .
, `. . . .~ . . .

.
.. . . . .
,. , . .. - , :
.. ; . ~ .:

- , ~ . .
` . , - . . . ~ ,, 2'~3~9 erythrocytes. Then the cell~ were washed three time wlth PBS
and finally resu6pended in RPMI medium supplemented with lOX
of fetal calf serum.

Culture conditions and determ~nation of PFC num~er:
To one milliliter of spleen cell suspension (S x 106 cells/ml) 0.1 ml of the reagent was added followed by addl-tion of 0.1 ml of 0.005X suspension of SRBC in the cell cul-ture medium, and incubated for 4 days at 37C, S~ of C02 and lOOX of humidity. The number of PFC was measured using the procedure of Mlshell-Dutton. As a control PBS or approprlate concentration of the 601vent was u6ed. The experiments were performed using NUNC 24-well Tissue Culture Plate.
The result6 are 6hown in Tables D and E.
Effect of CLA analoaues on delaved tY~e hvDersensitivitv (DTH) The re~ults and de~criptlon of the test are given ~n Table F.
ImmunosuDDresslve actlvitY of CLA analoaues in Graft-versus-host reaction (GvH) Anlmals: Hybride Fl (C3H~I~w x 86/Iiw~ and female 86 mlce, ~-10 weeks old.

Pr-para~lon of parental cells:

F-male B6 mice were killed, their popliteal nodes were pre--ed through a pla~tic ~creen lnto PBS. Then the cell~
were wa-hed three times with PDS ~nd re~u~pended in RPMI
medium.

SUBSTITUTE SHEET

. .
.. ~ . ~ - -.
.. . . . ~ . . i . . ...

. . . .

W092/05189 15 PCT/SE9~628 f i............................................................................. .

GvH test:

GvH reaction was perormed according to Twist and Barnes ~Twist V.S., Barnes R.D.: Transplantation, 197~, 15, l9B2).
Hybride mlce, F1 ~C3H x B6) were injected subcutaneously in the left hind footpad with 5 x 106 parental lymphoid cells tB). After 7 days, drainlng lymph node6 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 reactlon). The results are given ln 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 SR~C) of 1-Mpa-LA-D Cys-NH2, 1-Mpa-LA-Cys-NH2 and CLA dissolved in Intralipid ~ tint.) ~A fatty emul~ion for intravenous nutritlon from Kabi, Stockholm, Sweden) ~5 Reagent P
~s/mouse PFC~106 I SE Student test Control int. 3047 35~ -l-Mpa-LA-D-Cys-NH2 1.0 1139 162 0.01 I I 10.0 5~6 77 0.001 1-Mpa-LA-Cys-NH21.0860 64 0.001 ~ _ I10.0 798 32 0.001 CLA 1.0 1649 339 0.05 10.0 864 74 0.001 Preparation and ~ntroduction of the substance, see explana-tion after Table C.

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

SUBSTITUTE SHEET

~ . - .. ... . ..

, WO92/05189 17 PCT/SE91/00628 ~ I ' .
- .
~00~9 Table B

The number of PFC in the ~pleen of mice treated with two doses ~-3 hr and 24 hr after SRBC~ of CLA, CS-A and analogue6 ot the in~ention dl~solved in olive oil, introduced per o~
~po. ) Reagent p ~g/mouse PFC/106 + SE StudPnt test 15Control olive oil 21~3 166 CLA 10 1016 125 0.05 100 314 73 0.001 20 CS-A 10 1092 209 0.01 100 753 51 0.001 1-Mpa-D-LA-D-Cys-NH2 10 602 112 0.01 I I 100 680 86 O.OOl l-Mpa-LA-D-Cy~-NH2 lO 764 60 0.01 I I ~00 477 45 0.001 l-Mpa-LA-Cy~-NH2 10 687 136 0.01 30 1 î 1OO 501 109 0.001 The r--ults ~re expressed aY a mean ~ SE of 6 mice.

SUBSTITUTE SHEET

.-~ . . .. ~ . : . . : . .
. . . . ~ . . . . . . ' . . . . . .
.- . . . . . . . . . ..
. . . .. . ... . ..
- . . .

. , . . , . . - . ..
. ... ..... .. .. . . . ~ . .. .. . . .

WO92/05189 18 PCT/SE9l/00628 2~8~9 Table C

T~e number of PFC in the spleen of mi~e treated intravenou~ly tiv.) with two doses o~ 1-Mpa-LA-Cy~-NH2, CLA and CS-A dis-~olved in intralipid ~int.) I

Reagent p ~g/mouse PFC/106 + SE Stud~nt test Control P~S 19O5 B2 Control int. 2326 67 NS
1-Mpa-LA-Cys-NH2 0.01 1649 126 0.05 1 1 0.11364 43 0.01 1.0 903 39 0.001 CLA 0.011943 77 NS
0.11475 97 0.02 1.01203 85 0.01 CS-A 0.011042 122 NS
0.11633 lel o . 05 1.0115e 50 0.01 i Th- pr-parat-s in the volume of 0.2 ml were lntroduced twice, rirst dose 3 hrs befor- sens~tizatlon with antigen second 24 hrs later. Preparation o CLA and 1-Mpa-LA-Cys-NH2: 2 mg of th- substance was di-solv-d ~n 0.15 ml of hot ethanol and 0.35 ml of intralipid, then diluted to desired concentration in intral~pid.
CS-A was diluted directly ~n intralipid to desired concentra-~5 tion. In the control 0.2 ml of PBS or intralipid was intro-duced.
The re~ults are expres~ed a~ a mean + SE of 6 mic~.

.- : , : , ' .:: . .::': . . :. :, ' ,, : . ' : : ' : :

WO~2/05189 ~9 PCT/SE91/00628 2 1Sa ~
Table D

The number of PFC ln the spleen cell cultures treated with CLA, CS-A ~nd analogues of the invention dissolved in intralipid ~int.) .. . . . .. _ Reagent P
~g/well PFC/106 I SE Student test Control PBS 4212 404 Control int. 4495 26B NS

CLA 0.012879 236 NS
lS 0.~865 27 0.001 :
1.0537 ~0 0.001 CS-A 0.013020 184 NS
0.1892 31 0.001 1.0569 84 0.001 1-Mpa-D-LA-D-Cys-NH2 0.01 3047 370 NS
I 1 0.1217B 306 0.05 1.0841 B9 0.001 ZS
I-Mpa-LA-D Cys-NH2 0.01 29B5 272 NS
I 1 0.13100 279 NS
~.0119 ~5 0.001 1-Mpa-CLA-Cys-NH2 0.0123BB 226 0.05 0.122~6 ~01 0.05 1.0~36 70 0.001 ~9 - 65 17 The results are expressed as a mean + SE of 6 wells.
Bg - background . . .
.- .. .. ..

W092/05189 20 R~T~SE91~00628 Table E
2 ~ 9 Imunosuppressive activity of CS-A and analogues of the invention mea~ured by the number of PFC. ~n in vivo studie~, mice were tre~ted with two doses of the preparations dissolved in cremophor or in olive oil. First dose ~ hours before immunization, ~econd 24 hour~ later.

Reagent Route of P
~g/mouse introduc- PFC/106 + SE
tion Control "C" i.p. 1330 lD9 1-Mpa-LA-Cys-NH2 1 ~g l.p. 1263 124 NS
1 1 10 ~3 i.p. 700 90 0.01 1-Hmp-LA-Cys-NH2 1 ~g i.p. 1178 111 NS
I I 10 ~9 i.p. 831 58 0.05 20 Control PBS i.p. 1186 109 - i CS-A 1 ~9 ~.p. 1350 120 NS
10 ~9 i.p. 839 105 0.05 ___________________________________________________________ Control "C" i.p. 1122 144 1-Mpa-LA-Cys-NH2 10 ~9 ~.p. 238 23 0.001 30 Control PBS i.p. 1056 95 CS-A 10 ~9 i.p. 124 36 0.001 ___________________________________________________________ ~

Control "C" - cremophor in Appropriate concentr~tion need-d to prepare 10 ~9 of the pept~de.
CS-A - dissolved in PBS.
SUBSTITUTE SHEET

. . . ~..... . , .. :, ~ . . ; :-: . :

WO92/05189 21 PCT/SE91/~628 ~'~7.

Continued _________________________________________________________ Control "Ol" p.o.2428 329 1-M~a-LA-Cys-NH2 10 ~9 p.o.1214 91 0.02 I I100 ~9 p.o. 406 73 0.001 CS-A 10 ~g p.o.1888 272 NS
100 ~9 p.o. 834 142 0.01 Control 'Ol p.o.1693 133 1-Mpa-LA-Cys-NH2 100 ~g p.o. 444 66 0.001 1-Hmp-LA-Cys-NH2 100 ~9 p.o.672 74 0.001 Control PBS p.o.1788 219 CS-A 100 ~9 p.o.561 .88 0.001 _____________________________________________________________ Control "C" ln v~tro 3690 321 1-Mp~-LA-Cys-NH2 1 ~g/ml in vitro 65 11 0.001 CS-A 1~g/ml in vitro 20 12 0.001 _____________________________________________________________ Control "Ol' - olive oil SU~STITUTE SHEET

.. ~ . .:

- ~ . - ., . : ~ .
. .. - . ~ . .. . : . .- .
~ . ..

. : . .

W O 92/05189 22 PCT/SE91tO0628 ~ !
~ ,.,i ' 2 a ~ ~ 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 lntroduced per 08 ~po ) twice, first dose 3 hours before sensitization, second 48 hours later Reagent P
~g/mouse Vnits I SE Student test Control olive oil 22 50 1 60 t lS CS-A 10 13 5 0 86 0 01 1-Mpa-D-LA-D-Cys-NH2 10 12 6 0 70 0 01 1-Mpa-LA-D-Cy6-NH2 10 13 70 0 94 0 02 I 1 100 11 20 0 65 0 00l 1-Mpa-LA-Cys-NH2 10 11 7 0 50 0 01 l 1 100 8 50 0 71 0 001 On- unit = 0 1 mm DTH was induced ln m~c- accordin~ to Lagrange et al ~Lagrange P H , Mackaness G E , Miller T E , Pardon P J Immunol , 1975, 114, 447) Mice were sensitized intra~enously with 105 SRBC. After 4 d~ys the react~on was elicited by an intra-d-rmal introductlon of lo8 SRBC into left, hind foot pad The ma~nitude of the reaction was mea8ured as the increa~e of the foot pad thickness at 24 hrs following administratlon of the challenging dose The results are expressed as a mean ~ SE of 10 mice SUBSTITUTE SHEET
:~.

W092/05189 23 PCT/SE9t/~628 f.~,,,,., .
~03~!~
Table G

Immunosuppressive activity of 1-Mpa-LA-Cys-NH2 and 1-Hmp-LA-Cy~-NH2 in GvH reaction (cellular immunity). `, Preparations dissolved in cremophor were given i.p. -4 and 48 hrs before and after introduction of parental cells, respectively.
,' Reagent Route of GvH ~ SE P
~g~mouse introduc- (index) tion Control "C" i.p. 3.17 0.36 1-Mp~-LA-Cys-NH2 10 ~g i.p. 1.96 0.27 0.05 l-Hmp-LA-Cys-NH2 10 ~g i.p. 1.54 0.22 0.01 , Control PBS ~.p. 3.09 0.42 CS-A 10 ~g ~.p. 1.41 0.16 0.001 Control PBS - phorphat- buffer ~olution Control "C" - cremophor at a conc-ntration needed to di~olve 10 ~g of the peptide~ or 1 ~9 ~in in vitro) Control "Ol' - olive oil SUt~STlTUTE SHEET

.
.
.

. . .. - . .: . , . ;~

.

2~9V~ 24 ~ I

Description of the drawings:

Fig.1 Inhibition of PPIase activity by Cyclosporin A.
Catalysis of proline isomerization by PPIase:
- X -X - without CS-Ak=26 X 10 3s 1 - O -O - 0.25 ~g CS-A 12.9 , i o -O - 0.50 ~g CS-A 10.9 -A - 1.5 ~g 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 - O - O - with 1-M ~ ys-NH2 2~g 41 - - - LA - inactive 10~g 35 _ O - O - with CLA 20~g 24.1 without PPIase 7.5 SUBSTITUTE SHEET

, "
. .
.. . . . ~ . . ~ .. . -

Claims (11)

1 An anlogue of Cyclolinopeptide A, characterised by a cyclic formula derived from the sequence of Cyclolino-peptide A

which sequence is extended between any two amino acid residues with the following cystine derivative residue in which X is selected from -H and -OH, and Y is selected from and -H

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 , and all the amino acid residues are of L-configuration.

4. An analogue according to claim 2, wherein X is -H and Y
is , and 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 , and all the amino acid residues are of L-configuration.

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

WO 92/05189 PCT/SE9l/00628

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 immunosuppresive treatment comprising administering to said mammal a pharmacologically effective amount of an analogue according to anyone of claims 1-6.