IE66680B1 - The use of imexon as an immune suppressive - 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

The present invention concerns the use of imexon for the preparation of medicaments with immunosuppressive action,» In particular* the invention is concerned with the use of imexon for th® preparation of medicaments for the 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 treatment of diseases in which a pathophysiologically increased B~lymphocyte proliferation or B-lymphocyte activation is to be observed» Furthermore·, th® subject of the present invention is the use as combination preparation of the active material imexon and of a further effective chemotherapeutic j, for example of an additional immune suppressive or of an anti-retrovirally effective compound.

The systematic designation of imexon, the structural formula of which is illustrated in Fig» 1,, is 4-imino-1»3-diazabicyclo-(3.1.0)-hexan-2-one» Fig. 1 C C - NH NH With regard to its structure, imexon I® not comparable with any other active substance hitherto used in therapy· The surprisingly found preferred action on B-lymphocytes also show®, no parallels with other previously known imxaunosupprassively-acting compounds.

Imexon and processes for its preparation are known from US-A 4,083*987· The compound is thereby described as cancerostatically-active therapeutic -3which displays immune-stimulating properties. The cancerostatic action was demonstrated on the basis of the inhibition of the tumour growth of Walker sarcoma 256 after the administration of imexon to rata. The immune-stimulating action can be deduced from experiments in which an increase of the leukocytes , as well aa an increase of the 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, co be seen 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 cancerostatically20 active therapeutic, which shows immune»stimulating properties, by U. Bicker et al. in ICRS, volume 5 (19773, page 428.

In general, immune suppressives as such have been known for quite a long time from the prior art (Pharmazie unserer Zeit, 1., 2-8(1972) and 12, 20-21 (19833). The expression ‘immune suppression used in this connection thereby in general designates various types of non-specific suppression of the immune response, e.g. with the help of antisera, ionising irradiations ox special therapeutics. > The use of immune suppressive-acting chemotherapeutics can find use after the transplantation . of tissue or organ® and in the case of the therapy of autoimmune diseases. They inhibit the proliferation of lymphocytes by direct or indirect intervention into -4the DNA and RNA synthesis. To this class of compounds belong cyclosporins, folic acid antagonists, purine analogues, alkylating substances, such as cyclophosphamide , and certain corticosteroids. How,aver, the disadvantage of these previously used immunosuppressives is tha increased extent of observed susceptibility to infection of th® created organism since Che whole of the body’s immune system weakens and not only the humoral but also the cellular immune response ar® 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 ocher biological reagents, such as e.g. antilymphocyte antisera, by use of immunosuppressively-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 immunecompetent cells. These compounds inhibit not only the humoral but also the cellular immun© 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 & great interest for immunosuppressives which interfere specifically with pathologically strengthened or increased immune mechanisms but without influencing the immune reactions taking place normally in th® body. -5Only ciamexons, a z-cyanoaziridine» is previously described in the literature (Fortschritte der Medizin, voluiae 105, 1987, pages 509 - 512) as a compound which preferentially suppresses antibody-mediated immune reactions and does not suppress T-cell-mediated immune reactions» In addition» Bicker in Immunol- Ser-,, volume 25 (1984),, pages 447 - 473 describes 2-cyanoaziridines as immune modulators, the immune modulation of which depends on the fact that, in the case of simultaneous stimulation of the suppressor T-cells, inhibits the helper T-cell activity.

Therefore, the task forming the basis of the invention is to make available a further selectivelyacting 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-cells is of pathophysiological, symptomatic or clinical relevance, In this sense, for example, the treatment of the following diseases comes into question: autoimmune diseases, such as rheumatoid arthritis, diabetes mellitus Type I, psoriasis, lupus systemicus erythematosus; 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/ plasma cell neoplasias, such as e.g. plasmacytoma (multiple myeloma).

As autoimmune diseases, in the literature there are generally designated those diseases in which the -6formation of autoantibodies have pathogenic significance. These autoantibodies are directed against the body’s own antigens and thus bring about a destruction of tha body’s own organs, cells or proteins. It is an object co suppress these morbid overreactions of the immune system with specifically-acting immune suppressives.

Furthermore, it was, surprisingly, found that imexon inhibits the proliferation of B-lymphocytes dosage-dependently.

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

In the meaning of the present invention, the expression ’’immune suppression" is, in general, to comprise all aspects of the naturally-induced immunological non-responsiveness, the artificiallyinduced non-responsiveness and the pathologicallyinduced tolerance of an individual to auto- and foreign antigens.

The immune suppressive action of imexon could be demonstrated on th© basis of the inhibition of the proliferation of human B-lymphocytes, whereby the proliferation is induced experimentally by the B-cell growth factor (BCGF).

Furthermore, the pharmacological properties of imexon could b© characterised by concanavalin A (ConA)-induced proliferation of murine splenocytes (LTT),by phytohaemagglutinin (PHA)-iaduced proliferation of human lymphocytes, as well as by the tumour growth inhibition assay (TGI)., In order to stimulate dormant B-cells to proliferation, two signals axe necessary. The first signal is an activation signal which is mediated by an antigen or anti-p. The transmission of this activating signal finally has the result that „7receptors for the B-cell growth factor (BCGF) axe expressed on the B-cell surface. BCGF is a soluble lymphokine secreted by T-cells with a molecular weight of 17,,000 - 13„000 D. The expression of BCGF receptors on the B-cells makes it possible fox these to respond to the proliferation signal of BCGF.

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

Imexon now suppresses this procedure specifically insofar as the concanavalin A (ConA)~ and phytonaemagglutinin (PHA)-induced lymphocyte proliferation, as well as the spontaneous proliferation of methylcholanthrene-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 suppressives, such as e.g. cyclosporin A, ciamexon or azathioprine, as well as antiretrovixally-active substances „ such as e.g. azidothymidine (A2T).

A combination of imexon with cytostatics is also possible, e.g· with cis-platinum complexes, for example cis-diaminodichloroplatinum, or with adriamycin, cyclophosphamide,, vincristin, tamoxifen, methotrexate or 5-fluoxouracil etc. In this connection, the use of such combination preparations is of especial interest subsequent to a plasmaphaeresis for the monitoring of autoimmune diseases. -8Ia the case of the use of the combination therapy, it is possible to administer the active materials in a so-called fixed combination, i.e. in a single pharmaceutical formulation, in which both active materials are present simultaneously, or co choose a so-called free combination in which the active materials are administered in the form of pharmaceutical formulations simultaneously or, however, also successively in individually selectable dosage ratios.

For th® 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 water, 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 us.® 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, agaragar, 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· i 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 created» -9In principle* one can scare from the point: that 0.1 " 100 mg of imexon per kg of body weight can be administered daily orally* intravenously* subcutaneously or intramuscularly. However* amounts of -50 mg/kg of body weight* especially 5-20 mg/kg* are preferred. The amounts of active material can b© administered 1 to 3 times daily* The specific immunosuppressive action of imexon is demonstrated on the basis of the following Examples: Example 1 BCGF-dependent proliferation of human fi-lymphocytes The enrichment of peripheral human B-cells and the BCGF proliferation assay were carried out as follows (cf. Eur. J. Inrnm., 16* 350 (1986): Enriched human B-lymphocytes are washed twice with complete RPMI 1640 medium (streptomydin/ penicillin* L-glutamine* 2-asercaptoethanol * FCS) and adjusted to 3 x 105 cells/ml. 160 ml of this suspension are* in each case* pipetted per well into raicrocicre plates. As pseudosntigen* there are added thereto 10 ml of a solution of HFC yS-IgG (300 yg/ral) and* as growth factor* 20 pi BCGF (Cellular Products Incorporated). To this are pipetted 20 pi of the compound to be tested in 10 fold concentration. The cultures are incubated for a total of 140 h at 37‘SC* ¾ CO? aad 951 relative atmospheric humidity* 16 h before conclusion of the incubation time, each culture is pulsed with 1 pCi of a ί Hj-thymidine solution. At the end of the experiment* the cells axe collected with a harvester and the incorporated radio-activity determined in a liquid scintillation counter.

Example 2 Coacanavalin A (ConA)-induced proliferation of murine splenocytes Spleen cells (4 x IQ’) of CB6F* mice are -10incubated for a total of 48 h with 0.5 pg/ml CoaA ia microtitre plates (Nunc GmbH, Wiesbaden, FBLG) and various concentrations of imexon ia 6 fold batches. h before termination of the incubation time, the cultures are pulsed with pH]-thymidine and subsequently harvested on glass fibre filter platelets by means of a multi-sample harvester (Skatron A.S., Liar, Norway). The. filter platelets are dried and the radioactivity determined in a Packard scintillation spectrometer.

Example 3 Phytohaemagglucinin (PHA)-induced proliferation of human lymphocytes ml of human whole blood is diluted with 500 pg 15 PEA solution (500 pg/ml) and 48 ml DMEWt 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 Ϊ**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 metfeylcholanthrene-induced fibrosarcoma cell line (MethA.) was obtained from our own tumour cell bank and passed incraperitoneally Into CBSF^ mice. x 10 MethA cells are incubated with the imexon concentration to be tested in DMEM medium for 48 h. h before the end of the Incubation time, one pulses with (¾]-thymidine, harvests and evaluates as described in Example 2* The values given ia Table 1 show the result of a representative experiment. There are shown the results of the investigations with imexon in the TGI, LTT (ConA, PHA) as well as In the BCGF assay,, i.e. the influence of imexon on the MethA sarcoma cell, -11T-lymphocyte and B-cell proliferation. Imexon suppressed significantly and specifically the BCGFinduced B-cell proliferation at a concentration of 1 pg/ml, whereas the lymphocyte proliferation, induced either by ConA or PHA, are first significantly inhibited at concentrations of > 10 pg/ml. Furthermore, the spontaneous proliferation of MethA sarcoma cells is also first significantly suppressed from >10 pg/ml.

The results of the above experiments are summarised in the following Table 1: Tab* li Effect of imexon on the proliferation of various cell types Imexon (pg/ml) TGI (MethA) LTT (splenocytes, ConA) LTT (splenocytes, PHA) BOGE (human B= lymphocytes)3H=TdR ομη (n=6) 1 inhibition3H-TdR cpui (n=»6) X inhibition3H-TdR cpm in=6) X inhibition3lI-TdR cptn (n=6) X inhibition X SD X SD X SD X SD Control 33966 (ri"5) 3000 - 109879 12203 = 44283 6458 = 5541 1792 - 100 534 363 98** 903 62 99« 585 44 99« 562 44 90** 30 911 110 97« 2509 863 98« 573 59 99** 617 59 89« 10 21913 235735« 24895 6563 77** 4724 704 89« 574 50 90« 3 35473 3135 -4 118487 9494 =3 35850 13018 19 831 231 85« 1 35475 1753 -4 11.9120' 9172 =-8 49348 4168 -11 2096 455 62* 0,3 37593 3080 -11 134032 37682 -22 45542 9870 " 3 4201 1636 24 oa 31722 3991 7 109717 11192 0 41349 1892 5 4847 .1.146 13 * p < 0,002 ** p < 0.001 -13Example 5 Action of imexon in the 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 autoantlbodies. For th® investigation of the prophylactic effect of imexon on the development of these symptoms, 11 week old MRL mice were treated intrsperitoneally once daily with the given dosages of imexon and cyclophosphamide. The number of lymphadenomas and the concentration of antinuclear antibodies were documented- In th® case of the investigation for the therapeutic potency of imexon, MRL mice, after each animal had developed at least one lymphadenosis (about 14 week old animals), were also treated ones daily with the given dosages of imexon and cyclophosphamide. The measurement parameters were again the number of lymphadenomas, as well as autoantibody titre.

The results of the®® investigations have shown that imexon, in the case of very good compatibility, lowers the number of spontaneously arising lymphadenomas and the concentration of DMA-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. dosage, ss well as the titre of the autoantlbodies.

Example 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 b® a suitable medicinal composition: imexon weight/unit/mg 100.000 lactose.1Ή?0 63.000 poly-(0-carboxymethyl)-starch, Na salt 7.000 poly-(l-vinyl-2-pyrrolidone) 25,000 4.000 poly-(0-carboxymethyl)-starch, Na, salt 3.000 microcrystalline 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 mg, 200 mg or 500 mg of active material are produced in corresponding manner.

Claims (6)

CLAIMS:

1. Use of imexon for the production of medicaments with immunosuppressive action, fox the treatment of diseases which are involved with an increased 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, characterised in that imexon is used in an amount of 10 to 1000 mg/form of administration.

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

5. Use according to claim l s characterised in that imexon is used in combination with an anti-retroviral active material.

6. The use of imexon according to any of the preceding claims, substantially as hereinbefore described and exemplified.