EP0553133A1 - Analogues de cyclolinopeptide a et leur utilisation - Google Patents

Analogues de cyclolinopeptide a et leur utilisation

Info

Publication number
EP0553133A1
EP0553133A1 EP19910917536 EP91917536A EP0553133A1 EP 0553133 A1 EP0553133 A1 EP 0553133A1 EP 19910917536 EP19910917536 EP 19910917536 EP 91917536 A EP91917536 A EP 91917536A EP 0553133 A1 EP0553133 A1 EP 0553133A1
Authority
EP
European Patent Office
Prior art keywords
amino acid
cyclolinopeptide
acid residues
analogue according
configuration
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19910917536
Other languages
German (de)
English (en)
Inventor
Zbigniew Wieczorek
Z. Ignacy Siemion
Jerzy Trojnar
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ferring AB
Original Assignee
Ferring AB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ferring AB filed Critical Ferring AB
Publication of EP0553133A1 publication Critical patent/EP0553133A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • Cyclolinopeptide A was first isolated from linseed cake by Kaufmann H.P. and Tobschirbel A., in 1959 (Chem. Ber., 92.
  • Immunosuppressive treatment of a patient in need thereof requires large amounts of the immunosuppressive agent used.
  • 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.
  • Cyclolinopeptide A having a cyclic formula derived from the sequence of Cyclolinopeptide A
  • 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.
  • an analogue of the invention for use as a medicament.
  • an analogue of the invention for the preparation of a medicament for immunosuppressive treatment.
  • 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.
  • 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.
  • 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.
  • immunosuppressive treatment 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.
  • GHHD graf t-versus-host-disease
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • 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 .
  • 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.
  • linear linopeptide (LA) analogues 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.
  • 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 ⁇ (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 C 18 column
  • the peptide was prepared according to the general description of synthesis. 3-Mercaptopropionic acid CS-(p-methoxy-)benzyl] was used for position 1.
  • 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
  • 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 peptide was prepared according to the general description of synthesis using amino acids of D-configuration.
  • PPIase peptidyl-prolyl cis-trans isomerase
  • PPIase is probably identical to cyclophilm, a recently discovered mammalian protein which binds tightly to Cyclosporin A.
  • 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 first order rate constant k (s -1 ) was calculated from the resulting straight lines.
  • mice CBA/ I iw mice 8 - 10 weeks old
  • SRBC sin red blood cells
  • mice 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.
  • the activity of the derivatives studied was compared to the activity of CLA and CS-A.
  • 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.
  • 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 10 6 splenocytes. The results obtained are given in tables A, B and C.
  • 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 Cl 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:
  • Hybride F 1 (C 3 H/Iiw x B 6 /Iiw) and female B 6 mice
  • mice Female B 6 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:
  • Hybride mice F 1 (C 3 H x B 6 ) were injected subcutaneously in the left hind footpad with 5 x 10 6 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-NH 2 , 1-Mpa-LA-Cys-NH 2 and CLA dissolved in Intralipid® (int.) (A fatty emulsion for intravenous
  • mice treated intravenously (iv.) with two doses of 1-Mpa-LA-Cys-NH 2 , CLA and CS-A dissolved in intralipid (int.)
  • 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.
  • 0.2 ml of PBS or intralipid was introduced.
  • the results are expressed as a mean - SE of 6 wells .
  • Imunosuppressive activity of CS-A and analogues of the invention measured by the number of PFC.
  • 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.
  • mice DTH was induced in mice according to Lagrange et al (Lagrange P.H., Mackaness G.B., Miller T.E., Pardon P: J. Immunol.,
  • mice were sensitized intravenously with 10 5 SRBC. After 4 days the reaction was elicited by an intradermal 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.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

On décrit des analogues nouveaux de Cyclolinopeptide A ayant une formule cyclique dérivée de la séquence de Cyclolinopeptide A, (I) dont la séquence s'étend entre deux résidus d'acides aminés quelconques ayant le résidu de dérivation cystine (II) où X est choisi entre -H et -OH, et Y est choisi entre (a) et -H, et où les résidus d'acides aminés sont de configuration L- ou D-. Ces analogues peuvent être utilisés comme médicaments, notamment comme immunosuppresseurs. On décrit également l'utilisation de ces analogues dans la préparation d'un médicament pour le traitement par immunosuppresseurs et dans les préparations pharmaceutiques comprenant lesdits analogues. On peut traiter un mammifère, y compris l'être humain, nécessitant un traitement immunosuppresseur grâce à un tel médicament comprenant les analogues susmentionnés en quantité efficace du point de vue de la pharmacologie.
EP19910917536 1990-09-25 1991-09-18 Analogues de cyclolinopeptide a et leur utilisation Withdrawn EP0553133A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9003040A SE467410B (sv) 1990-09-25 1990-09-25 Analoger av cyklolinopeptid a och anvaendning daerav
SE9003040 1990-09-25

Publications (1)

Publication Number Publication Date
EP0553133A1 true EP0553133A1 (fr) 1993-08-04

Family

ID=20380440

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19910917536 Withdrawn EP0553133A1 (fr) 1990-09-25 1991-09-18 Analogues de cyclolinopeptide a et leur utilisation

Country Status (7)

Country Link
EP (1) EP0553133A1 (fr)
AU (1) AU8653191A (fr)
CA (1) CA2090859A1 (fr)
FI (1) FI931259A0 (fr)
HU (1) HU9300847D0 (fr)
SE (1) SE467410B (fr)
WO (1) WO1992005189A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006115509A2 (fr) 2004-06-24 2006-11-02 Novartis Vaccines And Diagnostics Inc. Immuno-potentialisateurs a petites molecules et analyses visant a detecter leur presence

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE8804640L (sv) * 1988-12-23 1990-06-24 Ferring Ab Laekemedel omfattande cyklolinopeptide a

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9205189A1 *

Also Published As

Publication number Publication date
SE467410B (sv) 1992-07-13
WO1992005189A1 (fr) 1992-04-02
CA2090859A1 (fr) 1992-03-26
FI931259A (fi) 1993-03-22
FI931259A0 (fi) 1993-03-22
SE9003040D0 (sv) 1990-09-25
SE9003040L (sv) 1992-03-26
AU8653191A (en) 1992-04-15
HU9300847D0 (en) 1993-06-28

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