WO2006005620A1

WO2006005620A1 - Use of manzamines in transplantation and autoimmune diseases

Info

Publication number: WO2006005620A1
Application number: PCT/EP2005/007678
Authority: WO
Inventors: Peter C. Hiestand; Frank Petersen; Silvio Roggo; Mark T. Hamann
Original assignee: Novartis Ag; Novartis Pharma Gmbh; The University Of Mississippi
Priority date: 2004-07-15
Filing date: 2005-07-14
Publication date: 2006-01-19

Abstract

The use of a manzamine for preventing, treating or inhibiting (a) acute or chronic graft rejection in a recipient of cell, tissue or organ allotransplant, (b) vein graft stenosis, restenosis and/or vascular occlusion following vascular injury or (c) an autoimmune disease is described.

Description

Use of manzamines in transplantation and autoimmune diseases

This research is supported in part by grants from the National Institutes of Health of the United States of America R01AI36596 and 5KO2AI01502. The Government of the United States may have certain rights in this invention.

The present invention relates to a new use of a group of sponge-derived alkaloids known as or closely related to the "manzamines" as well as rationally prepared and natural derivatives and analogs thereof, particularly as an immunosuppressive agent, e.g. for the prevention, treatment or inhibition of acute or chronic graft rejection or autoimmune diseases. Manzamines are defined as a class of alkaloids isolated from the Phylum Porifera and containing a sophisticated array of aromatic and aliphatic rings.

The manzamines are complex, polycyclic, marine-derived alkaloids first reported by Higa and coworkers in 1986 and isolated from the Okinawan sponge genus Haliclona. See, Sakai, R; Higa, T.; Jefford, C.W.; Bernardinelli, G. "Manzamine A; An Antitumor Alkaloid From a Sponge," J. Am. Chem, Soc. 1986, 108, 6404-6405. These compounds possess a fused and bridged tetra- or pentacydic ring system that is attached to a β-carboline moiety. Since the first report of manzamine A, an additional thirty manzamine- type alkaloids have been reported from the sponge genera Prianos, (see, Ohtani, I.I.; lchiba, T.; Isobe, M.; Kelly-Borges, M.; Scheuer, P.J. "Kauluamine: An Unprecedented Manzamine Dimer from an Indonesian Marine Sponge, Prianos sp. J. Am. Chem. Soc. 1995, 117, 10743-10744), Haliclona, (see, Sakai, R.; Kohmoto, S.; Higa, T.; Jefford, C.W.; Bernardinelli, G. "Manzamines B & C, Alkaloids From a Sponge, Haliclona sp. Tetrahedron Left. 1987, 28, 5493-5496; Higa, T.; Sakai, R. ; Kohomoto, S.; Lui, M.S., "Antitumor alkaloids from marine sponges," Eur. Pat. Appl. 1987, EP 87-310872871210; Kobayashi, M.; Chen, Y. J.; Aoki, S.; In, Y.; Ishida, T.; Kitagawa, I. Tetrahedron ^§5, 51, 3727-3736), Xestospongia, (see, lchiba, T.; Sakai, R.; Kohomoto, S.; Saucy, G.; Higa, T. "New Manzamine Alkaloids From A Sponge Of The Genus Xestospongia," Tetrahedron Lett. 1988, 29, 3083-3086; Edrada, RA; Proksch, P.; Wray, V.; Witte, L.; Muller, W.E.G.; Van Soest, R.W.M, J. Nat Prod. 1996, 59, 1056- 1060), Pachypeilha, (see, lchiba, T.; Corgiat, J.M.; Scheuer, P.J.; Kelly-Borges, M. "8-Hydraxymanzamine A, A β-carboline Alkaloid From a Sponge, Pachypellina sp.," J. Nat. Prod. 1994, 57, 168-170), Petrosia, (see, Crews, P.; Cheng, X.C.; Adamczeski, M.; Rodriguez, J.; Jaspars, M.; Schmte, F.J.; Traeger, S.C.; Prodesimo, E.G., "1 ,2,3,4-Tetrahydro-8-hydroxymanzamines, Alkaloids From Two Different Haplosderid Sponges," Tetrahedron 1994, 50, 13567-13574), Cribrochalina, Ircinia, (see, Kondo, K.; Shigemori, H.; Kikuchi, Y.; Ishibashi, M.; Sasaki, T.; Kobayashi, J. "Ircinals A and B From the Okinawan Marine Sponge Ircinia sp.: Plausible Biogenetic Precursors of Manzamine Alkaloids," J. Org. Chem. 1992, 57, 2480-2483), Amphimedon (see, Tsuda, M.; Kawasaki, N.; Kobayashi, J., "Ircinols A and B, First Antipodes of Manzamine-Related Alkaloids from an Okinawan Marine Sponge," Tetrahedron 1994, 50, 7957-7960; Tsuda, M.; Kawasaki, N.; Kobayashi, J. "Keramaphidin C and Keramamine C, Plausible Biogenetic Precursors of Manzamine C from an Okinawan Marine Sponge," Tetrahedron Lett. 1994, 35, 4387-4388; Kobayashi, . J.; Tsuda, M.; Kawasaki, N.; Matsumoto, K; Adachi, T. "Keramaphidin B, a Novel Pentacyclic Alkaloid From a Marine Sponge Amphimedon sp. : A Plausible Biogenetic Precursor of Manzamine Alkaloids," Tetrahedron Lett. 1994, 35, 4383-4386; Tsuda, M.; Inaba, K.; Kawasaki, N.; Honma, K.; Kobayashi, J. "Chiral Resolution of ±keramaphidin B and Isolation of Manzamine L, a Newp- Carboline Alkaloid From a Sponge Amphimedon sp.," Tetrahedmn 1996, 52, 2319-2924; Kobayashi, J.; Watanabe, D.; Kawasaki, N.; Tsuda, M. J. Org. Chem. 1997, 62, 9236-9239; Tsuda, M.; Watanabe, D.; Kobayashi, J., "Ma'eganedin A, a New Manzamine Alkaloid from Amphimedon Sponge, Tetrahedron Lett. 1998, 39, 1207-1210) and Pellina (see, Nakamura, H.; Deng, S.; Kobayashi, J. ; Ohizumi, Y.; Tomotake, Y.; Matsuzaki, T., "Keramamine-A and -B, Novel Antimicrobial Alkaloids from the Okinawan Marine Sponge Pellina sp., 'Tetrahedron Lefti987, 28, 621-624).

The manzamine alkaloids can be isolated from a diversity of unrelated species . For example, the manzamines can be produced by a microorganism from the actinomycete class based on WO2004013297, Microbial Production of Manzamines, Russell, T. Hill, Mark T. Hamann, Olivier Peraud and Noer Kasanah, The Center of Marine Biotechnology, University of Maryland Biotechnology Institute, The University of Mississippi, and the National Center for the Development of Natural Products.The Diversity of Reported Manzamine Producing Sponges

Orders Family Genus

Halichondrida Halichondriidae Prianos

Haplosclerida Chalinidae Haliclona

Niphatidae Amphimedon.

Cribnochalina

Petrosida Petrosiidae Petrosia

Xestospongia.

Oceanapiidae Pellina

Pachypellina

Dictyoceratida Irciniidae Irυinia

In the context of the present invention the term "manzamine" comprises, unless specified differently, a manzamine or a manzamine derivative or analog or an optical isomer or racemate or tautomer thereof or a pharmaceutically acceptable salt thereof or a mixture of any of these. In the context of the present invention, a manzamine may also be defined as the product obtained by a process comprising the use of marine sponge extracts, preferably as described in detail above.

Preferred manzamines are manzamines which may be represented by the following general formulae (I) to (VIII)

In one embodiment, the subject invention relates to the use of manzamines of the formula (I):

wherein any of R₁ to R₂₃ are, independently, hydrogen, halogen, hydroxy, C₁-C₆ alkoxy, C₁-C₆ acyloxy, or C₁-C₆ alkyl, C₁-C₆ alkyl, di-CrC₆ alkyl amino. Each of the rings in the compound can range from 3 to 18-membered of carbon, nitrogen, oxygen and/or sulfur.

In another preferred embodiment of the invention, the invention pertains to the use of manzamines of the formula (II): -A-

wherein any of R₁ to R₂₂ are, independently, hydrogen, halogen, hydroxy, C₁-C₆ alkoxy, C₁-C₆ acyloxy, or C₁-C₆ alkyl, C₁-C₆ alkyl, di-CrC₆ alkyl amino. Each of the rings in the compound can range from 3 to 18-membered of carbon, nitrogen, oxygen and/or sulfur.

In another preferred embodiment of the invention, the invention pertains to the use of manzamines of the formula (III):

wherein any of R-i to R₂₂ are, independently, hydrogen, halogen, hydroxy, C₁-C₆ alkoxy, C₁-C₆ acyloxy, or C₁-C₆ alkyl, C₁-C₆ alkyl, di-C-i-C₆ alkyl amino. Each of the rings in the compound can range from 3 to 18-membered of carbon, nitrogen, oxygen and/or sulfur. In one embodiment, at least one of R₁ to R₂₃ in formula (I)₁ (II) or (III) is C₁-C₆ alkylcarbonylamino or substituted C₁-C₆ alkyl, more particularly, sulfonyl or aminosulfonyl C₁-C₆ alkyl.

In one embodiment, the subject invention relates to the use of manzamines of the formula (IV):

wherein X¹, X², X³, X⁴, X⁵, and X⁶ are, independently, hydrogen, halogen, hydroxy, CrC₆-alkoxy, C₁- C₆-acyloxy, or mono-C_rC₆-alkyl- or di-C_rC₆-alkyl-amino; R¹ is hydrogen, C_rC₆-alkyl, or C_rC₆-acyl group; R² is hydrogen, hydroxy, C_rC₆-alkoxy, or C_rC₆-acyloxy.

In a preferred embodiment of the invention, the invention pertains to manzamine A having the following structure:

In a further embodiment, the subject invention relates to the use of manzamines of the formula (V):

wherein X¹, X², X³, X⁴, X⁵, and X⁶ are the same or different and are hydrogen, halogen, hydroxyl, C_rC₆-alkoxy, CrCe-acyloxy, thiol, C_rC₆-alkylthiol, nitro, amino, C_rC₆-alkylsulfonyl, aminosulfonyl, hydroxy sulfonyl (-SO₃H), CrC₆-acylamino, C_rC₆-alkyl, or mono-C_rC_δ-alkyl- or di-C_rC₆-alkyl-amino and R¹ and R² are the same or different and are hydrogen, C₁-C₆-alkyl, or C_rC₆-acyl.

In a preferred embodiment of the invention, the invention pertains to manzamine B having the following structure:

(B)

In a further embodiment, the subject invention relates to the use of manzamines of the formula (Vl):

wherein X¹, X², X³, and X⁴ are the same or different and are hydrogen, halogen, hydroxyl, C₁-C₆- alkoxy, C_rC₆-acyloxy, thiol, C_rC₆-alkylthiol, nitro, amino, C_rC₆-aIkylsulfonyl, aminosulfonyl, hydroxy sulfonyl (-SO₃H), CrC₆-acylamino, CrC₆-alkyl, ormono-CrCe-alkyl- or diC_rC₆-alkyl-amino; and R¹ is hydrogen, CrC₆-alkyl, or CrC₆-acyl. Each of the rings in the compound can range from 3 to 18- membered consisting of carbon, nitrogen, oxygen and/or sulfur.

In a preferred embodiment of the invention, the invention pertains to manzamine C having the following structure:

(C)

In a further embodiment, the subject invention relates to the use of manzamines of the formula (VII):

wherein X¹, X², X³, and X⁴ are the same or different and are a hydrogen, halogen, hydroxyl, CrC₆- alkoxy, C_rC₆-acyloxy, thiol, CrC₆-alkylthiol, nitro, amino, C_rC₆-aIkylsulfonyl, aminosulfonyl, hydroxy sulfonyl ( — SO₃H), C_rC₆-acylamino, CrC₆-alkyl, or mono-CrC₆-alkyl- or di-C_rC₆-alkyl-amino; R¹ and R² are the same or different and are hydrogen, Ci-C₆-alkyl, or Ci-C₆-acyl; and Y is hydrogen, hydroxyl, C_rC₅-alkoxy, or CrC₆-acyloxy.

In a preferred embodiment of the invention, the invention pertains to manzamine D having the following structure:

In a further embodiment, the subject invention relates to the use of manzamines of the formula (VIII):

wherein R is hydrogen, halogen, hydroxy, or d-Cβ-acyloxy; and X is a double bonded oxygen, or is the same or different and is any two of hydrogen, hydroxy, C_rC₆-alkyl, CrC₅-alkoxy, or C₁-C₆- acyloxy.

In a preferred embodiment of the invention, the invention pertains to manzamine E and F having the following structures:

In other embodiments of the invention, the double bonds in the general formulae (I)-(VIII) are partially or fully reduced. In further embodiments of the invention, the manzamine is in the form of a pharmaceutically acceptable salt which includes salts with a mineral acid (e.g., HCI, HBr, H₂SO₄, H₃PO₄, HNO₃, etc.) or with an organic acid, such as acetate, fumarate, maleate, benzoate, citrate, malate, methanesulfonate and benzenesulfonate salts. The manzamine compounds of the invention also encompass hydrate and solvate forms. Methods for obtaining these compounds are described in, for example, U.S. Patent Nos.4,895,852; 4,895,853; and 4,895,854, which are herein incorporated in their entirety by reference thereto.

Skilled chemists having the benefit of the present disclosure of the structure of these manzamines can readily use procedures to prepare the subject compounds from sponge/microbial extracts or through synthetic or biocatalytic transformations. In carrying out such operations, suitable filtration, chromatographic, crystallization and other purification techniques well known in the art may be used. These techniques may include, for example, reversed phase (RPLC)₁ column, vacuum flash, medium pressure (MPLC) and high performance liquid chromatography (HPLC) with a suitable column such as silica gel, Sephadex LH-20, ammonia-treated silica gel, bonded phase RP-18, RP-8 and amino columns. Such columns are eluted with suitable solvents such as hexanes, ethyl acetate, acetone, methylene chloride, methanol, isopropanol, acetonitrile, water, trifiuoroacetic acid (TFA) and various combinations thereof. A specific example of an isolation/extraction of manzamine from a marine sponge is described in detail in WO 02/17917, pages 34 to 38, incorporated herein by reference.

As used herein, the term "graft" refers to organs and/or tissues and/or cells which can be obtained from first a mammal (or donor) and transplanted into a second mammal (or recipient), preferably a human. The term "graft" encompasses, for example, skin, eye or portions of the eye (e.g., cornea, retina, lens), muscle, bone marrow or cellular components of the bone marrow (e.g., stem cells, progenitor cells), heart, lung, liver, kidney, pancreas (e.g., islet cells, p-cells), parathyroid, bowel (e.g., colon, small intestine, duodenum), neuronal tissue, bone and vasculature (e.g., artery, vein). A graft can be obtained from a suitable mammal (e.g., human or pig), or under certain circumstances a graft can be produced in vitro by cuituring cells, for example embryonal, skin or blood cells and bone marrow cells. A graft is preferably obtained from a human.

Organ transplants of kidney, heart, liver and lung are now regularly performed as treatment for endstage organ disease. Allograft as well as xenograft transplants have been performed. However, because of problems with long-term chronic rejection, organ transplantation is not yet a permanent solution to irreversible organ disease. There is also a need for improved agents for treatment of acute rejection.

Chronic rejection, which manifests itself as progressive and irreversible graft dysfunction is the leading cause of organ transplant loss. The clinical problem of chronic rejection is clear from transplantation survival times; about half of kidney allografts are lost by around 10 years after transplantation, and a similar value is observed in patients with a heart allograft. Chronic rejection is considered as a multifactorial process in which not only the immune reaction towards the graft but also the response of the blood vessel wall in the grafted organ to injury ("response- to-injury" reaction) plays a role. The variant of chronic rejection with the worst prognosis is an arteriosclerosis-like alteration, also called transplant vasculopathy, graft vessel disease, graft atherosclerosis, transplant coronary disease, etc. This vascular lesion is characterized by migration and proliferation of smooth muscle cells under influence of growth factors that are amongst others synthesized by endothelium. A contribution of blood-borne stem cells as precursors of neo-intimal tissue has recently been demonstrated. It appears to progress also through repetitive endothelial injury induced amongst others by host antibody or antigen-antibody complexes, through intimal proliferation and thickening, smooth muscle cell hypertrophy repair, and finally to gradual luminal obliteration. Also so-called non-immunological factors like hypertension, hyperlipidemia, hypercholesterolemia etc. play a role.

Chronic rejection appears to be inexorable and uncontrollable because there is no known effective treatment or prevention modality. Thus, there continues to exist a need for a treatment effective in preventing, controlling or reversing manifestations of chronic graft vessel diseases.

The manzamines are useful in the treatment and/or prevention of diseases or disorders mediated by T lymphocytes and/or B lymphocytes, e.g. acute or chronic rejection of organ, tissue or cell allografts, atherosclerosis, vascular occlusion due to vascular injury such as angioplasty, restenosis, autoimmune diseases e.g. rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, Hashimoto's thyroidis, myasthenia gravis, diabetes type I and the disorders associated therewith, skin diseases such as psoriasis, atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis and further eczematous dermatitises, seborrhoeic dermatitis, Sjoegren's syndrome, keratoconjunctivitis, uveitis or myocarditis and multiple sclerosis (similar neuroinfammatory conditions).

In accordance with the particular findings of the present invention, there is provided:

1.1 A method for preventing, treating or inhibiting acute graft rejection in a recipient of cell, tissue or organ allotransplant, comprising the step of administering to said recipient a therapeutically effective amount of a manzamine;

1.2. A method of preventing, treating or inhibiting chronic rejection, e.g. to avoid, reduce or restrict chronic rejection, in a recipient of tissue or organ allotransplant, comprising the step of administering to said recipient a therapeutically effective amount of a manzamine;

1.3. A method of preventing, treating or inhibiting graft vessel diseases, e.g. transplant vasculopathy, arteriosclerosis or atherosclerosis, in a recipient of tissue or organ allotransplant, comprising the step of administering to said recipient a therapeutically effective amount of a manzamine;

1.4 A method of preventing, treating or inhibiting vein graft stenosis, restenosis and/or vascular occlusion following vascular injury, e.g. caused by catherization procedures or vascular scraping procedures such as percutaneous transluminal angioplasty, in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a manzamine;

1.5 A method for preventing, treating or inhibiting an autoimmune disease in a subject in need thereof, comprising the step of administering to said subject a therapeutically effective amount of a manzamine;

2. 1 Use of a manzamine for preventing, treating or inhibiting (a) acute or chronic graft rejection in a recipient of cell, tissue or organ allotransplant, (b) vein graft stenosis, restenosis and/or vascular occlusion following vascular injury or (c) an autoimmune disease;

2. 2 Use of a manzamine for the preparation of a medicament for preventing, treating or inhibiting (a) acute or chronic graft rejection in a recipient of cell, tissue or organ allotransplant, (b) vein graft stenosis, restenosis and/or vascular occlusion following vascular injury or (c) an autoimmune disease.

3. A pharmaceutical composition for use in any method as defined under 1.1 to 1.5 above comprising a manzamine, together with one or more pharmaceutically acceptable diluents or carriers thereof.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Figure 1 shows the effect of Manzamine A in the inhibition of knee swelling in the antigen- induced arthritis in mice as described in the Examples.

Filled square: Vehicle (saline) alone 5 ml/kg Empty square: Dexamethasone 1 mg/kg Filled triangle: Manzamine A 15 mg/kg Empty triangle: Manzamine A 30 mg/kg EXAMPLES

Utility of a manzamine as defined herein, e.g. Manzamine A, in treating diseases or disorders as hereinbefore specified, may be demonstrated in animal test methods as well as in Clinic, for example in accordance with the methods hereinafter described.

A. In Vitro

Allogeneic Mixed Lymphocyte Reaction (MLR)

The two-way MLR is performed according to standard procedures (J. Immunol. Methods, 1973, 2, 279 and Meo T. et al., Immunological Methods, New York, Academic Press, 1979, 227-39). Briefly, spleen cells from CBA and BALB/c mice (1.6 x 10⁵ cells from each strain per well in flat bottom tissue culture microtiter plates, 3.2 x 10⁵ in total) are incubated in RPMI medium containing 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μM 2-mercaptoethanol, non-essential amino- acids, sodium pyruvate (2 mM), sodium selenite (22 ng/ml), ethanolamine (20 μM), bovine insulin (5 μg/ml), human transferring (32 μg/ml), human albumin 0.1%, lecithine 0.0024%, L-gluta-mine (2 mM) and sodium bicarbonate (23.8 mM) and serially diluted compounds. Seven three-fold dilution steps in duplicates per test compound are performed. After four days of incubation 1 μCi ³H-thymidine is added. Cells are harvested after an additional five-hour incubation period, and incorporated ³H-thymidine is determined according to standard procedures. Background values (low control) of the MLR are the proliferation of BALB/c cells alone. Low controls are subtracted from all values. High controls without any sample are taken as 100% proliferation. Percent inhibition by the samples is calculated, and the concentrations required for 50% inhibition (IC₅₀ values) are determined. In this assay, Manzamine A has an IC₅₀ of less than 5 nM.

B. In Vivo

Systemic graft - versus Host reaction in mice

This assay measures in vivo alloreactivity of donor T-cells transferred into an allo-mismatched recipient chosen to be incapable of mounting a host-versus- graft response, i.e. an immunodeficient recipient. The model is performed according to the published method ["Effect of immunosuppressants on T-cell subsets observed in vivo using carboxy-fluorescein diacetate succinimidyl ester labeling" Hu H, Dong Y, Feng P, Fechner J, Hamawy M and Knechtle SJ. Transplantation. 2003 75, 1075-7]. In the current model, lymphocytes from wild type B6 mice are transferred to fully-allo-mismatched CB17 SCID/beige (BALB/C congenic) mice. Donor alloreactivity is reflected by T cell expansion ( numbers of T cells retrieved from recipient spleens) and T cell proliferation. To visualize T cell proliferation, donor spleen cells are stained with the fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) prior to transfer into recipients. This dye binds covalently and non-specifically to cellular proteins. As cells divide, daughter cells 'inherit' half of the stained protein, thereby displaying half of the parental cell's fluorescence intensity. This allows quantifying the number of cell divisions and cells per cycle by flow cytometry. Four days after transfer of CFSE-stained B6 splenocytes, CB17 SCID/beige recipients are sacrificed, and their spleens processed for FACS analysis.

Cyclosporine A is dissolved in 20%KZI vehicle 80% PBS. Manzamine A is dissolved in corn oil 1% ethanol. The results are as follows:

Antigen-induced arthritis in mice

Mice were sensitised intradermal^ on the back at two sites to methylated bovine serum albumin (mBSA) homogenised 1 :1 with complete Freund's adjuvant on days -21 and -14 (0.1 ml containing 1 mg/ml mBSA). On day 0, the mice were anaesthetised using a 5% isoflurane/air mixture and maintained using isoflurane in a face mask for the intra-articular. The right knee received 10μl of 10mg/ml mBSA in 5% glucose solution (antigen injected knee), while the left knee received 10μl of 5% glucose solution alone (vehicle injected knee). The diameters of the left and right knees were then measured using calipsers immediately after the intra-articular injections and again on days 2, 4, 7, 9, 11 and 14.

Treatments were administered daily as follows by oral gavage; vehicle (saline) at 5ml/kg, Dexamethasone at 1 mg/kg and Manzamine A at 15 and 30 mg/kg.

Right knee swelling was calculated as a ratio of left knee swelling, and the R/L knee swelling ratio plotted against time to give Area Under the Curve (AUC) graphs for control and treatment groups. The percentage inhibition of the individual treatment group AUCs were calculated vs the control group AUC (0% inhibition) using an Excel spreadsheet. On day 14, the mice were killed by CO₂ inhalation and the right and left knees removed for histology.

The knees were processed for decalcified histology using a Histodur plastic embedding method (Leica AG, Germany). Sections (5μm) from both the control and arthritic knees were cut on a RM 2165 rotation microtome (Leica AG, Germany). After staining adjacent sections with toluidine blue (metachromatic stain for cartilage proteoglycans) and Haemotoxylin and Eosin (general morphology stain), the slides were number coded as left knee/right knee pairs from each animal. The slides were read in a blinded fashion. The left knee joint from each animal was examined for comparison, immediately prior to scoring the arthritic right knee joint from the same animal, to establish the anatomy of the non-arthritic knee in each case. A subjective scoring system was used to assess inflammatory cell infiltrate/hyperplasia, cartilage proteoglycan loss and erosive damage to bone and cartilage, where 1 = the minimum score and 5 = the maximum score for each parameter compared to the control sections (left knee) in each case. The results are shown in figure 1.

Dosages required in practicing the method of the present invention using a manzamine will of course vary depending on the mode of administration, the particular condition to be treated and the effect desired. In general, satisfactory results are indicated to be obtained systemically at daily dosages of from about 0.01 to about 200 mg/kg body weight. An indicated daily dosage in the larger mammal, e.g. humans, is in the range from about 0.5 mg to about 100 g p.o., conveniently administered, for example, in divided doses up to four times a day or in retard form.

The manzamines may be administered by any conventional route, in particular enterally, e.g. orally, e.g. in the form of tablets or capsules, or parenterally, e.g. in the form of injectable solutions or suspensions, topically, e.g. in the form of lotions, gels, ointments or creams, or in a nasal or a suppository form. Pharmaceutical compositions comprising a manzamine in free form or in pharmaceutically acceptable salt form in association with at least one pharmaceutical acceptable carrier or diluent may be manufactured in conventional manner by mixing with a pharmaceutically acceptable carrier.

The manzamines to be used in the present invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations are described in detail in a number of sources that are well known and readily available to those skilled in the art. For example, Remington's Phatπiaceutical Scien∞ by E.W. Martin describes formulations that can be used in connection with the subject invention. In general, the compositions of the subject invention will be formulated such that an effective amount of the bioactive compound(s) is combined with a suitable carrier in order to facilitate effective administration of the composition.

The manzamine may be administered as the sole active ingredient or together with other drugs in immunomodulating regimens or other anti-inflammatory agents e.g. for the treatment or prevention of allograft acute or chronic rejection or autoimmune disorders or malignant proliferative diseases. For example, a manzamine may be used in combination with a calcineurin inhibitor, e.g. cyclosporine A, cyclosporine G, FK-506, ABT-281 , ASM 981; an mTOR inhibitor, e.g. rapamycin, 40-O-(2-hydroxy)ethyl-rapamycin, CCI779, ABT578, AP23573, AP23464, AP23675, AP23841, TAFA-93, biolimus-7 or bioimus-9; a corticosteroid; cyclophosphamide; azathioprine; methotrexate; a S1P receptor agonist, e.g. FTY 720 or an analogue thereof; leflunomide or analogs thereof; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualine or analogs thereof; immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CDS, CD4, CD11a/CD18, CD7, CD25, CD27, B7, CD40, CD45, CD58, CD137, ICOS, CD150 (SLAM), OX40, 4-1BB or their ligands, e.g. CD154; or other immunomodulatory compounds, e.g. a recombinant binding molecule having at least a portion of the extracellular domain of CTLA4 or a mutant thereof, e.g. an at least extracellular portion of CTLA4 or a mutant thereof joined to a non-CTLA4 protein sequence, e.g. CTLA4lg (for ex. designated ATCC 68629) or a mutant thereof, e.g. LEΞA29Y, or other adhesion molecule inhibitors, e.g. mAbs or low molecular weight inhibitors including LFA-1 antagonists, Selectin antagonists and VLA-4 antagonists.

When a manzamine is administered in conjunction with other drugs, dosages of the co¬ administered drug will of course vary depending on the type of co-drug employed, on the condition to be treated, and so forth.

In accordance with the foregoing the present invention provides in a yet further:

4. A pharmaceutical combination for use in any method as defined under 1.1 to 1.5 above comprising a) a first agent which is a manzamine, e.g. Manzamine A, and b) a co-agent, e.g. a second drug agent as defined above, in particular a drug used in immunomodulating regimens or an anti- inflammatory agent.

5. A method as defined above comprising co-administration, e.g. concomitantly or in sequence, of a therapeutically effective amount of a manzamine, e.g. manzamine A, and at least one second drug substance, e.g. as indicated above. The terms "co-administration" or "combined administration" or the like as utilized herein are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.

The term "pharmaceutical combination" as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients, e.g. a compound of formula I and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, e.g. a compound of formula I and a co-agent, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the 2 compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of 3 or more active ingredients.

Claims

1. A method for preventing, treating or inhibiting (a) acute or chronic graft rejection in a recipient of cell, tissue or organ allotransplant, (b) vein graft stenosis, restenosis and/or vascular occlusion following vascular injury or (c) an autoimmune disease in a subject in need thereof, comprising the step of administering to said subject a therapeutically effective amount of a manzamine.

2. Use of a manzamine for preventing, treating or inhibiting (a) acute or chronic graft rejection in a recipient of cell, tissue or organ allotransplant, (b) vein graft stenosis, restenosis and/or vascular occlusion following vascular injury or (c) an autoimmune disease.

3. Use of a manzamine for the preparation of a medicament for preventing, treating or inhibiting (a) acute or chronic graft rejection in a recipient of cell, tissue or organ allotransplant, (b) vein graft stenosis, restenosis and/or vascular occlusion following vascular injury or (c) an autoimmune disease

4. A pharmaceutical composition for use in a method according to claim 1 comprising a manzamine, together with one or more pharmaceutically acceptable diluents or carriers therefore.

5. A pharmaceutical combination for use in a method according to claim 1 comprising a) a firstagent which is a manzamine, and b) a co-agent, in particular a drug used in immunomodulating regimens or an anti-inflammatory agent.

6. A method, use, a pharmaceutical composition or a pharmaceutical combination according to any of claims 1 to 5, wherein the manzamine is a manzamine of formula (I) to (VIII) or manzamine A, B₁ C₁ D, E or F, as hereinbefore defined and described.

7. A method, use, a pharmaceutical composition or a pharmaceutical combination according to any of claims 1 to 5, wherein the manzamine is manzamine A.