MEDICAMENT COMPRISING ANTAMANIDE

Abstract: Antamanide having cyclic formula (I), and/or a functional linear derivative thereof, for use as an immunosuppressive agent is disclosed. Furthermore, the use of Antamanide and/or a functional linear derivative thereof for the preparation of a medicament for immunosuppressive treatment, and a method of treating a mammal, including man, in need of immunosuppressive treatment comprising administering to said mammal a pharmacologically effective amount of Antamanide and/or a functional linear derivative thereof, are disclosed. Disclosed is also a pharmaceutical preparation comprising Antamanide and/or a functional linear derivative thereof, together with pharmaceutically acceptable carrier(s), excipient(s) and/or diluent(s).

Description: MEDICAMENT COMPRISING ANTAMANIDE The present invention relates to Antamanide and/or a functional linear derivative thereof for use as an immunosuppressive agent, to the use of Antamanide and/or a functional linear derivative thereof for the preparation of a medicament for immunosuppressive treatment, to a method of treating a mammal, including man, in need of immunosuppressive treatment and to a pharmaceutical preparation comprising Antamanide and/or a functional linear derivative thereof. Background Antamanide is a cyclic decapeptide isolated from the mushroom Amanita phalloides ( ieland T, et. al. Angew. Chem. Int. Ed. Engl. ]_, 204-208, (1969)). The chemical structure of Antamanide is Phe-Pro-Pro-Phe-Phe Phe-Ala-Pro-Pro-Val It is well known that Antamanide protects liver cells against poisons, such as ethanol, dimethyl sulfoxide, cysteamine, or phalloidin (Primmer, M. and Petzinger, E., Artzneim.-Forsch. _25>, 1423 (1975)). Kessler, H. , et al (Ang. Che ie Int. Ed. Engl. 2_5, 997-999 (1986)) showed that Antamanide in vitro inhibits the uptake of cholate into hepatocytes. We are not aware of any biological studies effected on linear forms of Antamanide. Neither are we aware of any medicaments comprising Antamanide or linear forms thereof. Description of the invention The present invention is based on the finding that Antamanide (ANTA) having the cyclic formula Phe-Pro-Pro-Phe-Phe Phe-Ala-Pro-Pro-Val and a linear form thereof, i.e. H-Val-Pro-Pro-Ala-Phe-Phe- -Pro-Pro-Phe-Phe-OH (ANTA-L) exhibit immunosuppressive activity. In one aspect of the invention there is provided Antamanide and/or a functional linear derivative thereof for use as an immunosuppressive agent. The expression "a functional linear derivative thereof" in conjunction with Antamanide, is intended to comprise all linear forms of Antamanide and C-termi- nal amides and esters of linear forms of Antamanide and/or N-acylated linear forms of Antamanide, as long as said derivatives exhibit an immunosuppressive activi¬ ty. An example of such a functional linear form of Antamanide, which has proved to be immunosuppressive, is H-Val-Pro-Pro-Ala-Phe-Phe-Pro-Pro-Phe-Phe-OH. In another aspect of the invention there is pro¬ vided the use of Antamanide and/or a functional linear derivative thereof 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 Antamanide and/or a functional linear derivative thereof. In yet another aspect of the invention there is provided a pharmaceutical preparation comprising, as an active ingredient, Antamanide and/or a functional linear derivative thereof together with pharmaceutically acceptable carrier(s), excipient(s) and/or diluent(s). The amount of Antamanide and/or a functional linear derivative thereof to be administered to a mammal in need of immunosuppressive treatment has to be decided by a physician who is experienced in immunosuppressive therapy. The pharmaceutical preparation comprising Antamanide and/or a functional linear derivative thereof can be formulated into oral preparations or preparations for infusion using pharmaceutically acceptable carrier(s), excipient(s) and/or diluent(s) suitable for such pre¬ parations, but keeping in mind that Antamanide and linear forms thereof are insoluble in water. The pharmaceutical preparation can e.g. comprise Antamanide and/or a linear derivative thereof in a pharmacologically effective amount suspended in an olive oil. The present invention reveals that Antamanide and/or a functional linear derivative thereof exhibit an immuno¬ suppressive activity, which is comparable to that of Ciclosporin (also called Cyclosporin A). Accordingly, Antamanide and/or a functional linear derivative thereof 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, pro- phylactic or therapeutic treatment of graft-versus-host- -disease (GVDH), and autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and diabetes mellitus. Cyclic Antamanide having the formula Phe-Pro-Pro-Phe-Phe Phe-Ala-Pro-Pro-Val can -be obtained by isolation as described by Wieland T, et al (ibid.), or can be synthesized by any conven¬ tional method in the art of peptide chemistry. The amino acid residues are coupled to each other by known techni- ques, by using solid phase technique or in solution, forming a linear peptide (a linear form of Antamanide), which is then cyclized, also in per se known manner by activating the C-terminal and keeping the activated linear peptide in high dilution and low temperature for cyclization. The cyclization is performed e.g. ac¬ cording to the well known azide method. The crude peptide can be purified by crystallization. Synthesis of linear and cyclic Antamanide H-Val-Pro-Pro-Ala-Phe-Phe-Pro-Pro-Phe-Phe-OH The above linear peptide was synthesized by solid phase technique in a Beckman model 9090 peptide synthe- sizer using tert-butoxycarbonyl-protected amino acids (BocAA) . BocPhe was attached to a chloromethylated resin (1% DVB) by the method of Gisin (Helv. Chim. Acta 5j5, 1481 (1973)). The Boc-Phe-resin was subjected to nine cycles of deprotection (TFA/CH 2 C1 2 ) , neutralization [(i-Pr)-,- EtN/CH 2 Cl 2 J, coupling (BocAA-3 eq. ; DCC-3 eq/CH 2 Cl 2 ), recoupling (BocAA-3 eq.; DCC-3 eq/CH-Cl- ) and termi¬ nation ( [CH 3 C0] 2 0/CH 2 C1 2 ) performed in a peptide syn- thesizer. The Boc-decapeptide-resin was deprotected (TFA/CH-- Cl 2 ), neutralized [ (i-Pr) 2 EtN/CH 2 Cl 2 ] and washed (CH 2 C1 2 ) on-line. The peptide was then cleaved from the resin in its linear form, followed by isolation and purification or transformation to its cyclic form by a suitable pro¬ cedure in solution. The crude product of cyclization was then isolated and purified by the reversed phase high performance liquid chromatography. The purity of the cyclic product was determined by analytical HPLC. Column: Bondapak C18 flowrate 1.0 ml/min a) retention time 41.6 min Solvent system: 60 min gradient from 0 to 100% A in B A: 80% acetonitrile in B B: 0.1% TFA in H 2 0 b) retention time 7 min Solvent system: 60 min gradient from 0 to 100% A in B A: methanol B: 0.1% TFA in H 2 0 The structure of the linear product and the cyclic prod¬ uct, i.e. Phe-Pro-Pro-Phe-Phe Phe-Ala-Pro-Pro-Val were confirmed by amino acid analysis and FAB-MS (fast atom bombardment mass spectrometry) . Immunosuppressive activity of cyclic Antamanide (ANTA) I. Effect of ANTA on the humoral immune response Materials and methods Animals: CBA/Iiw mice 8-10 weeks old Antigen: SRBC (sheep red blood cells) Reagents: ANTA, Cyclosporin A (CS-A) (Sandimmun. ® Sandoz. Basel Switzerland) Solvents: Mixture of Cremophor EL (SIGMA) [A trademark for a derivative of castor oil and ethylene oxide] and 94% ethanol (6.5:3.5); olive oil, PBS (phosphate buffer solution) ϊ£§§ϊπ . §2£_2 : _ : _™iS§_w th . _ OT ANTA was dissolved in the mixture of Cremophor and ethanol (cremophor), diluted at a desired concen¬ tration in PBS, and 0.2 ml of it was introduced i.p. to the animal twice; the first dose 3 hours before the antigen, and the second dose 24 hours later. The immunosuppressive activity of ANTA was compared with that of CS-A dissolved in PBS. Treatment_of_mice_with_ANTA_ger_os_^direct stomach! : One ml of ANTA dissolved in cremophor at a desired concentration was added to two ml of olive oil and intro¬ duced per os to the animal; the first dose 3 hours before the antigen, and the second dose 24 hours later. The immunosuppressive activity of ANTA was compared with that of CS-A dissolved in PBS. Immunization_of_mice_with_SRBC_^ § * 5P§£iS D£ : Mice were injected intraperitoneally with 0.2 ml of 10% SRBC in PBS 3 hours after the introduction of the first dose of reagents. After 4 days, the number of plaque forming cells (PFC) in the spleen was deter¬ mined according to Mishell-Dutton (Mishell R.I., Dut- ton R.W. : J. Exp. Med. , 1967, 126, 423.) The magni¬ tude of the humoral immune response was expressed as the number of PFC per 10 splenocytes. Each group of experimental animals consisted of 7 mice. The results obtained are shown in Tables 1 and 2. TABLE 1 The number of PFC in the spleens of mice treated with two doses of Antamanide (ANTA), or Cyclosporin A (CS-A) i.p. + SE (Student test) 165 88 260 <0.01 98 >0.001 126 <0.001 49 <0.001 Control I - mice were treated with 0.2 ml of PBS Control II - mice were treated with appropriate concen¬ tration of cremophor needed to dissolve 10 μg of ANTA The results are expressed as a mean + SE of 7 mice. TABLE 2 The number of PFC in the spleens of mice treated with two doses of Antamanide (ANTA) or Cyclosporin A (CS-A) per os + SE p (Student test] 137 243 <0.01 51 <0.001 184 <0.001 47 <0.001 As a Control olive oil was used. The results are expressed as a mean + SE of 7 mice. II. Effect of ANTA on cellular immune response in vivo Effect of Antamanide (ANTA) on graft-versus-host reaction (GvH) Materials and methods Animals: Hybride F, (C3H/Iiw x B,/Iiw) and female B, mice, 8-10 weeks old Reagents: ANTA, CS-A Solvents: Mixture - Cremophor EL (Sigma): 94% ethanol (6.5:3.5) -(cremophor), PBS *£ §§£5§Dέ_2-![_2i_:§_^i£?- ^!£ : ANTA dissolved in cremophor was introduced into F.1 (C3HxB6,) mice. The first dose was introduced 3 hours before injection of parenteral cells, and the second dose was introduced 48 hours later intraperitoneally. The immunosuppressive activity of ANTA was compared with that of CS-A. 2E E5*£§£i2D_2^_0§£§2£§I_2§I . : Female B fi mice were killed, their popliteal nodes were pressed through a plastic screen into PBS. Then, the cells were washed three times with PBS and resus- pended 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, F, (C-.HxB fi ) were injected sub- cutaneously in the left hind footpad with 5x10 parental lymphoid cells (B fi ). After 7 days, draining lymph nodes were isolated and weighed. As a control, the weight of popliteal node isolated from the right leg was measu¬ red. 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 3. TABLE 3 GvH reaction in mice treated with two doses of Antamanide (ANTA) or Cyclosporin A (CS-A) i.p. Reagent GvH (index) + SE p μg/mouse (Student test) Control* 3.21 0.340 ANTA 1 μg 1.74 0.133 0.01 10 μg 1.27 0.169 0.001 CS-A 1 μg 2.09 0.379 0.02 10 μg 1.29 0.109 0.001 As a Control cremophor was used at a concentration needed to dissolve 10 μg of the peptide. The results are expressed as a mean + SE of 9 mice. Immunosuppressive activity of linear Antamamide (ANTA-L) I. Effect of linear Antamanide (ANTA-L) on the humoral immune response Materials and methods Animals: CBA/Iiw mice 8-10 weeks old Antigen: SRBC (sheep red blood cells) Reagents: ANTA-L, Cyclosporin A (CS-A) (Sandimmun. ® Sandoz, Basel Switzerland) Solvents: Mixture of Cremophor EL (SIGMA) and 94% ethanol (6.5:3.5); olive oil, PBS (phosphate buffer solution) T 2§£5_=D£_2_ _5i22_^i£h_ANTA-L_ger_os_ d stomachic One ml of ANTA-L dissolved in cremophor at a desire concentration was added to two ml of olive oil and 0.2 ml of it was introduced per os to the animal; the first dose 3 hours before the antigen, and the second dose 24 hours later. The immunosuppressive activity of ANTA-L was compared with that of CS-A dissolved in PBS. Immunization_of_mice_with_SRBC_and_£ §2B2Ξϊπ.§S£ : Mice were injected intraperitoneally with 0.2 ml of 10% SRBC in PBS 3 hours after the introduction of the first dose of reagents. After 4 days, the number of plaque forming cells (PFC) in the spleen was deter- mined according to Mishell-Dutton (Mishell R.I., Dut- ton R.W.: J. Exp. Med. , 1967, 126, 423.) The magnitude of the humoral immune response was expressed as the number of PFC per 10 splenocytes. Each group of experimental animals consisted of 7 mice. The results obtained are shown in Table 4. TABLE 4 The number of PFC in the spleens of mice treated with two doses of linear Antamanide (ANTA-L), or Cyclosporin A (CS-A) per os. Reagent PFC/10 + SE μg/mouse (Student test) Control I 1788 219 Controll II 1693 133 ANTA-L 100 μg 501 150 0.001 CS-A 100 μg 561 88 0.001 Control I - mice were treated with 0.2 ml of PBS Control II - mice were treated with appropriate concen¬ tration of cremophor needed to dissolved 10 μg of ANTA-L The results are expressed as a mean + SE of 7 mice, II. Effect of ANTA-L on cellular immune response in vivo Effect of linear Antamanide (ANTA-L) on qraft-versus reaction (GvH) Materials and metόds Animals: Hybride F, (C3H/Iiw x B g /Iiw) and female B β mice, 8-10 weeks old Reagents: ANTA-L, CS-A Solvents: Mixture - Cremophor EL (Sigma): 94% ethanol (6.5:3.5) -(cremophor), PBS _!?ϊ_=__iέ5L.§D-_:_ £_Di22_5ϊi£l_}_ * ^_!-_: l£. : ANTA-L dissolved in cremophor was introduced into F, (C3HxB g ) mice. The first dose was introduced 3 hours before injection of parenteral cells, and the second dose was introduced 48 hours later intraperitoneally. The immunosuppressive activity of ANTA-L was compared with that of CS-A. E£§E§ϊ§£i2D_2__:_E§£2ϊϊέ§I_2!='!:_i= : Female B fi mice were killed, their popliteal nodes were pressed through a plastic screen into PBS. Then, the cells were washed three times with PBS and resus- pended 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, F, (C-.HxBg) were injected sub- cutaneously in the left hind footpad with 5x10 parental lymphoid cells (Bg)« After 7 days, draining lymph nodes were isolated and weighed. As a control, the weight of popliteal node isolated from the right leg was measu- red. 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 5. TABLE 5 GvH reaction in mice treated with two doses of linear Antamanide (ANTA-L), or Cyclosporin A (CS-A) i.p. Reagent GvH (index) + SE p μg/mouse (Student test) Cremophor 3.17 0.36 ANTA-L 10 μg 1.48 0.16 0.01 CS-A 10 μg 1.41 0.16 0.001 As a Control cremophor was used at a concentration needed to dissolve 10 μg of the peptide. The results are expressed as a mean + SE of 10 mice.

Claims: CLAIMS 1. Antamanide having the cyclic formula Phe-Pro-Pro-Phe-Phe Phe-Ala-Pro-Pro-Val and/or a functional linear derivative thereof, for use as an immunosuppressive agent. 2. Use of Antamanide and/or a functional linear derivative thereof, for the preparation of a medica¬ ment for immunosuppressive treatment. 3. A method of treating a mammal, including man, in need of immunosuppressive treatment comprising admi- nistrering to said mammal a pharmacologically effective amount of Antamanide having the cyclic formula Phe-Pro-Pro-Phe-Phe Phe-Ala-Pro-Pro-Val and/or a functional linear derivative thereof. 4. A pharmaceutical preparation comprising, as an active ingredient, Antamanide having the cyclic formula Phe-Pro-Pro-Phe-Phe I Phe-Ala-Pro-Pro-Val and/or a functional linear derivative thereof, together with pharmaceutically acceptable carrier(s), excipient(s) and/or diluent(s).

Inventors: SIEMION IGNACY Z, TROJNAR JERZY, WIECZOREK ZBIGNIEW, SIEMION, IGNACY, Z., TROJNAR, JERZY, WIECZOREK, ZBIGNIEW
Applicants: FERRING AB
Application year: 1990
Main classification: A61K37
IPC codes: A61K38, A61P37, C07K7, C07K14