IE892448L - The use of imexon as an immune suppressive and¹pharmaceutical compositions containing imexon - Google Patents

Abstract

The present invention relates to the use of 4-imino-1,3-diazabicyclo[3.1.0]hexan-2-one (imexon) as an immunosuppressive agent. Imexon selectively suppresses beta -lymphocyte activation and can be employed for the treatment of B-cell or plasma-cell leukaemia and neoplasms. Imexon is used in the treatment of those disorders in which polyclonal B-cell proliferation is of pathophysiological, symptomatic or clinical relevance such as, for example, for autoimmune diseases.

Description

-2- The present invention concerns che use of imexon for che preparation of medicaments with immuno-suppressive action.

In particulars, the invention is concerned with 5 the use of imexon for the preparation of medicaments for che treatment of autoimmune diseases* 3 cell or plasma cell neoplasias» lymphoblastic lymphomas and rejection reactions after tissue or organ transplants-In general, imexon can be used in the case of the 10 treatment of diseases in which a pathophysiologically increased B~lymphocyte proliferation or B-lymphocyte activation is to be observed- Furthermore* the subject of the present invention is the use as combination preparation of the 15 active material imexon and of a further effective chemocherapeucic, for example of an additional immune suppressive or of an anti-retrovirally effective compound.

The systematic designation of imexon, the 20 structural formula of which is illustrated in Fig. 1, is 4-imino-l,3-diazabicyclo-(3.1.0)-hexan-2-one.

H.,C C C - NH Fig- 1 0 c nh * With regard to its structure* imexon is not comparable with any other active substance hitherto used 25 is therapy* The surprisingly found preferred action oa B-lymphocytes also shows no parallels with other previously known immunosuppressively-acting compounds.

Imexon and processes for its preparation are known from US-A 4,083*987* The compound is thereby 30 described as csncerostatlcally-accive therapeutic which displays imsnune~stimulating properties. The cancerostatic action was demonstrated on the bast® of the inhibition of the tumour growth of Walker sarcoma 256 after the administration of imexon to rats* The immune~stimulating action can be deduced from experiments in which an increase of the leukocytes , as well as an increase of che number of the antibody-forming spleen cells could be observed after administration of imexon.. The pharmacological importance of imexon given therefrom is, according to this U.S. Patent Specification,, to be sees, in the fact that imexon so strongly impairs the growth of the rapidly dividing cancer cells that, under certain circumstances, a regression of the tumours is possible. According to US-A 4,083,987, the advantageous action of imexon lies in the simultaneous strengthening of the per se weakened immune defence system inherent in the body which accompanies the concerostatic action.

Imexon is also described as cancerostatically- active therapeutic,,, which shows immune-stimulating properties, by U. Bicker et al. in ICRS, volume 5 (1977), page 428.

In general, immune suppressives as such have been known for quite a long time from the prior art (Pharmazie unserer Zeit, JU 2-8(1972) and 12, 20-21 (1983)),. The expression "immune suppression" used in this connection thereby in general designates various types of non-specific suppression of the immune response, a*g,» with the help of antisera, ionising irradiations or special therapeutics„ The use of immune suppressive-actlng chemo-therapeutics can find use after the transplantation of tissue or organs and in the case of the therapy of autoimmune diseases., They inhibit the proliferation of lymphocytes by direct or indirect Intervention into che DMA and RNA synthesis. To this class of compounds belong cyclosporins, folic acid antagonists » purine analogues® alkylating substances,, such as cyclophosphamide » and certain corticosteroids. However, the disadvantage of these previously used immunosuppressives is the increased extent of observed susceptibility to infection of the created organism since Che whole of the body's immune system weakens and not only che humoral but also the cellular immune response are, suppressed.

The hitherto known artificially induced immune suppression could be achieved in various ways: by administration of antigens, administration of specific antisera or antibodies, use of other biological reagents, such as e.g. antilymphocyte antisera, by use of immunosuppresslvely~active compounds» by radiation or by the surgical removal of lymphoid tissue.

The immunosuppressive properties of most of the immunosuppressives at present known, such as e.g. cytostatics and corticosteroids, are dosage-dependent but non-selective, i.e. they act upon all immunocompetent cells. These compounds inhibit not only the humoral but also the cellular immune response to a plurality of antigens and act non-specifically on T- or B-lymphocytes,. Cyclosporin A, the at present most selective medicament, suppresses not only the proliferation of T~lymphocytes but also immune processes which are not T-cell dependent.

Therefore, there is a great interest for immuno~ suppressives which interfere specifically with pathologically strengthened or increased Immune mechanisms but without influencing the immune reactions taking place normally in the body.

Only ciamexone» a z-cyanoaziridine, is previously described in the literature (Fortschritte der Medizin, volume 1055I 1987j, pages 509 - 512) as a compound which preferentially suppresses antibody-mediated iimmune reactions and does not suppress T-cell-caediated immune reactions® In addition* Bicker in Immunol- Ser*„ volume 25 (1984), pages 447 - 473 describes 2-cyano-aziridines as immune modulators, che immune modulation of which depends on the fact that, in che case of simultaneous stimulation of the suppressor T-cells, inhibits the helper T-cell activity.

Therefore,, che task forming che basis of the invention is to make available a further seleccively-acting immunosuppressive agent - Surprisingly,, it has now been found that imexon solves this task and can be used as advantageous immune suppressive. It specifically suppresses the B-cell proliferation or the B-cell activation. It can be advantageously used in the case of the treatment of all diseases in which a polyclonal activation or proliferation of B-ealls is of pathophysiological, symptomatic or clinical relevance.

In this sense„ for example, che treatment of the following diseases comes into question: autoimmune diseases„ such as rheumatoid arthritis,» diabetes mellitus Type I, psoriasis, lupus systemicus erythematosusi rejection reactions after tissue or organ transplants» e.g. of skin,, bone marrow and kidneys; B-cell leukaemias and lymphomas, such as e.g* chronic lymphatic leukaemia, lymphoblastic lymphoma (e.g. Burkitt"s lymphoma etc.) or B-cell/ Pi asma cell neoplasias, such as e.g. plasmacytoma (multiple myeloma).

As autoimmune diseases,, in the literature there are generally designated those diseases in which the formation of autoantibodies have pathogenic significance . These autoantibodies ar® directed against the body's own antigens and thus bring about a destruction * of che bodyas own organs, cells or proteins,. It is 5 an object to suppress these morbid overreactions of che immune system with specifically-acting immune suppressives Furthermore, it was, surprisingly, found that imexon inhibits the proliferation of B-lymphocytes 10 dosage-dependently.

Imexon can be used itself directly or in the form of its physiologically acceptable addition salts.

In the meaning of che present inventions, the expression "immune suppression" is, in general, to 15 comprise all aspects of the naturally-induced immunological non-responsiveness, che artificially-induced non-responsiveness and the pathologically-induced tolerance of an individual to auto- and foreign antigens - 20 The immune suppressive action of imexon could be demonstrated on the basis of the inhibition of the proliferation of human B-lymphocytes, whereby che proliferation is induced experimentally by the B-cell growth factor (BCGF). 25 Furthermore, the pharmacological properties of imexon could b© characterised by concanavalin A (ConA)-induced proliferation of murine splenocytes (LTT), by phytchaemagglutinin (PHA)-induced proliferation of human lymphocytes, as well as by the tumour * 30 growth inhibition assay (TGI)., In order to stimulate dormant B-cells to proliferation, two signals are necessary. The first signal is an activation signal which is mediated by an antigen or anti-y. The. transmission of this 35 activating signal finally has che result that receptors for che® B-cell growth factor (BCGF) are expressed on the B-cell surface, BCGF is a soluble lymphokine secreted by T-cells with a molecular weight of 1/j.000 - 18^000 D„ The expression of BCGF receptors on the B-cells makes it possible for these to respond to the proliferation signal of BCGF.

Normally, B-cells are converted by these two signal processes from the formant state into the proliferative phase.

Imexon now suppresses this procedure specifically insofar as the concanavalin A (ConA)- and phyto-haamsgglutinin (PHA)-induced lymphocyte proliferation,, as well as the spontaneous proliferation of methyl-cholanthrene-induced fibrosarcoma cells (MethA), are not influenced or only in the case of 10 - 30 times higher concentrations.

The influence of imexon on the spontaneous formation of lymphomas and the synthesis of antinuclear autoantibodies in the case of the mouse (Example 5) proves the effectiveness on an animal model for autoimmune diseases.

Imexon can also be used as a combination preparation with other immune suppressive® „ such as e.g. cyclosporin A, ciamexon or azathioprine, as well as antiretrovirally-active substances, such as e,g. azidothymidine (AZT), A combination of imexon with cytostatic© is also possible,,, e.g. with cis-pistinuni complexes» for example cis-aiaminadichloroplatinum» or with adriamycin, cyclophosphamide, vincristine tamoxifen, methotrexate or 5~fluorouracil etc., In this connection® the use of such combination preparations is of especial interest subsequent to a plasmaphaeresis for the monitoring of autoimmune diseases.

In the case of the use of che combination therapy., it is possible to administer the .active, materials in a so-called fixed combinations, i.e. in a single pharmaceutical formulation, in which both active materials are present simultaneously, or to choose a so-called free combination in which the active naaterials are administered in the form of pharmaceutical formulations simultaneously or,,, however,, also successively in individually selectable dosage ratios„ For the preparation of pharmaceutical agents, imexon is mixed in per se known manner with suitable pharmaceutical carrier substances, possibly granulated and pressed, for example® into tablets or dragee cores. A filling of the mixture into hard capsules is also possible. With addition of appropriate adjuvants„ a solution or suspension in ^ater® an oil (e.g. olive oil) or high molecular polymers (e.g. polyethylene glycol) can also be produced and administered as injection solutions, soft gelatine capsules„ syrups or drops.

As solid carrier materials, there can find use e.g. starches or starch derivatives, sugars,, sugar alcohols, celluloses or cellulose derivatives, tensides, talc, highly dispersed silicic acids, high molecular fatty acids or their salts, gelatine, agar-agar, calcium phosphate, animal or vegetable fats or waxes and solid high molecular polymers (such as polyethylene glycols or polyvinylpyrrolidones). Compositions suitable for oral administration can„ if desired, contain flavouring and sweetening materials., The dosage of the active material imexon depends upon the age and sex of the individual, as well as upon the nature of the indication to be treated. -9- In principle, one cas stare £xom che point that 0*1 - 100 mg of imexon per kg of body weight can be administered daily orally® intravenously, subcutaneous ly or intramuscularly* However, amounts of 5 5 - 50 sag/kg of body weight;, especially 5-20 mg/kg, are preferred. The amounts of active material caa be administered 1 to 3 times daily* The specific immunosuppressive action of imexon is demonstrated on the basis of the following Examples: 10 Example 1 BCGF-dependent proliferation of human B-lymphocytes The enrichment of peripheral human B-celis and the BCGF proliferation assay were carried out as follow© (cf. Eur- J. Immun., 16, 350 (1986): 15 Earichad human B-lymphocytes are washed twice with complete RPMI 1640 medium (streptomydin/ penicillin, L~glutamine, 2-mercaptoethanol, PCS) and adjusted to 3 x 105 cells/ml* 160 ml of this suspension are, in each case, pipetted per well into 20 microtitre plates- As pseudoantigen, there are added thereto 10 ml of a solution of HFC pS-IgG (300 pg/ml) and, as growch factor, 20 pi BCGF (Cellular Products Incorporated)- To this are pipetted 20 pi of the compound to be tested in 10 fold concentration. The 25 cultures are incubated for a total of 140 h at 3/"sC, 5% C07 and 95% relative atmospheric humidity. 16 h before conclusion of the incubation time, each culture »3 is pulsed with 1 pCi of a [ H3-thymidine solution. At the end of the experiment, the cells are collected 30 with a harvester and the incorporated radio-activity determined in a liquid scintillation counter.

Example 2 Concanavalin A (ConA)-induced proliferation of murine splenocytes 35 Spleen cells (4 x 10^) of CB6F, mice are -10- incubated for a total of 48 h with 0.5 pg/sal ConA ia microcitra plates .(Nunc GmbH, Wiesbaden, FB.G) end various concentrations of imexon in 6 fold batches. 5 h before termination of che incubation tins©,, the. cultures are pulsed with []-thymidine and subsequently harvested oa glass fibre, filter platelets by eCti cfs a multi-sample harvester (Skatron A„S.» Lier, Norway). The filter platelets are dried, and the radioactivity determined in a Packard scintillation spectroine ter.

Example 3 Phytohaem&gglutinin (PHA)-induced proliferation, of human lymphocytes 1 si of human whole blood is diluted with 500 pg PHA solution (500 pg/ml) and 48 ml DMEM medium. In each case» 200 pi of this batch are mixed with 20 pi of the. Imexon concentration co be tested in 6 fold batches and incubated for 4 days. After pulsing with "X I**H|-thymidine, one incubates for a further 24 h, harvests and evaluates as described in Example 2.

Example 4 Tumour growth inhibition assay (TGI) A methylcholanthrene-inducad fibrosarcoma cell line CMetb.,4) was obtained from our owe c°ow©ur call bank assd passed intraperitoneally into CBSF^ mice. 4 1 x 10 MetthA cells are incubated with the imexon concentration to be tested in DMEM medium for 48 h. 3 h before the end of the Incubation time, one pulses with C 3-thymidine, harvests and evaluates as described ia Example 2* The values given in Table 1 show the result of a representative experiment. There are shown the results of the investigations with imexon in ch© TGI, LTT (ConA, PHA) as well as in the BCGF assay, i.e. the influence of on the MethA sarcoma cell» -11- T-lymphocyte and B-cell proliferation. Imexon suppressed significantly and specifically the BCGF-induced B-cell proliferation at a concentration of 1 pg/ral, whereas che lymphocyte proliferation,, 5 induced either by ConA or PHA* are first significantly inhibited at concentrations of > 10 pg/ml. Furthermore;, the spontaneous proliferation of MechA sarcoma cells is also first significantly suppressed from >10 pg/ml. 10 The results of the above experiments are summarised in the following Table 1: Tab., 1» Effect of imexon on the proliferation of various cell types Imexon ({jg/uil} TGI (MethA) LTT (spleriocytes* ConA) LTT (aplenocytes, PHA) BOGF (human. B= lyuiphocytes) 3H=TdR cpJi (n«6) % inhibition 3H-TdR cptn (rv-6) 1 inhibition 3 cpai FdR (n=6} X Inhibition 3H-TdR cptu (n=6) X inhibition rr> V SD X SD X SD Control 33966 (n»5) 3000 « 109879 12203 = 44283 6458 ~ 5541 1.792 - 100 534 363 98** 903 62 gg-A-A" 585 44 99** 562 44 90** 30 911 110 97** 2509 863 98** 573 59 99** 617 59 89** 10 21913 2357 35** 24895 6563 77** 4724 704 89** 574 50 90** 3 35473 3135 -A 118487 9494 =8 35850 13018 19 831 231 85** 1 35475 1753 ~4 11.9120 9372 -8 49348 4168 -11 2096 455 62* 0,3 37593 3080 -11 134032 37682 -22 45542 9870 o 4201 1636 24 0,1 31722 3991 7 109717 11192 0 41349 1892 5 4847 1146 13 * p < 0,002 ** p < 0.001 -13- Exaraple 5 Action of imexon ia che case of autoimmune diseases.

With increasing age, the mouse strain MRL lpr/lpr develops increasingly spontaneously lymphadenomas and SLE-like symptoms, e.g., synthesis of antinuclear autoantibodies. For the investigation of the prophylactic effect of imexon on. the development of these symptoms, 11 week old MRL mice were treated intraperitoneally oace daily with the given dosages of imexon and cyclophosphamide. The number of lymphadenomas and the concentration of antinuclear antibodies were documented- In the case of the investigation for the therapeutic potency of imexon, MRL m.ice„ after each animal had developed at least one lymphadenosis (about 14 week old animals), were also treated once daily with che given dosages of imexon and cyclophosphamide.., The measurement parameters were again che number of lymphadenomas, as well as autoantibody titre.

The results of these investigations have shown that imexon, in the case of vary good compatibility, lowers che number of spontaneously arising lymph-adenomas and the concentration of DNA-specific antibodies. The effectiveness of imexon is also shown in the case of therapeutic use with animals already having lymphomas. The number of lymphadenomas decreased dependent upon the dosag®,, as well as the titre of the autoantibodies,.

Sxaaaple 6 Preparation of a pharmaceutical formulation of imexon A film tablet with, for example, 100 g of active material„ which is composed as follows, has proved to be a suitable medicinal compositions -14- weight/unit/mg imexon 100.000 lactose.1H90 63.000 poly-(O-carboxymethyl)-starch, Na 5 salt 7.000 poly~<1-vinyl-2-pyrrolidone) 25,000 4.000 poly-(0-carboxynae chyl)-starch, Na salt 3.000 10 raicrocrystelline cellulose 20.000 silicon dioxide,, highly dispersed 1*500 magnesium stearate 1.500 core weight 200.000 Th® film tablets are then produced in the usual 15 way by film drageeing of the imexon cores obtained.

Film tablets with e.g. 10 mg» 50 sag, 200 mg or 500 mg of active material are produced in corresponding manner. ~15~

Claims (6)

CLAIMS:

1. Use of imexon for the production of medicaments with immunosupprassive action, fox the treatment of diseases which are involved with an increased 5 ' B~lymphocyte activation.

2. » Use of imexon according to claim 1 for the production of medicaments for the treatment of autoimmune diseases.

3. « Use according to one of claims 1 or 2? 10 characterised in that imexon is used in an amount of 10 to 1000 sag/form of administration.

4. « Use according to one of claims 1 or 2, characterised in that imexon is used in combination with a further immunosuppressive active material- 15

5. Use according to claim 1, characterised in that imexon is used in combination with an anti-retrovixal active material.

6. The use of imexon according to any of the preceding claims, substantially as hereinbefore 20 described and exemplified. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS.