AU619027B2 - Method for suppressing immune responses by administering 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

619027 COMMONWVEALTH OF AUSTRALIA PATENTS ACT 1952

COLEMECCATQ

NAMAE ADDRESS OF APPLICANT: Boebringer Mannheim GmbH Sandhofer Strasse 112-132 D-6800 Mannbtim-Waldhof Federal Republic of Germany NAME(S) OF INVENTOR(S): 9 Diete.r HERRMANN Raier HAAG Elmar BOSIES Uwe BICKER Wolfgang KAMPE ADDRESS FOR SERVICE: DAVIES COLLISON Patent Attoineys 1 Little Collins Street, Melbourne, 3000.

COMPLETE SPECIFICATION FOR THE INVENTION ENITTLED: "METHOD FOR SUPPRESSING IMMUNE RESPONSES By ADMINISTERING IMEXON".

The following statement is a full description of this invention, including the best method of performing it known to me/us:i rll 2 The present invention is concerned with a method for suppressing immune responses by administering imexon to a patient.

In general imexon can be used for the treatment of diseases in which a pathophysiologically S* increased B-lymphocyte proliferation or B-lymphoe..

cyte activation is to be observed. In particular, the present invention is concerned with the treatment of autoimmune diseases, B cell and plasma cell I neoplasias, lymphoblastic lymphomas, rejection ji reactions after tissue and organ transplants and 15 viral and retroviral infections, for example AIDS or ARC (AIDS-related complex) by administering g imexon to a patient.

Imexon, which has the systematic designation 4imino-1,3-diazabicyclo-(3.1.0)-hexan-2-one, has the *20 following structural formula:- H2C C C =NH

N

C NH 0 With regard to its structure, imexon is not comparable with any other active compounds used therapeutically. The surprisingly found prefer" ed -3action on B-lymphocutes also has no parallel with other previously known immunosuppressively-acting compounds.

Imexon and processes for the preparation thereof are known from U.S. Patent Specification No.4,083,987.

The compound is thereby described as being a cancerostatically-active therapeutic which displays immunestimulating 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 rats. The immune-stimulating action can be deduced from experiments in which an 00 0 0 000 Sincrease of the leukocytes, as well as an increase of 00 the number of the antibody-forming spleen cells could be observed after the administration of imexon. The i 15 pharmacological importance of imexon is, according to this U.S. Patent Specification, to 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.

I. 20 According to U.S. Patent Specification No. 4,083,987, the advantageous action of imexon lies in the simultaneous strengthening of the weakened 'immune defence system inherent in the body which accompanies the cancerostatic action.

In general, immune suppressives as such have been known for a long time from the prior art (Pharmazie unserer Zeil, 1, 2-8/1972 and 12 20-29/1983). The 4. M M I- 1- -4expression "immune suppression" used in this connection generally designates the various types of non-specific suppression of the immune response, for example with the help of antisera, ionising irradiations and special therapeutics.

The use of immune suppressive-acting chemo- ;therapeutics can be employed after the transplantation of tissue or organs and in the therapy of autoimmune diseases. They inhibit the proliferation of lympho- 10 cytes by direct or indirect intervention into the synthesis of DNA or RNA. To this class of compounds belong cyclosporins, folic acid antagonists, purine analogues, alkylating compounds, such as cyclophosphamide, and certain corticosteroids. However, a disadvantage of these previously used immunosuppressives is the increased extent of observed susceptibility to infection of the treated organism which weakens the whole of the body's immune system and suppresses not only the humoral but also the cellular immune response.

The previously known artificially induced immune I suppression could be achieved in various ways: by the administration of antigens, administration of specific antisera or antibodies, the use of other biological reagents, for example antilymphocyte antisera, by the use of immunosuppressively-active compounds, by radiation or by the surgical removal of lymphoid tissue.

5 The immunosuppressive properties of the immunosuppressives at present known, for example cytostatics and corticosteroids, are dosage-dependent but nonselective, i.e. they act upon all immune-competent cells. These compounds inhibit not only the humoral but also the cellular immune response to a plurality Sof antigens and act non-specifically on T- and Blymphocytes. Cyclosporin A, which at present is the L o most selective medicament, suppresses not only the 10 proliferation of T-lymphocytes but also immune processes which are not T-cell-dependent.

S Therefore, there is a great interest for immunosuppressives which interfere specifically with patho- Slogically strengthened or increased immune mechanisms i 15 but without influencing the immune reactions which take place normally in the body. Hitherto, such specifically-active immunosuppressive substances are I .not known.

Therefore, it is an object of the present 20 invention to provide such an immunosuppressivelyactive agent, suitable in performing such a method.

Surprlisingly, we have now found that imexon is an active agent which can be used as an advantageous immune suppressive. It specifically suppresses the B-cell proliferation or the B-cell activation. It can be advantageously used in the treatment of all diseases in which a polyclonal activation or proliferation of -6- B-cells is of pathophysiological, symptomatic or clinical relevance.

In this sense, the treatment of the following diseases can, for example, be considered: autoimmune diseases, for example rheumatoid arthritis, diabetes mellitus Type I, psoriasis, lupus systemicus erythematosus; rejection reactions after tissue or organ transplants, for example of skin, bone marrow and kidneys; viral or retroviral infections of any

S

10 genesis, for example ARC (AIDS-related complex) and i AIDS, as well as their preliminary stages; B-cell .leukaemias and lymphomas, for example chronic lymphatic leukaemia, lymphoblastic lymphoma, for example Burkitt's lymphoma and the like, or B-cell/ plasma cell neoplasias, for example plasmacytoma (multiple myeloma).

As autoimmune diseases, in the literature there *are generally designated those diseases in which the formation of autoantibodies have a pathogenic significance. These autoantibodies are directed against the body's own antigens and thus bring about a destruction of the body's own organs, cells or proteins. It is an object to suppress these diseased overreactions of the immune system with specificallyacting immune suppressives.

Furthermore, we have, surprisingly, found that imexon inhibits the proliferation of B-lymphocytes in r a dosage-dependent manner.

Imexon can be used itself directly or in the form of 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 Simmunological non-responsiveness,artificially-induced non-responsiveness and pathologically-induced tolerance to auto- and foreign antigens.

The immune suppressive action of imexon could be i demonstrated on the basis of the inhibition of the S proliferation of human B-lymphocytes, the proliferi ation being induced experimentally by the B-cell growth I factor (BCGF).

j i 15 Furthermore, the pharmacological properties of I imexon could be characterised by concanavalin A I (ConA)-induced proliferation of murine splenocyte, S (LTT), by phythaemagglutinin (PHA)-induced proliferi ation of human lymphocytes, as well as by tumour growth 20 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 brought about by an antigen or anti-p. The transmission of this activating signal finally has the result that receptors for the B-cell growth factor (BCGF) are expressed on thp B-cell surface. BCGF is a soluble lymphokine secreted by -8- T-cells with a molecular weight of 17,000 to 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 this two-signal process from the dormant state into the proliferative phase.

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

The antiretroviral action of imexon could be demonstrated on the basis of the Rauscher virus leukaemia model (cf. Example The influence of imexon on the spontaneous formation of lymphomas and Sthe synthesis of antinuclear autoantibodies in the mouse *(Example 6) proves the effectiveness on an animal model for autoimmune diseases.

Imexon can also be used as a combination preparation with other immune suppressives, for example cyclosporin A, ciamexon or azathioprine, as well as antiretrovirally-active substances, for example azidothymidine (AZT).

A combination of imexon with cytostatics is also possible, for example with cis-platinum complexes,

A

i a i -9- I such as cis-diaminodichloroplatinum, or with adriamycin, cyclophosphamide, vincristin, tamoxifen, methotrexate or 5-fluorouracil and the like. In this connection, the use of such combination preparations is of especial interest subsequent to a plasmaphaeresis for the *J monitoring of autoimmune diseases.

In the case of the use of a combination therapy, it is possible to administer the active materials in a so-called fixed combination, i.e. in a single pharma- 10 ceutical formulation, in which both active materials i are present simultaneously, or to use a so-called free combination in which the active materials are adminism. tered in the form of pharmaceutical formulations simultaneously or also successively in individually i 15 selectable dosage relationships.

For the preparation of pharmaceutical agents, imexon is mixed in known manner with appropriate pharmaceutical carrier substances, possibly granulated and pressed, for example, into Lablets or dragee cores.

A filling of the mixture into hard capsules is also I possible. With the addition of appropriate adjuvants, Sre a solution or suspension in water, an oil, for example olive oil, or a high molecular weight polymer, for example polyethylene glycol, can also be produced and administered iu the form of injection solutions, soft gelatine capsules, syrups or drops.

As solid carrier materials, there can be used, for example, starches or starch derivatives, sugars, sugar alcohols, celluloses or cellulose derivatives, tensides, talc, highly dispersed silicic acids, high molecular weight fatty acids or the salts thereof, gelatine, agar-agar, calcium phosphate, animal or vegetable fats or waxes and solid high molecular weight polymers (such as polyethylene glycols or polyvinylpyrrolidones). Compositions suitable for oral 10 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 indications to be treated.

In principle, the treatment can be based on the fact that 0.1 to 100 mg. of imexon per kg. body weight can be administered daily orally, intravenously, subcutaneously or intramuscularly. However, it is preferred to use amounts of from 5 to 50 mg./kg. body' weight and especially 5 to 20 mg./kg. body weight.

I The dosages of the active material can be administered 1 to 3 times daily.

The specific immunosuppressive action of imexon is demonstrated by the following Examples: Example 1.

BCGF-dependent proliferation of human B-lymphocytes.

The enrichment of peripheral human B-cells and

I

i_ i I -11the BCGF proliferation assay were carried out as follows (Cf. Eur. J. Immun., 16, 350/1986): Enriched human B-lymphocytes were washed twice with complete RPMI 1640 medium (streptomycin/penicillin, L-glutamine, 2-mercaptoethanol, FCS) and adjusted to 3 x 105 cells/ml. 160 ml. of this suspension were pipetted into each well of microtitre plates. As pseudoantigen, there were added thereto 10 ml. of a solution of HFC pS-IgG (300 pg./ml.) and, as growth 10 factor, 20 pl. BCGF (Cellular Products Incorporated).

To this were pipetted 20 pl. of the compound to be tested in 10 fold concentration. The cultures were incubated for a total of 140 hours at 37°C. with 0 carbon dioxide and 95% relative atmospheric humidity.

16 hours before the conclusion of the incubation period, each culture was pulsed with 1 pCi of a 3 H]-thymidine solution. At the end of the experiment, the cells were collected with a harvester and the incorporated radio- *0 activity decermirned in a liquid scintillation counter.

Example 2.

I Concanavalin A (ConA)-induced proliferation of murine l splenocytes.

Spleen cells (4 x 10 of CB6F 1 mice were incubated for a total of 48 hours with 0.5 pg./ml. ConA in microtitre plates (Nunc GmbH, Wiesbaden, Federal Republic of Germany) and various concentrations of imexon in 6 fold batches. 5 hours before the I I -12termination of the incubation period, the cultures were 3 pulsed with [3H]-thymidine and subsequently harvested on glass fibre filter platelets by means of a multisample harvester (Skatron Lier, Norway). The filter platelets were dried and the radioactivity was determined in a Packard scintillation spectrometer.

Example 3.

Phythaemagglutinin (PHA)-induced proliferation of human lymphocytes.

10 1 ml. of human whole blood was diluted with 500 pg. PHA solution (500 pg./ml.) and diluted with 48 ml. DMEM medium. 200 pl. amounts of this batch were mixed with 20 pl. of the imexon concentration to be tested in 6 fold batches and incubated for 4 days.

i 15 After pulsing with H]-thymidine, incubation was Scontinued for a further 24 hours, followed by harvesting and evaluation as described in Example 2.

*I Example 4.

Tumour growth inhibition assay (TGI).

A methylcholanthrene-induced fibrosarcoma cell line (MethA) was obtained from our own tumour cell bank and passed intraperitoneally into CB6F 1 mice.

1 x 104 MethA cells were incubated with the imexon concentration to be tested in DMEM medium for j hours. 3 hours before the end of the incubation time, pulsing was carried out with 3 H]-thymidine, followed by harvesting al, evaluated as described in Example 2.

i _l~l-l.il-

D

-13- The values given in the following Table 1 show the results of a representative experiment. They are 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 proliferation of the MethA sarcoma cell, T-lymphocytes and B-cells is shown. Imexon suppressed significantly and specifically the BCGF-induced B-cell proliferation at a concentration of 1 pg./ml., whereas the lymphocyte proliferation induced either by ConA or PHA was only significantly inhibited at concentrations of 10 pg./ml. Furthermore, the spontaneous proliferation of MethA sarcoma cells was also only significantly suppressed at 10 pg./ml.

The results of the above experiments are summarised in the following Table 1: S o o LI S S p o a a a a a a a a a Table 1 Effect of imexon on the proliferation of various cell types Irmexon TGI (MethA) LTT (Splenocytes, ConA) LTT (Splenocytes, PHA) BOGF (human B- (pg/ml) lymphocytes) 3H-IdR inhib- 3H-TdR inhib- 3 H-TdR inhib- 3 H-TdR inhibcpm ition cpm ition cpm ition cpnm ition x SD x SD x SD x SD Control 33966 3000 109879 12203 44283 6458 5541 1792 100 534 363 98** 903 62 99** 585 44 99** 562 44 911 110 97** 2509 863 98 7 573 59 99** 617 59 89** 21913 2357 35 1 24895 6563 77** 4724 704 89** 574 50 3 35473 3135 -4 118487 9494 -8 35850 13018 19 831 231 1 1 35475 1753 -4 119120 9172 -8 49348 4168 -11 2096 455 62* 0.3 37593 3080 -11 134032 137682 -22 45542 9870 -3 4201 1636 24 0.1 31722 3991 7 109717 11192 0 41849 1892 5 4847 1146 13 1892_5_4847_1146 p 0.002 p 0.001 i_; Example Antiretroviral action of imexon in the Rauscher virus leukaemia model.

8 to 9 week old female Balb/c mice were infected with 0.2 ml. of spleen homogenate of infected animals (diluted 1:2 in PBS). From day 0 (or day up to day 13, the animals were treated intraperitoneally daily with the given dosage of the active material.

On day 7 and on day 14, animals of the individual 0 treatment groups were sacrificed and the spleen weight o** determined as a measure of the viraemia.

In the following Table 2 are summarised the results of the investigations. Imexon controlled the virus-caused increased weight of the spleen in the same dosage range as azidothymidine.

-Ltl be.

a a a a a a a a a a a a.

a a *a.

a a a a a a AZT) in the ions of 5 or Table 2 Results of a comparative Rauscher virus leukaemia investigation c the action of imexon and azidothymidine model. There are -ven average values and standard deviat fold determinations (Experiment R 17) day 7 placebo placebo dose (mg/kg x d, i.p.) Virus) Virus) Imexon 90 Imexon 120 AZT 100 Ribavirin 100 spleen weight 0.112 0.291 0.248 0.190 0.185 0.116 0.019 0.045 (10) 0.030 (10) 0.031 (10) 0.017 (10) 0.012 animal weight 20.2 20.7 21.4 20.0 20.0 18.8 1.1 1.3 (10) 1.7 (10) 1.4 (10) n,8 (10) 1.2 day 14 placebo placebo dose (mg/kg x d, i.p.) Virus) Virus) Imexon 90 Imexon 120 AZT 100 Ribavirin 100 spleen weight 0.165 0.670 0.306 0.238 0.316 0.260 0.013 0.201 (10) 0.121 (10) 0.076 (10) 0.089 (10) 0.060 animal weight 20.5 19.6 19.6 20.9 20.9 19.2 0.3 0.9 (10) 1.9 (10) 0.8 (10) 1.1 (10) 1.1 i 3 animals died because of toxicity I i II- -17- Example 6.

Action of imexon in the case of autoimmune diseases.

With increasing age, the mouse strain MRL Ipr/lpr develops increasingly spontaneously lymphadenoma and SLE-like symptoms, for example the 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 once 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 of the therapeutic potency of imexon, MRL mice, after each i animal had developed at least one lymphadenoma (about 14 week old animals), were also treated once daily with the given dosages of imexon and cyclophosphamide.

The measurement parameters were again the number of .o lymphadenomas, as well as the autoantibody titre.

The results of these investigations have shown that imexon, in the case of very good compatibility, lowers the number of spontaneously arising lymphadenomas S* and the concentration of DNA-specific antibodies. The effectiveness of imexon was also shown in the case of therapeutic use with animals already having lymphomas.

The number of lymphadenomas decreased dependent upon the dosage, as well as the titre of the autoantibodies.

A*

-il l;' -18- Example 7.

Preparation of a pharmaceutical formulation of imexon.

A film tablet with, for example, 100 g. of active material and which has the following composition has proved to be an appropriate pharmaceutical composition: 0

S

S

S

0

S

S

imexon lactose monohydrate 10 poly-(0-carboxymethyl)-starch, sodium salt poly-(l-vinyl-2-pyrrolidone) 25,000 poly-(0-carboxymethyl)-starch, 15 sodium salt microcrystalline cellulose highly dispersed silicon dioxide magnesium stearate weight/unit/mg.

100.000 63.000 7.000 4.000 3.000 20.000 1.500 1.500 core weight 200.000 i tj

S.

S

S..

000* 0 S S 0 S 50 The film tablets were then produced in the usual way by the film drageeing of the'imexon cores obtained.

Film tablets with, for example 10 mg., 50 mg., 200 mg. and 500 mg. of active material were produced in a corresponding manner.

Claims (11)

1. Method of treating diseases which involve an increased B-lymphocyte activation or proliferation respectively comprising administering imexon or a physiologically acceptable salt thereof to a patient.

2. Method according to claim 1, wherein autoimmune diseases are treated.

3. Method according to claim 1, wherein leukaemias or lymphomas are treated.

4. Method according to claim 1, wherein rejection reactions after tissue or organ transplantations are suppressed.

5. Method according to claim 1, wherein viral or retroviral infections are treated or prevented.

6. Method according to claim 5, wherein AIDS or ARC are prevented or treated.

7. Method according to claim 1, wherein imexon or the salt thereof acts cytostatically. I: i V -I- 1. i 20

8. Method according to any of the preceding claims, wherein the imexon or the salt thereof is administered to the patient in the form of a tablet.

9. Method according to any of the preceding claims, wherein imexon or the salt thereof is administered to the patient in an amount from 10 to 1000 mg per unit form of administration o.

10. Method according to any of claims 1 to 8, wherein the imexon or the salt thereof is administered to the patient daily in a dosage of 0.1 to 100 mg/kg body weight. 0

11. Method according to any of the preceding claims, subsiantially as hereinbefore described and exemplified. S Co DATED this 25th day of October, 1991 BOEHRINGER MANNHEIM GmbH By Its Patent Attorneys DAVIES COLLISON L1.4E