Field of the invention The invention relates to the use of topoisomerase I inhibitors for the prevention and treatment of autoimmune diseases.

Background of the invention Autoimmune diseases are associated with abnormal lymphoproliferation as a result of defects in the termination of lymphocyte activiation and growth. Often, such diseases are associated with inflammation like rheumatoid arthritis, insulin dependent diabetes mellitus, multiple sclerosis, systemic lupus erythematosus and the like. Systemic lupus erythematosus is a chronic autoimmune disorder that predominantly affects women of childbearing age. It is estimated that in the United States up to 275Ό00 of adult women suffer from lupus erythematosus. The disease affects the skin, joints, cardiac and pulmonary system, the central nervous system, and kidneys, while lupus- associated glomerulonephritis is a significant cause of mortality in these patients. The 5- year survival increased from 50% in 1955 to a 15-year survival of 76% recently. The current treatment for systemic lupus erythematosus consists of an induction therapy with cyclophosphamide and prednisolone followed by a maintenance therapy with

cyclophosphamide or azathioprine or mycophenolate mofetil. However, despite clear advances in the treatment of this autoimmune disease the mean age of death of patients dying from systemic lupus erythematosus is 44 years. Death is caused by active lupus with 26.5%, or by side effects of current treatment such as infections with 25%. Therefore, new treatment options are required to overcome these obstacles.

Summary of the invention

The invention relates to topoisomerase I inhibitors, in particular camptothecin and camptothecin derivatives, for use in the prevention and treatment of autoimmune diseases, for example systemic lupus erythematosus. Furthermore the inventions relates to a method of prevention or treatment of autoimmune diseases, for example systemic lupus erythematosus, comprising administering a topoisomerase I inhibitor, in particular camptothecin or a camptothecin derivative, to a patient in need thereof. Brief description of the Figures

Figure 1. Frequency of proteinuria grade≥2+ (≥100 mg/dl) in NZB/W F1 mice treated with irinotecan from week 13 every fourth week compared with saline control. x-axis: age of mice in weeks; y-axis: frequency of mice with proteinuria grade≥2+.

Open squares: control group treated with saline, filled circles: mice treated with 25 mg/kg irinotecan, filled triangles: mice treated with 50 mg/kg irinotecan.

P<0.001 (analysis of variance followed by Bonferoni's correction) for irinotecan-treated groups versus saline group.

Figure 2. Survival of NZB/W F1 mice treated with irinotecan from week 13 every fourth week compared with saline control. x-axis: age of mice in weeks, y-axis: survival in percent

Open squares: control group treated with saline, filled circles: mice treated with 25 mg/kg irinotecan, filled triangles: mice treated with 50 mg/kg irinotecan.

P<0.0001 (Kaplan-Meier, logrank test) for irinotecan-treated groups versus saline group.

Figure 3. Body weight of NZB/W F1 mice treated with irinotecan from week every fourth week compared with saline control. x-axis: age of mice in weeks, y-axis: body weight in gram.

Open squares: control group treated with saline, filled circles: mice treated with 25 mg/kg irinotecan, filled triangles: mice treated with 50 mg/kg irinotecan. Mice that had already died were counted according to their last body weight.

P<0.001 (analysis of variance followed by Bonferoni's correction) for irinotecan-treated groups versus saline group. P<0.01 (analysis of variance followed by Bonferoni's correction) for 25 mg/kg irinotecan versus 50 mg/kg irinotecan.

Figure 4. Survival of NZB/W F1 mice with established lupus nephritis treated with irinotecan compared with saline control. x-axis: age of mice in weeks, y-axis: survival in percent

Open squares: treatment with saline started from proteinuria grade≥3+, filled circles: treatment with 50 mg/kg irinotecan initiated from proteinuria grade≥3+, filled triangles: treatment with 50 mg/kg irinotecan initiated from proteinuria grade≥4+. The treatment of relapsed disease was performed as the initial treatment. P<0.0001 (Kaplan-Meier, logrank test) for irinotecan-treated groups versus saline group.

Detailed description of the invention

The invention relates to topoisomerase I inhibitors, in particular camptothecin and camptothecin derivatives, for use in the treatment of autoimmune diseases, for example systemic lupus erythematosus.

Topoisomerases are isomerase enzymes that act on the topology of DNA by breaking and rejoining the phosphodiester backbone of DNA strands. The double-helical configuration of DNA is difficult to separate, and yet the strands must be separated when enzymes are transcribing the sequences that encode proteins, or if chromosomes are to be replicated. In so-called circular DNA, in which double helical DNA is bent around and joined in a circle, the two strands are topologically linked. Otherwise identical loops of DNA having different numbers of twists are topoisomers, and cannot be interconverted by any process that does not involve the breaking of DNA strands. Topoisomerases catalyze and guide the unknotting of DNA by creating transient breaks in the DNA using a conserved tyrosine as the catalytic residue.

Type I topoisomerase (EC 5.99.1 .2) cuts one strand of a DNA double helix and then reanneals the cut strand. Type I topoisomerases are subdivided into two subclasses: type IA topoisomerases which share many structural and mechanistic features with the type II topoisomerases, and type IB topoisomerases, which utilize a controlled rotary mechanism. Type IA topoisomerases form a covalent intermediate with the 5' end of DNA, while the IB topoisomerases form a covalent intermediate with the 3' end of DNA. Topoisomerase I inhibitors are agents designed to interfere with the action of

topoisomerase I. Topoisomerase I induces single strand breaks into the DNA in order to unknot overwind DNA followed by re-ligation of the DNA strand. It is thought that topoisomerase I inhibitors block the re-ligation step, generating single stranded breaks that harm the integrity of the genome. Introduction of these breaks subsequently lead to apoptosis and cell death. Therefore, topoisomerase I inhibitors are well known for the treatment of different types of cancer by chemotherapy. Given this type of mechansim it is surprising that topoisomerase I inhibitors should be useful also in the treatment of autoimmune diseases.

Typical examples of topoisomerase I inhibitors are the quinoline alkaloid camptothecin (4-ethyl-4-hydroxy-1 H-pyrano[3' _! 4':6 _! 7]indolizino[1 _! 2-b]quinoline-3 _! 14-(4H,12H)-dione) _! and camptothecin derivatives, for example derivatives known under the name irinotecan, iritenum, orathecin, hydroxycamptothecin, aminocamptothecin, topotecan, topotecin, gimatecan, lipotecan, and diflomotecan. Camptothecin derivatives considered herein are derivatives which, for example, carry substituents in position 7, 9, 10, 1 1 , and 12, i.e. in the positions of the quinoline structural unit, in particular in position 7, 9, 10, and 1 1.

Substituents considered are alkyl, haloalkyl, hydroxyalkyl, silylalkyl, aminoalkyl including nitrogen containing heterocyclylalkyl, aminocarbonyloxy including nitrogen containing heterocyclylcarbonyloxy, iminoalkyl, oximinoalkyl, halo (bromo, chloro or fluoro), hydroxy, amino, alkylsilyl, and cyclic substituents such as methylenedioxy or ethylenedioxy. Such substituents may influence the potency, hydrophilicity or lipophilicity, and stability of the derivative compared to the basic camptothecin. A further camptothecin derivative considered is one wherein the lactone (oxo-pyrano) ring is expanded by one methylene group to give a seven-membered lactone ring. Inhibitors of topoisomerase I specifically considered in the present invention are irinotecan, iritenum, orathecin, topotecan, topotecin, gimatecan, lipotecan, diflomotecan, and compounds named ALS 423, TP300, DTS 108, MRX952, RTA 501 and 502, SP1060C and CO, DB67, MEN4901 , T0128, PEP02, CPT417, Xin Pu Jin, INX007, GEM231 , GI14721 1 , INX-0076, DRF 1644, TAS103, INOC010, IHL305, and BN80927, and further lamellarin D.

Preferred inhibitors of topoisomerase I are camptothecin and camptothecin derivatives, in particular irinotecan and pharmaceutically acceptable salts thereof. Autoimmune diseases are associated with abnormal lymphoproliferation as a result of defects in the termination of lymphocyte activiation and growth. Often, such diseases are associated with inflammation.

It has now surprisingly been found that topoisomerase I inhibitors are active for the prevention and treatment of systemic lupus erythematosus in a representative mouse model. Topoisomerase I inhibitors may therefore be used in the treatment of autoimmune diseases, for example systemic, discoid or subacute cutaneous lupus erythematosus, rheumatoid arthritis, antiphospholipid syndrome, CREST, progressive systemic sclerosis, mixed connective tissue disease (Sharp syndrome), Reiter's syndrome, juvenile arthritis, cold agglutinin disease, essential mixed cryoglobulinemia, rheumatic fever, ankylosing spondylitis, chronic polyarthritis, myasthenia gravis, multiple sclerosis, chronic

inflammatory demyelinating polyneuropathy, Guillan-Barre syndrome, dermatomyositis / polymyositis, autoimmune hemolytic anemia, thrompocytopenic purpura, neutropenia, type I diabetes mellitus, thyroiditis (including Hashimoto's and Grave' disease), Addison's disease, polyglandular syndrome, pemphigus (vulgaris, foliaceus, sebaceous and vegetans), bullous and cicatricial pemphigoid, pemphigoid gestationis, epidermolysis bullosa acquisita, linear IgA disease, lichen sclerosus et atrophicus, morbus Duhring, psoriasis vulgaris, guttate, generalized pustular and localized pustular psoriasis, vitiligo, alopecia areata, primary biliary cirrhosis, autoimmune hepatitis, all forms of glomerulonephritis, pulmonal hemorrhage (goodpasture syndrome), IgA nephropathy, pernicious anemia and autoimmune gastritis, inflammatory bowel diseases (including colitis ulcerosa and morbus Crohn), Behcet's disease, Celic-Sprue disease, autoimmune uveitis, autoimmune myocarditis, granulomatous orchitis, aspermatogenesis without orchitis, idiopatic and secondary pulmonary fibrosis, and other inflammatory diseases with a possibility of autoimmune pathogensesis.

In particular, topoisomerase I inhibitors are considered for the prevention and treatment of systemic, discoid or subacute cutaneous lupus erythematosus, rheumatoid arthritis, antiphospholipid syndrome, CREST, progressive systemic sclerosis, multiple sclerosis, and all forms of glomerulonephritis. Most preferred is the use of topoisomerase I inhibitors for the prevention and treatment of systemic lupus erythematosus.

In particular, it was found that the topoisomerase I inhibitor irinotecan (marketed under the trade name Campto® by Pfizer) prevents and cures a systemic lupus erythematosus type disease in mice. Based on preliminary observations, prevention studies were performed in a mouse model of spontaneous systemic lupus erythematosus, which demonstrated that administration of irinotecan completely prevents the onset of the disease. Furthermore, administration of irinotecan to mice with established disease induced remission from lupus-nephritis in a substantial number of mice as demonstrated by proteinuria reversed to normal level. Furthermore, survival of mice with established lupus nephritis which were treated with irinotecan was significantly prolonged compared with saline controls.

Importantly, a previous study indicates that the current standard therapy for lupus erythematosus using the combination of cyclophosphamide, azathioprine and

methylprednisolone failed to prolong survival of mice when treatment started from established disease [Steinberg A.D. et al., Arthritis Rheum. 1975, 18(1 ):9-14]. These data indicate that the use of topoisomerase I inhibitors provide a complete new and effective treatment option for systemic lupus erythematosus and related autoimmune diseases.

Furthermore the invention relates to a method of prevention or treatment of autoimmune diseases, for example systemic lupus erythematosus, comprising administering a prophylactically or therapeutically effective amount of a topoisomerase I inhibitor, in particular camptothecin or a camptothecin derivative, to a patient in need thereof.

The topoisomerase I inhibitor can be administered alone or in combination with one or more other therapeutic agents, possible combination therapy taking the form of fixed combinations, or the administration of a compound of the invention and one or more other therapeutic agents being staggered or given independently of one another, or the combined administration of fixed combinations and one or more other therapeutic agents. The topoisomerase I inhibitor is preferably used in the form of a pharmaceutical composition that comprises the active ingredient and that can be used especially in the treatment of the diseases mentioned above. Compositions for enteral administration, such as nasal, buccal, rectal or oral administration, and especially for parenteral administration, such as intravenous, intramuscular or subcutaneous administration, to warm-blooded animals, especially humans, are especially preferred. The compositions comprise the active ingredient alone or, preferably, together with a pharmaceutically acceptable carrier. The dosage of the active ingredient depends upon the disease to be treated and upon the species, its age, weight, and individual condition, the individual pharmacokinetic data, and the mode of administration.

Preference is given to the use of solutions of the active ingredient, and also suspensions or dispersions, especially isotonic aqueous solutions, dispersions or suspensions which, for example in the case of lyophilized compositions comprising the active ingredient alone or together with a carrier, for example mannitol, can be made up before use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes. The said solutions or suspensions may comprise viscosity-increasing agents, typically sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, or gelatins, or also solubilizers, e.g. Tween 80 ^® (polyoxyethylene(20)sorbitan mono-oleate).

Suspensions in oil comprise as the oil component the vegetable, synthetic, or semisynthetic oils customary for injection purposes. In respect of such, special mention may be made of liquid fatty acid esters that contain as the acid component a long-chained fatty acid having from 8 to 22, especially from 12 to 22, carbon atoms. The alcohol component of these fatty acid esters has a maximum of 6 carbon atoms and is a monovalent or polyvalent, for example a mono-, di- or trivalent, alcohol, especially glycol and glycerol. As mixtures of fatty acid esters, vegetable oils such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and groundnut oil are especially useful.

The manufacture of injectable preparations is usually carried out under sterile conditions, as is the filling, for example, into ampoules or vials, and the sealing of the containers.

The preferred dosage of the toposiomerase I inhibitors are decided by the medical doctor taking into account the type of compound, type of disease, its severity, the age and condition of the patient, and the mode of administration. Usual doses are between 0.1 mg/kg to 10 mg/kg, preferably 0.5 mg/kg to 5 mg/kg daily, three times weekly, twice weekly, or preferably, once weekly, preferably given as an intravenous infusion. It may also be considered to give higher doses during one or two weeks followed by a period without medication, and repeating the treatment cycles as required.

Complete prevention of murine systemic lupus erythematosus by administration of irinotecan.

Administration of 25 mg/kg and 50 mg/kg irinotecan from week 13 three times a week and repeated every fourth week completely prevents the onset of lupus nephritis in NZB/W F1 mice. Whereas 92% of the saline treated control group possessed severe proteinuria, none of the animals of the irinotecan-treated groups demonstrated signs of lupus nephritis in week 49 of animal live (figure 1 ). More importantly, 83% of mice from the control group died until week 49, while all of the irinotecan-treated animals were alive at that time (figure 2). The course of body weight of irinotecan-treated groups (figure 3) suggests that the used concentration of irinotecan did not induce striking side effects. In contrast to contracted animals of the control group, mice of irinotecan-treated groups appeared completely normal.

Reversal of established lupus erythematosus by administration of irinotecan.

Treatment of NZB/W F1 mice was initiated with 50 mg/kg irinotecan three times a week for two weeks (6 applications), when proteinuria achieved grade 3+ (≥ 300 mg/dL ) or grade 4+ (≥ 2000 mg/dL) both indicating a highly advanced stage of systemic lupus erythematosus. Control group was treated with saline starting from grade 3+. Treatment with irinotecan resulted in complete remission (defined as 30 mg/dL of proteinuria or less for at least 3 weeks) in 70% of animals with grade 3+ and 55% of animals with grade 4+ proteinuria. None of the mice in the control group showed reversal of proteinuria. Life span of irinotecan-treated animals was significantly prolonged both in the grade 3+ and in the grade 4+ groups. Whereas only 10% of saline-treated mice with proteinuria survived until week 49, 90% of mice with proteinuria treated with irinotecan from grade 3+ and 73% of mice with proteinuria treated with irinotecan from grade 4+ were alive (figure 4). Median time to relapse was 8.0 weeks for grade 3+ group and 7.5 weeks for grade 4+ group, respectively.

Notably, except for one mouse with diarrhoea, no toxic side effects related to the treatment with irinotecan were observed both in the preventive study and in the study with established disease.

Examples

Irinotecan is a semisynthetic water-soluble analogue of the natural alkaloid camptothecin which is approved by the U.S. Food and Drug Administration for the treatment of metastatic colorectal cancer. Irinotecan is a prodrug that is converted to the biologically active metabolite 7-ethyl-10-hydroxy-camptothecin (SN-38) by a carboxylesterase- converting enzyme. One thousand-fold more potent than its parent compound irinotecan, SN-38 inhibits topoisomerase I activity by stabilizing the cleavable complex between topoisomerase I and DNA, resulting in DNA breaks that inhibit DNA replication and trigger apoptotic cell death.

NZB/W F1 , generated as the F1 hybrid of New Zealand Black and New Zealand White mice, is a mouse model well known to spontaneously develop an autoimmune disease referred to as systemic lupus erythematosus. These mice resemble most likely human lupus disease with high levels of antinuclear antibodies, hemolytic anemia, proteinuria, and severe progressive immune complex glomerulonephritis leading to death. This animal model was used to study the influence of the topoisomerase I inhibitor irinotecan

(Campto®, Pfizer) on the course of systemic lupus erythematosus. Experimental setup has been composed of two main parts, at first a study of preventive- type and secondly, treatment of established murine lupus erythematosus by irinotecan.

Study 1 : Prevention of murine lupus erythematosus by irinotecan Six weeks old NZB/W F1 were obtained from Jackson Laboratories, USA, and were kept in isolated ventilated cages. From week 12 all mice were measured for proteinuria using Albustix® (Bayer Diagnostics) and body weight once a week. Treatment started from week 13, treatment was performed every fourth week (week 13, 17, 21 , 25, 29, 33, 37, 41 , 45, 49) three times a week.

3 groups, 12 animals per group

Group 1 : Saline 0.9% 10 ml/kg

Group 2: Irinotecan (Campto®, Pfizer) 25 mg/kg 10 ml/kg

Group 3: Irinotecan (Campto®, Pfizer)) 50 mg/kg 10 ml/kg

Criteria for sacrifice due to the severity of the disease were proteinuria grade 4+ (≥2000 mg/dl) and a weight loss of >25% calculated from the initial beginning of the disease. The beginning of the disease was defined as two times proteinuria grade 4+ three days apart. Study 2: Treatment of established murine lupus erythematosus by irinotecan

Six weeks old NZB/W F1 were obtained from Jackson Laboratories, USA, and were kept in isolated ventilated cages. Starting from week 18, proteinuria and body weight were measured twice a week.

3 groups, 12 animals per group, application volume all groups 10 ml/kg

Group 1 : treatment from proteinuria grade 3+ (≥300 mg/dl), Saline 0.9%

Group 2: treatment from proteinuria grade 3+ (≥300 mg/dl), Campto ^® 50 mg/kg

Group 3: treatment from proteinuria grade 4+ (≥2000 mg/dl), Campto ^® 50 mg/kg The beginning of the disease was defined as two times proteinuria grade 3+ (group 1 and 2) or proteinuria grade 4+ (group 3) three days apart. Treatment was performed three times a week for two weeks (6 applications). Relapse of the disease was defined as two times proteinuria grade 3+ (group 1 and 2) or proteinuria grade 4+ (group 3) three days apart. The treatment of relapsed disease was performed as the initial treatment.

Complete remission was defined as 30 mg/dL or less of proteinuria maintained for at least 3 weeks.

Criteria for sacrifice due to the severity of the disease were a proteinuria 4+ (≥2000 mg/dl) and a weight loss of >25% calculated from the beginning of the disease. Animals showing remarkable weight loss but no proteinuria before any treatment was initiated were excluded from the study.