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  • C07J—STEROIDS
  • C07J13/00—Normal steroids containing carbon, hydrogen, halogen or oxygen having a carbon-to-carbon double bond from or to position 17
  • C07J13/007—Normal steroids containing carbon, hydrogen, halogen or oxygen having a carbon-to-carbon double bond from or to position 17 with double bond in position 17 (20)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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• • A—HUMAN NECESSITIES
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  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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• • A—HUMAN NECESSITIES
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  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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  • C07J—STEROIDS
  • C07J31/00—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring
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  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J31/00—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring
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  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
  • C07J41/0005—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring the nitrogen atom being directly linked to the cyclopenta(a)hydro phenanthrene skeleton
  • C07J41/0027—Azides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
  • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
  • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
  • C07J41/0061—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives one of the carbon atoms being part of an amide group

Definitions

• the present invention relates to a new pharmaceutical use of fusidic acid and derivatives thereof, in particular the use for prevention and/or treatment of diseases, the pathogenesis of which is related to the production and/or function of certain immunoinflammatory mediators, especially cytokines.
• the invention relates to the use of fusidic acid and derivatives thereof and to the prevention and/or treatment of diabetes mellitus (type 1) (insulin-dependent diabetes mellitus (IDDM)), thyroid diseases, rheumatoid diseases, inflammatory diseases of the gut, skin diseases, conditions related to transplant rejection, endogenous uveitis and conditions related to opthalmological interventions.
• diabetes mellitus type 1
• IDDM insulin-dependent diabetes mellitus
• autoimmune diseases develop as as result of abnormalities in the immune system, especially in T lymphocyte-mediated immunity.
• Many signs and symptoms of infectious, inflammatory and neoplastic diseases evolve as a result of stimulation of cellular immunity.
• immunocompetent cells may not necessarily be involved at the initial stage, abnormal regulation of otherwise appropriate cellular immune reactions may lead to acute and chronic diseases.
• diseases are generally of unknown etiology and include systemic rheumatic diseases (e.g. rheumatoid arthritis (RA)), organ-specific endocrine diseases (e.g. insulin-dependent diabetes mellitus (IDDM)) and inflammatory diseases of the gut (e.g. Crohn's disease) and skin (e.g.
• disorders of cell-mediated immunity contribute to many other immunoinflammatory and proliferative diseases as will be described in further detail in the following.
• the treatments available in relation to said diseases are usually symptomatic or palliative treatments, i.e. most of the drugs prescribed in connection with said diseases are directed at the allaying of symptoms and usually have no curative effect.
• Other treatments are so-called substitution therapies which involve life-long supplying to the patient of substances, e.g. hormones, needed due to a reduced/insufficient internal production of said substance. Said treatments are often unsatisfactory and imply unwanted and often serious side-effects.
• improved methods of treatments and improved pharmaceutical compositions are needed.
• Fusidic acid and certain derivatives thereof can be isolated from the fermentation broth of certain strains of Fusidium coccineum and have for a number of years been known as efficient and pharmaceutically acceptable antibiotics.
• fusidic acid inhibits bacterial protein synthesis and it may be bacteriostatic and/or bactericidal. It is especially active against Staphylococcus aureus, including strains that are resistant to the penicillins or to other antibiotics.
• the drug is used clinically both in its acid form, and as the sodium and di- ethanolamine salts.
• the sodium salt of fusidic acid has the actions and uses of fusidic acid. It is more soluble in water, and is readily absorbed from the gastro-intestinal tract. It is also used in topical preparations. For severe staphylococcal infections the antibiotic may be given intravenously as e.g. diethanolamine fusidate.
• fusidic acid A description of chemical and microbiological modifications of fusidic acid is given in Structure-Activity Relationship in Fusidic Acid-Type Antibiotics (45), and fusidic acid and derivatives thereof are described in Pharmacopoieas and in Martindale, The Extra Pharmacopoeia, 28th edition (1982).
• Fusidic acid and derivatives thereof are described in PCT application No. WO 86/03966, published examined Danish application No. 148390 (US 4,119,717), DK 96349 (GB 930786), DK 99802 (AT 252446), DK 101687 (US 3,334,014), DK 104399 (US 3230240), DK 105981 (US 3,230,240), DK 107349 (GB 999794), DK 116940 (US 3499012), DK 131348 (US 3,867,413), DK 142852 (US 4100276, US 4162259 and US 4315004), U.S. Patent Nos.
• fusidic acid and derivatives thereof can be used for the prevention and/or treatment of certain forms of inflammatory diseases or processes, epecially forms related to the immune and/or hormone system. It is contemplated (as described in detail in the following description of immunological mechanisms) that the action mechanism is via interference with the action of mediators of the immune system, in particular cytokines such as monokines and lymphokines, i.e. that fusidic acid interferes with/suppresses the action of certain cytokines and thus inhibits pathological processes leading to tissue damage.
• cytokines such as monokines and lymphokines
• the present invention relates to the use of fusidic acid or a derivative thereof for substantially inhibiting a biological effect related to a cytokine such as a lymphokine, inter leukin, monokine, interferon or colony-stimulating factor for the prophylaxis or treatment of a condition related to a disturbance of a cytokine system such as the lymphokine, interleukin, monokine, interferon or colony-stimulating factor system.
• the broadest aspect of the invention also relates to a method of treating in a human a condition related to a disturbance in a cytokine system which method comprises administering to the subject an effective amount of fusidic acid or a functional derivative thereof.
• the use according to the present invention is not based on the use of fusidic acid or functional derivatives thereof as antibiotics.
• vasculitis e.g. polyarteritis nodosa, Wegener's granulomatosis, giant-cell arteritis, fever, malaise, anorexia (e.g. in acute and chronic inflammatory and infectious diseases)
• Cachexia e.g. in cancer, chronic infectious and chronic inflammatory diseases
• T lymphocytes govern the induction and regulation of cell-mediated Immune reactions, and proteins and glycoproteins produced by T lymphocytes (lymphokines) initiate and control the immune response (1,2).
• lymphocytes proteins and glycoproteins produced by T lymphocytes (lymphokines) initiate and control the immune response (1,2).
• antigen activation of T cells requires physical interaction with antigen-presenting cells such as macrophages (M ⁇ ), and these cells also produce mediator molecules crucial for T cell activation and the subsequent triggering of B lymphocytes to become antibody-producing plasma cells.
• M ⁇ macrophages
• mediators are also responsible for the recruitment and activation of many other cell types which build up inflammatory infiltrates in the diseased tissues (1-4).
• cytokines are essential transmitters of cell-to-cell communication in both physiological and pathophysiological infectious, Immunoinflammatory, and growth processes. In many cases they also function as hormones providing information between the immune system and other tissues and organs. Furthermore, many cytokines are produced by cells outside the Immune system (e.g. in the skin (5) and in blood vessels), and cytokines may therefore act as autocrine or paracrine hormones without necessarily involving immunocompetent cells.
• Cytokines are active at extremely low concentrations (10 -10 to 10 - 15 M) via binding to specific receptors on a large number of target cells. Most cytokines probably act in the vicinity of the production site but, as mentioned above, some of the mediators modulate functions of cells at distant sites via blood and lymph circulation.
• Fig. 1 shows the dynamics of production and function of cytokines during an immune reaction in a vascularized tissue. Macrophages
• Macrophages play a central role in the defence against microbial and neoplastic diseases, and they are important in a wide variety of tissue repair processes (2).
• M ⁇ functions are performed continuously, such as removal of aged erythrocytes. Other functions are performed periodically, and these functions need prior activation of the M ⁇ .
• the cells may be activated directly, for instance by contact with microorganisms or their products, such as endotoxins (lipopolysaccharides), muramyl peptides, and other cell wall components.
• endotoxins lipopolysaccharides
• muramyl peptides and other cell wall components.
• biologically active molecules including prostaglandins, proteolytic enzymes, complement components, etc.
• IL-1 ⁇ interleukin 1 ⁇
• IL-6 interleukin 6
• TNF ⁇ tumour necrosis factor- ⁇
• M ⁇ cytokines The molecular biology of the best characterized human M ⁇ hormones has recently been extensively reviewed (2,4,6-8). They are all members of a growing family of cytokines which includes the interferons and some hemopoietic growth factors. The M ⁇ cytokines are also produced during conditions of "stress", and they are important mediators of fever, the acute-phase reaction, and the generation of cachexia as a result of chronic infectious, inflammatory, autoimmune, and neoplastic diseases.
• TL-1 ⁇ / ⁇ , IL-6 and TNF ⁇ are produced primarily by activated M0, the capacity to elaborate these molecules, particularly TL-1 ⁇ / ⁇ and IL-6, is not confined to M ⁇ (2).
• Natural killer cells (NK cells) and B lymphocytes are already known to produce TL-1 ⁇ / ⁇ , and fibroblasts were the cells originally shown to produce IL-6
• the mature mediator molecules are single-chain polypeptides consisting of 169 (IL-1 ⁇ ), 153 (IL-1 ⁇ ), 184 (IL-6), and 157
• TNF ⁇ Native human IL-6 is glycosylated, and the resuiting molecular weight of this cytokine species range between 17 and 28 kD.
• TNF ⁇ and TNF/3 are both located in the class III region of the major histocompatibility complex, i.e. the LA region in man.
• the HLA-DR2 type is positively associated with diseases such as multiple sclerosis and narcolepsy and negatively associated with rheumatoid arthritis and IDDM.
• IL-1 ⁇ and IL-1 ⁇ are thought to bind to the same receptor on T and B lymphocytes, and both peptides are capable of down-regulating the expression of the CD4 structure on human T cells (10).
• the CD4 structure Is directly engaged in antigen presentation, possibly through interaction with MHC class II molecules on antigen-presenting cells (Fig. 1). It is there fore possible that the T lymphocyte IL-1 receptor binds to the major membrane-associated IL-1 species (IL-1 ⁇ ) on the antigen-presenting cell, and that this receptor is physically or functionally related to the CD4 molecule (Fig. 1 and ref. 2).
• IL-6 has been shown to be identical with B cell-stimulating factor 2 and a factor necessary for the growth of plasmacytomas and B cell hybridomas (2, 7). IL-6 is also a cofactor in T cell activation, probably because it induces IL-2 receptors and functions as a second signal for IL-2 production (2). Modulation of immune reactions and tolerance induction
• T cell activation may lead to antigenspecific tolerance.
• triggering the T cell receptor by a properly processed antigen while at the same time preventing the ensuing IL-1-induced activation of these specifically reactive cells, prevents the selected clone(s) from responding to the same antigen at a later time, both in vitro (11) and in vivo (12).
• any treatment that blocks the production or the immunological functions of these mediators would be expected to suppress the entire cellular immune system. This may be especially beneficial in the management of patients receiving transplants.
• Fever is often part of the acute-phase reaction usually seen in conjunction with acute and chronic infectious and immunoinflammatory diseases, and in cancer (2-4, 8).
• TL-1 ⁇ / ⁇ and IL6 and to a lesser extent TNF ⁇ , induce hepatocytes to synthesize acute-phase proteins, including serum amyloid A, C-reactive protein, fibrinogen, haptoglobin, complement components and clotting factors.
• TL-1 ⁇ / ⁇ and TNF ⁇ are potent inducers of IL-6 in many cell types, including hepatocytes, it is possible that IL-6 is a second mediator of the acute-phase response elicited by TL-1 ⁇ / ⁇ and TNF ⁇ .
• Cachexia disseminated intravascular coagulation (DIC) and shock
• DIC intravascular coagulation
• shock The above mentioned clinical picture is often associated with disturbances in carbohydrate-, lipid- and protein metabolism eventually resulting in wasting (cachexia). In rare situations, clotting abnormalities and shock may occur (6).
• LPS lipopolysaccharide
• TNF ⁇ is probably also responsible for other phenomena associated with LPS-induced disease, such as metabolic acidosis, DIC and cachexia (2,6). It is likely that some of these phenomena are mediated through TNF ⁇ -induced production of TL-1 ⁇ / ⁇ or IL-6, or one or several other (unknown) hormones.
• TNF ⁇ is a potent inducer of TL-1 ⁇ / ⁇ in endothelial cells, and all three cytokines trigger the release of procoagulant factor(s) from these cells. It is therefore likely that both mediators may be involved in the pathogenesis of some vascular diseases with or without accompanying clotting abnormalities.
• Neoplastic diseases Unregulated production of IL-6 may be pathogenetically involved in the manifestation of several human diseases, including cardiac myxoma and various lymphoid malignancies such as multiple myelomas and various T and B cell lymphomas (6). It is interesting, that myeloma cells constitutively produce IL-6 and express IL-6 receptors, and that the proliferation of myeloma cells in vitro is inhibited by specific antibodies to human IL-6 (13).
• IL-6 and, possibly, TNF ⁇ may also be involved in other proliferative disorders.
• TNF ⁇ and IL-6 in skin biopsies (5, 5A).
• the expression of both mediators was considerably augmented by UV- irradiation, and relatively large amounts of IL-6 and TNF ⁇ were found in psoriatic lesions.
• local treatment of the psoriatic lesions with a vitamin D3 analogue resulted in clinical improvement, and biopsies performed after treatment showed unaltered expression of TNF ⁇ , but an almost complete elimination of the expression of IL-6 in the upper epidermal layers. It is therefore possible that IL-6 may be involved in the uncontrolled proliferation of keratinocytes seen in psoriasis.
• TL-1 ⁇ / ⁇ , IL-6 and TNF ⁇ have many biological effects which qualify them as important in the pathology leading to rheumatic diseases (2,8).
• the most relevant actions of IL-1 ⁇ and - ⁇ are most likely their stimulatory effects on T and B cells (nonspecific production of inflammatory lymphokines and immunoglobulins), M0 (e.g. MHC class II antigen expression and production of eicosanoids), chondrocytes (production of collagen type II), fibroblasts (production of collagen types I and II and eicosanoids), and osteoclasts (bone resorption).
• TNF ⁇ and IL-6 share many of these effects with IL-1, although IL-6 does not cause bone resorption (Pedersen JG & Bendtzen K, unpublished).
• IL-6 does not cause bone resorption (Pedersen JG & Bendtzen K, unpublished).
• Several cellular sources of the cytokines are present in the arthritic joint, including M0, B lymphocytes, fibroblasts and vascular endothelial cells (2,8).
• M0 M0, B lymphocytes, fibroblasts and vascular endothelial cells (2,8).
• IL-1 ⁇ / ⁇ in synovial fluids from rheumatoid and osteoarthritic joints further suggests that IL-1 in particular may play an important role in the pathogenesis of arthritis and, possibly, in osteoarthrosis (2, 14).
• An unbalanced reaction between immunocompetent cells and accessory cells such as M ⁇ , NK cells and polymorphonuclear leukocytes might continually induce TL-1 ⁇ / ⁇ , IL-6 and other inflammatory molecules, for example prostaglandins and leukotrienes. This might contribute to the chronic nature of diseases such as RA.
• TL-1 ⁇ The arthritogenic activity of TL-1 ⁇ is most clearly seen from in vivo experiments using a rabbit arthritis model (15) .
• Human rIL-1 ⁇ induces M0 and neutrophil accumulation 24 h after injection in the knee, and this is associated with depletion of proteoglycan from the articular cartilage and an increase in the glycosaminoglycan content of the joint fluid.
• the same signs of cartilage damage by TL-l ⁇ are seen in joints of rabbits previously depleted of polymorphonuclear leukocytes and M0 by systemic administration of nitrogen mustard. This suggests that TL-1 ⁇ itself is capable of stimulating resident cells of the joint, such as the chondrocytes, to cause proteoglycan depletion.
• TL-1 ⁇ may be mediated via local, TL-l ⁇ -induced production of other cytokines, particularly IL-6 and IL-1 ⁇ .
• IL-1 may contribute to the manifestations of gout.
• IDM Insulin-dependent diabetes mellitus
• IL-1 ⁇ insulin-dependent diabetes mellitus
• IDM insulin-dependent diabetes mellitus
• IL-1 ⁇ the predominant species of human IL-1, IL-1 ⁇ , was shown to be a potent suppressor of insulin production in vitro , possibly as a result of a direct and selective cytotoxic effect on pancreatic islet ⁇ -cells (17).
• IL-1 ⁇ also reduces insulin production, but it is 10 times less potent than IL-1 ⁇ .
• TNF ⁇ itself fails to affect ⁇ -cell function
• the suppressive effect of IL-1 ⁇ is augmented significantly by TNF ⁇ (3,8).
• IL-6 augments the release of insulin from normal rat islets and that IL-1 ⁇ and IL-1 ⁇ induce IL-6 in the islets themselves (17A).
• this cytokine causes structural changes in ⁇ -cells similar to those caused by IL-1 (17A) .
• local IL-1-induced production of IL-6 may underly the biphasic insulin response observed with various concentrations of IL-1 (17).
• IL-6, in conjunction with IL-1 may contribute to the immunoinflammatory processes eventually leading to IDDM.
• TL-1 ⁇ is a very potent inducer of IL-6 in thyrocytes (17A).
• cytokines Prolonged exposure of cells to increased levels of these cytokines might lead to structural or functional damage to the cells (e.g., in IDDM), for instance if the target cells for unknown reasons are surrounded by cytokine-producing M ⁇ , NK cells and T and B lymphocytes, as is the case In the early stage of IDDM development. Endothelial cells, fibroblasts and other cells in the connective tissue may also contribute in these situations through an inappropriate production of cytokines such as TL-1 ⁇ / ⁇ and IL-6.
• TL-1 ⁇ / ⁇ and IL-6 may accumulate in target tissues by diffusion from the blood during conditions of stress, and the cytokines may therefore have important physiological functions by potentiating the secretion of insulin, thyroid- and, possibly, pituitary- and adrenocortical hormones under these circumstances (Fig. 1).
• HLA-DR2 As described above, the negative association between HLA-DR2 and the production of TNF ⁇ may be implicated in some HIA-associated diseases such as IDDM and RA (3,8). In the latter two, HIA-DR2 is associated with resistance to disease, and this might be explained if TNF ⁇ is involved, directly or indirectly, in the destruction of islet ⁇ -cells in IDDM and of cartilage and bone in RA and osteoarthrosis.
• IL- 1 ⁇ / ⁇ , IL-6 and TNF ⁇ intervention with the production or action of these hormones might be of great benefit in a number of infectious and immunoinflammatory diseases and, possibly, in certain neoplastic diseases.
• the production of the mediators is prevented by high doses of glucocorticoids, which directly suppress gene transcription and, If the genes have been transcribed, the mobilization of mRNA, at least in the case of TL-1 ⁇ / ⁇ and TNF ⁇ (6,8).
• Fusidic acid is a tetracyclic triterpenoic acid with a steroid-like primary structure as described above.
• the drug is used clinically both in its acid form and as the sodium and diethanolamine salts as an antibiotic particularly active against Staphylococcus aureus (22). It has been indicated that fusidic acid might have an inhibitory effect on T-lymphocytes in a mouse model (23A). Fusidic acid inhibits in vivo and in vitro protein synthesis in both prokaryotic and eukaryotic cells by inhibiting the attachment of transfer RNA to the 50S part of the ribosomes (23). Fusidin has few and only trivial side-effects, even during long-term treatment.
• fusidin acid act as an immunosuppressive agent and that its mode of action as an immunosuppressive agent closely mimicks that of cyclosporin (see the following).
• fusidin therapy should be investigated in all situations where cyclosporin treatment is advocated.
• cytokines polypeptide hormones
• M ⁇ polypeptide hormones
• IL-1 ⁇ and IL-1 ⁇ polypeptide hormones
• TNF ⁇ polypeptide hormones
• IL-6 polypeptide hormones
• IL- 1 ⁇ / ⁇ ac tivates cells to cell growth (e.g. fibroblasts, keratinocytes and glial cells) and/or increased activity (e.g.
• IL-1 appears to inhibit cell function.
• IL-1 is a potent suppressor of pancreatic islet ⁇ -cells and thyrocytes, and of melanoma cells
• IL-1 also induces a broad spectrum of systemic changes, including fever, slow wave sleep, and changes in blood levels of trace metals, albumin and acute-phase reactants such as fibrinogen and serum amyloid A (2,8,25).
• TL-la/ ⁇ are therefore considered of central importance in many if not all adaptive changes of the organism in response to various exogenous and, possibly, endogenous stimuli.
• IL-1 ⁇ / ⁇ have been recognized to share many activities with another cytokine, IL-6 (3,7). Despite this, IL-6 and TL-1 ⁇ / ⁇ do not show any sequence homology, and the cytokines act on different receptors.
• IL-6 has been shown to be identical with B cell-stimulating factor 2 (7) and a factor necessary for the growth of plasmacytomas and B cell hybridomas (26-28).
• Unregulated production of TL-1 ⁇ / ⁇ and IL-6 may be pathogenetically involved in the manifestation of several human diseases as described above, and the molecules have many biological effects which qualify them as important in the pathology leading to rheumatic diseases, some endocrine disorders, and various skin diseases (see Table 1).
• a dysregulated production and/or function of IL-6 may be a key event in the pathogenesis of at least some lymphoid proliferative diseases.
• Drugs that interfere with the function of the cytokine such as fusidic acid or derivatives thereof are therefore contemplated to be clinically useful. It is also contemplated that interference with the functions of TL-1 ⁇ / ⁇ and IL-6 with fusidin or fusidic acid derivatives may be of therapeutic importance in several types of arthritic and degenerative joint diseases.
• fusidic acid and derivatives thereof are useful for preventing effects of cytokines known to be pathogenetically involved in the previous described pathological conditions.
• the term "fusidic acid or a derivative thereof” comprises any pharmaceutically active and acceptable compound being identical or structurally similar to fusidic acid and exhibiting relevant biological actions similar to those of fusidic acid, including derivatives of fusidic acid, especially pharmaceutically acceptable salts, esters and solvates as well as conjugates of fusidic acid or of the fusidic acid
• Suitable salts are salts with pharmaceutically acceptable bases, such as alkaline earth metal salts, alkali metal salts (especially sodium salts), ammonium salts or amine salts (especially the diethanolamine salts).
• Suitable esters are easily hydrolysable esters, e.g. fusidic acid acetoxymethyl ester.
• Suitable solvates are e.g. hydrates, in particular hemihydrates.
• Suitable conjugates are especially conjugates with taurine or glycine.
• physiologically acceptable derivatives including functional derivatives, which are disclosed, as well as their preparation, in the patents and the references listed above In the section "Background of the invention", e.g. the derivatives disclosed in U.S. Patent No. 4,004,004. Some of these derivatives are Insufficient as antibacterial agents - a feature which may be beneficial in the use according to the present invention.
• A is selected from the group consisting of OH, OR', and NHR", wherein R' is selected from the group consisting of
• aralkyl such as phenyl-C 1 -C 4 -alkyl
• aryl such as phenyl or o- , m- or p-tolyl, and R" is CH 2 COOH or CH 2 CH 2 SO 3 H,
• R 1 is hydrogen, halogen or a nitrile group, or R 1 is selected from the group consisting of
• R 3 is hydrogen or is an organic group such as acyl, in particular C 1 -C 4 - alkylcarbonyl, alkyl, in particular C 1 -C 4 -alkyl, aralkyl, such as phenyl-C 1 -C 4 - alkyl, or aryl, such as phenyl or o-, m- or p-tolyl,
• R 2 is hydrogen or halogen or a nitrile group or a group
• R 3 Is hydrogen or is an organic group such as acyl, in particular C 1 -C 4 - alkylcarbonyl, alkyl, in particular C 1 -C 4 -alkyl, aralkyl, such as phenyl-C 1 -C 4 -alkyl, or aryl, such as phenyl or o-, m- or p-tolyl, and salts thereof.
• C 1 -C 4 -alkyl examples are as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sek.butyl, and tert. butyl.
• C 1 -C 4 -alkyl and the other groups mentioned above may optionally be substituted where appropriate, such as disclosed in the patents and other literature referred to in the above section "Background of the invention"
• preferred compounds of the general formula II are, e.g., compounds wherein A is OH, a double bond is present in the position C(17)-C(20) and in the position C(24)-C(25), the hydrogen atom at C(9) is in the ⁇
• R is a hydroxy group in the ⁇ configuration with respect to the C(19) methyl group
• R 1 is a C 1 -C 4 -alkylcarbonyloxy group such as an acetoxy group or a C 1 -C 4 -alkylcarbonylthio group, an arylcarbonylthio group such as a phenylcarbonylthio group, or a C 1 -C 4 -alkoxy group such as a methoxy or ethoxy group
• R 2 is a nitrile group, a C 1 -C 4 -alkylsulfonyloxy group, or, in particular, a hydroxy group
• fusidic acid and fusidic acid salts such as the sodium salt (fusidin).
• Cytokine is a general term for a proteinaceous mediator released primarily but not exclusively by a cell population of the immune system as a response to a specific stimulating agent, e.g. a specific antigen or an alloantigen; or a nonspecific, polyclonal activator, e.g. an endotoxin or other cell wall components of a gram-negative bacteria;
• a specific stimulating agent e.g. a specific antigen or an alloantigen
• a nonspecific, polyclonal activator e.g. an endotoxin or other cell wall components of a gram-negative bacteria
• lymphokine is a general term for a proteinaceous mediator released by sensitized lymphocytes as a response to a stimulating agent, e.g. a specific antigen or an alloantigen; or by a lymphocyte challenged by a polyclonal activator, e.g. an endotoxin or other cell wall component of a gram-negative bacteria;
• a stimulating agent e.g. a specific antigen or an alloantigen
• a lymphocyte challenged by a polyclonal activator e.g. an endotoxin or other cell wall component of a gram-negative bacteria
• interleukin is a general term for a proteinaceous mediator released primarily but not exclusively by a macrophage, T, B or NK cell as a response to a stimulating agent, e.g. a specific antigen or an alloantigen or by a cell challenged by a polyclonal activator, e.g. an endotoxin or other cell wall component of a gram-negative bacteria;
• a stimulating agent e.g. a specific antigen or an alloantigen or by a cell challenged by a polyclonal activator, e.g. an endotoxin or other cell wall component of a gram-negative bacteria
• monocyte is a general term for a proteinaceous mediator released by a mononuclear phagocyte (e.g. a monocyte or a macrophage or a Kupffer cell (liver) or a Langerhans' cell (skin)) as a response to any stimulating agent;
• a mononuclear phagocyte e.g. a monocyte or a macrophage or a Kupffer cell (liver) or a Langerhans' cell (skin)
• interferon is a general term for a proteinaceous antiviral and/or monocyte/macrophage activating factor released by all cells as response to a virus or an interferon-inducer such as a
• polynucleotide in particular cells of the immune system as a response to a specific stimulating agent, e.g. a specific antigen or an alloantigen, or a nonspecific, polyclonal activator, e.g. an endotoxin or other cell wall components of a gram-negative bacteria;
• a specific stimulating agent e.g. a specific antigen or an alloantigen
• a nonspecific, polyclonal activator e.g. an endotoxin or other cell wall components of a gram-negative bacteria
• colony-stimulating factor is a general term for a proteinaceous, haematopoietic colony-stimulating mediator released primarily but not exclusively by a cell population of the immune system as a response to a specific stimulating agent, e.g. a specific antigen or an al loantigen; or a nonspecific, polyclonal activator, e.g. an endotoxin or other cell wall components of a gram-negative bacteria.
• a specific stimulating agent e.g. a specific antigen or an al loantigen
• a nonspecific, polyclonal activator e.g. an endotoxin or other cell wall components of a gram-negative bacteria.
• One aspect of the invention is the use of fusidic acid or a functional derivative thereof for the manufacture of a pharmaceutical composition for substantially inhibiting a biological effect in a human related to a cytokine, such as a lymphokine, interleukin, monokine, interferon or colony-stimulating factor, for the prophylaxis or treatment of a condition related to a disturbance of a cytokine system such as the lymphokine, interleukin, monokine, interferon or colony-stimulating factor system.
• a cytokine such as a lymphokine, interleukin, monokine, interferon or colony-stimulating factor
• pharmaceutical composition comprises any composition suitable for human use.
• a cytokine for the prophylaxis or treatment of a condition related to a disturbance of a cytokine system; and/or - for substantially inhibiting a biological effect in a human
• lymphokine for the prophylaxis or treatment of a condition related to a disturbance of a lymphokine system
• a monokine for the prophylaxis or treatment of a condition related to a disturbance of a monokine system, and/or - for substantially inhibiting a biological effect in a human
• substantially inhibiting comprises a therapeutically relevant prevention of the harmful biological actions of said mediator, said prevention being based on fusidic acid or derivatives thereof or combinations with other treatments/drugs exerting a form of antagonistic effect on the actions of the mediator in question.
• Said conditions have been described above, and the conditions described comprise diseases occuring in humans which diseases demand therapeutical intervention as well as conditions in which a suppression of a cytokine system is desirable such as in connection with transplantation or eye surgery.
• clini- cally important interleukins in this context are in particular interleukin 1 ( ⁇ or ⁇ ) , and interleukin 6.
• the invention further relates to the use of fusidic acid or its derivatives as a substantially selective immunosuppressive drug and/or as a drug the action of which is via an antagonistic effect on the action of one or more cytokines.
• the administration may be orally, rectally and/or parenterally, the administration being dependent on the patient's age and weight and the particular condition being treated as well as the severity of the disease.
• the active ingredient or combinations thereof may be contained in any appropriate amount in a composition, and are generally contained in an amount of 1-95% by weight, based on the total weight of the preparation.
• the composition may be in any appropriate unit dosage form.
• Parenteral administration may comprise suitable injection, e.g.
• Formulations for parenteral use may be presented in unit dose forms, e.g. ampoules, or in multi-dose containers with an added suitable preservative.
• the composition may be in form of a solution, a suspension, an emulsion or a delivery device for implantation or may be presented as a dry powder to be reconstituted with water or another suitable solvent before use.
• the compositions may comprise suitable pharmaceutically acceptable carriers and/or exipients.
• the composition may, in addition, conveniently comprise suspending, stabilizing, pH-adjusting agents and/or dispersing agents.
• compositions for oral or rectal use may be formulated according to conventional pharmaceutical practice and may be in the form of tablets, capsules, pills, powders, ampoules, granulates, dragees, pastes, gels, suppositories or enemas; or liquid formulations such as suspensions (oily or aqueous), solutions, elixirs, emulsions or drenches and the like.
• auxiliary additives of the pharmaceutical compositions may be any conventional pharmaceutical additives and carriers:
• Binding agents such as cellulose derivatives, starch, gelatin and polyvinylpyrrolidine; fillers such as sugar, mannitol, lactose, microcrystalline cellulose, potato starch, calcium phosphate; suitable absorption promotors; disintegrants such as potato starch, alginic acid; lubricants such as magnesium stearate, stearic acid, talc; emulsifying agents such as lecithin, sorbitan monooleate;
• wetting agents such as lecithin, polyoxyethylene esters; or buffering agents such as acetate, citrate, phosphate.
• the solid compositions may by means of specially adapted coating techniques be provided with a coating adapted to protect the composition from unwanted chemical changes prior to the release of the active compound.
• the coating may be adapted to release the active compound in a predetermined pattern e.g. in order to achieve a controlled release formulation.
• the dosage of fusidic acid or its derivatives depend on the administration method, the disease, the severity of the disease and of the weight and age of the patient.
• the dosage is often 3.5 mg-1 g administered 2-6 times daily or for instance 0.5 g x 5 daily or in certain cases 1 g every second or third day.
• oral administration may comprise ranges from approximately 1 mg to 75 mg per kg body weight per day or in appropriate cases 0.1 mg to 20 mg per kg body weight per day.
• a preferred dosage regime is normally 0.5 g 3 times daily. In certain cases, e.g. in the treatment of graft-versus-host disease the dosage is normally doubled.
• fusidic acid or a derivative thereof is administered rectally, a somewhat higher amount such as from about 1 mg to about 100 mg per kg body weight per day is usually preferred.
• a dosis of about 0.1 mg to about 50 mg per kg body weight per day, most preferably in an amount from about 1 mg to about 20 mg per kg body weight per day is convenient.
• an amount from about 0.1 mg to 20 mg per kg body weight per day is usually preferable.
• an aqueous solution of e.g. 0.5-2% or more of the active Ingredient may be employed.
• fusidic acid or a derivative thereof When fusidic acid or a derivative thereof is administered to the eye, an amount from about 0.1 mg to about 50 mg per kg body weight per day is usually preferred.
• the dosage should be carefully adapted so as to imply the specific action in the cytokine system, i.e. attainment of optimal total dosages, dosage forms and dosage frequency. In certain cases it is relevant to administer a relatively high unit dosis, a so-called "booster dosis".
• compositions suitable for topical use such as compositions suitable for application to the skin according to the present invention are suitably creams, gels, ointments, lotions, liniments, suspensions, solutions, pastes, sticks, sprays, soaps, shampoos, powders, suppositories and enemas.
• the topical administration should be onto or close to the pathological changes in the body.
• the compositions may be any suitable medicated mass adapted for direct application or for introduction into the relevant orifice of the body, e.g. the rectal, urethral or vaginal orifice.
• the compositions may simply be applied directly onto the diseased part, e.g. the skin or mucosa.
• Other relevant formulation adaptations for application to the eye may e.g.
• eye lotions eye ointments, eye-drops or drug delivery systems adapted for administration to the eye such as compositions suitable for implantation administration.
• compositions suitable for implantation administration may be applied by means of special medical devices such as dressings including occlusive dressings, or alternatively plasters, pads, sponges, strips, or other forms of suitable flexible pieces of material.
• Formulations for topical administration may comprise pharmaceutically acceptable carriers and/or exipients such as ointment bases (e.g. paraffin, polyethylene glycols, Tween ® , Span ® , vegetable oils), suspending or emulsifying agents (e.g. lecithin, sorbitan monooleate, gelatin, methyl cellulose, gum acacia, sorbitan monooleate derivatives), gelforming agents (e.g. Carbopol, alginates, gelatin), preservatives (e.g. methyl or propyl p-hydroxybenzoates, benzalkonium chloride), antioxidants (e.g. tocopherol, ascorbic acid, butylated hydroxy anisol), humectants (e.g. glycerin, propylene glycol, urea), and perfumes and skin protective agents.
• ointment bases e.g. paraffin, polyethylene glycols, Tween ® , Span
• Formulations for topical administration to the eye may comprise pharmaceutically inert vehicles or be in form of a suitable carrier system.
• Pharmaceutically acceptable carriers and/or exipients for the preparation of eyedrops include for example buffering agents such as boric acid or borates, pH adjusting agents to obtain optimal stability or solubility of the actuve drug substance, agents to adjust the tonicity such as sodium chloride or borates, viscosity altering agents such as hydroxypropyl cellulose, methylcellulose, polyvinyl- pyrrolidone, polyvinyl alcohols or polyacrylamide, oily vehicles such as vehicles comprising arachis oil, castor and mineral oils.
• buffering agents such as boric acid or borates
• agents to adjust the tonicity such as sodium chloride or borates
• viscosity altering agents such as hydroxypropyl cellulose, methylcellulose, polyvinyl- pyrrolidone, polyvin
• Eyedrops presented as emulsions or suspensions may furthermore comprise stabi lizing, dispersing, wetting, emulsifying and/or suspending agents.
• Eye lotions and eye ointments may comprise pharmaceutically acceptable carriers and/or exipients such as used in an eyedrop composition or in other relevant topical composition, e.g. ointments, creams, lotions and the like.
• An example of a general method for preparing aqueous eyedrops comprising an active drug substance is to dissolve the compound (preferably as the soluble sodium salt) in sterile water in a given concentration, optionally adjust the pH to a suitable value with a suitable buffer or with hydrochloric acid or sodium hydroxide, optionally add a preservative like phenethanol or chlorobutanol, optionally add a viscous altering excipient like methylcellulose, and sterilize the final solution by e.g. autoclavation by use of one of the generally accepted cycles or by membrane filtration.
• the topical compositions for use according to the present invention comprise e.g. 0.001-60% w/w of the active ingredient, preferably 0.1- 20%, especially 0.5-10% or 1-5%.
• compositions may be applied several times a day, e.g. 1-5 times a day, depending on the condition in question and the severity thereof and furthermore on the absorption conditions on the site in question.
• the particular dosis will depend on obtaining an appropriate absorption of the fusidic acid or derivative thereof in a sufficient form and amount.
• said amount and form should be adapted so as to exert its specific action in the cytokine system. Hence, it is very important - e.g. when application onto the skin is performed - to ensure that the patient applies a relatively welldefined layer of the composition.
• the invention relates to the use of fusidic acid or a derivative thereof for prophylaxis or treatment of diabetes mellitus, in particular insulin-dependent diabetes mellitus (type 1), in particular for the prevention of progression of diabetes mellitus (type 1), especially substantially immediately after the first diagnostic establishment of diabetes mellitus, or for prophylaxis after establishment of being in a high risk group of developing diabetes mellitus.
• diabetes mellitus in particular insulin-dependent diabetes mellitus (type 1)
• type 1 insulin-dependent diabetes mellitus
• prophylaxis after establishment of being in a high risk group of developing diabetes mellitus especially substantially immediately after the first diagnostic establishment of diabetes mellitus, or for prophylaxis after establishment of being in a high risk group of developing diabetes mellitus.
• cyclosporin a cyclic peptide metabolite of the fungi cylindrocarpon lucidum and trichoderma polysporum
• the remission rate was greater in diabetic patients who entered after less than six weeks with symptoms and two weeks of insulin therapy, than in those who entered later.
• the findings demonstrate that an acute process affecting the ⁇ -cells can be modulated by initiation of the cyclosporin immunosuppressive therapy within a critically brief time interval after onset of overt disease, and that continued treatment can maintain partial or complete remission through a period of at least one year.
• substantially immediately after comprises a period after the first diagnostic establishment of the disease or a high risk of developing the disease within a limited number of months or years which period should be as short as possible, so as to enable intervention with the progression of irreversible changes in the pathological body tissues, said invention being obtained by means of the use according to the invention of fusidic acid or derivatives thereof.
• cyclosporin increased the remission rate in diabetes mellitus (type 1) of recent onset, and enhanced and preserved ⁇ -cell function through the first year after diagnosis. It is inferred that these effects were induced by virtue of the immunosuppressive action of cyclosporin.
• the findings strongly support the hypothesis that an immune process is involved in ⁇ 3-cell damage in the human disease.
• the effect of duration of diabetes on the early outcome suggests that although the immune processes affecting ⁇ -cells may be chronic and precede diagnosis through a long period of time, the development of overt disease is associated with an acceleration of events.
• fusidic acid and derivatives thereof may be used according to the present invention for the prevention of progression of irreversible pathological processes in the pancreatic tissues which otherwise lead to diabetes mellitus (type 1).
• the therapy of the invention is preferably instituted as soon as possible, i.e. very shortly after the onset of diabetes or, preferably, immediately after diagnosis of pathological processes in the islets of pancreas (e.g.
• the therapy of the invention may also apply to certain identified high risk groups the identification being e.g. obtained by means of a screening program employing markers relatively specific for pathological changes seen in diabetes mellitus and/or preliminary stages thereof, especially markers of ongoing pathological changes in predisposed individuals (e.g. in HIA-DR 3/4-positive individuals).
• a person is contemplated to be in a high risk group of developing diabetes mellitus (type 1) if his risk Is about 10-100%, such as about 50-100%.
• the present invention relates to the use of fusidic acid or a derivative thereof for prophylaxis or treatment of diabetes mellitus (type 1) for oral or rectal use.
• the invention relates to the use of fusidic acid or a derivative thereof for the prophylaxis or treatment of
• thyroiditis acute, subacute or chronic
• thyroiditis chronic fibrous thyroiditis
• de Quervain's thyroiditis subacute granulomatous thyroiditis
• the inflammatory diseases of the gut, arthritis urica, multiple sclerosis, B and T cell lymphoma such as multiple myeloma treatment with fusidic acid or derivatives thereof can be beneficial to the patient also in a period of inapparant disease, in the way that the treatment with fusidic acid or derivatives thereof can prevent or postpone a relapse of the condition in question.
• treatment is therefore generally also meant prophylactic treatment designated to prevent or postpone a relapse of the disease.
• the invention relates to the use of fusidic acid or a derivative thereof for the prophylaxis or treatment of a condition related to hypofunction of the adrenal glands, in
• the invention relates to the use of fusidic acid or a derivative thereof for the treatment of endogenous uveitis.
• endogenous uveitis e.g. non-infectious uveitis, such as non-infectious uveitis anterior and uveitis posterior.
• the present invention relates in particular to the manufacture of a composition suitable for treatment of the eye, such as an eye drop composition, an eye ointment composition, an eye lotion composition or an injectable composition for intraocular or retroocular
• the invention relates to the use of fusidic acid or a derivative thereof for the prevention of the inflammation after eye surgery such as cataract operation or laser surgery.
• This treatment, as well as treatment in other conditions, can in some cases be prophylactic in the way the the goal of the treatment is to diminish or avoid an inflammation not to actually treat the
• the present invention relates in particular to a
• composition suitable for treatment of the eye such as an eye drop composition, an eye ointment composition, an eye lotion composition or an injectable composition for intraocular or retroocular
• the invention relates to the use of fusidic acid or a derivative thereof for treatment of progression of arthritis such as rheumatoid arthritis including juvenile rheumatoid arthritis, psoriatic arthritis or Reiter arthritis substantially immediately after the first diagnostic establishment of arthritis.
• the therapy of the invention is preferably instituted as soon as possible, i.e. very shortly after the onset of arthritis or, preferably, immediately after diagnosis of pathological processes (e.g. before clinical manifestations).
• the therapy of the invention may also apply to certain identified high risk groups the identification being e.g. obtained by means of a screening program employing markers relatively specific for arthritic changes and/or preliminary stages of
• the invention relates in particular to the manufacture of a composition for oral use or for use as parenteral or intraarticular injections.
• the Invention relates to the use of fusidic acid or a derivative thereof for the prophylaxis or treatment of
• the invention relates to the use of fusidic acid or a derivative thereof for the prophylaxis or treatment of arthritis urica (gout). In another aspect, the invention relates to the use of fusidic acid or a derivative thereof for prophylaxis or treatment of a condition related to transplant rejection, such as a graft-versus-host reaction, or
• the treatment often requires higher doses of fusidic acid than usual and is often instituted as a prophylactic treatment to avoid transplant rejection or graft-versus-host disease.
• the present invention relates to a such composition for oral or parenteral administration.
• the invention relates to the use of fusidic acid or a derivative thereof for the prophylaxis or treatment of a condition related to cornea transplantation, in particular to a composition suitable for treatment of the eye, such as an eye drop composition, an eye ointment composition, an eye lotion composition or an injectable composition for intraocular or retroocular
• the invention relates to the use of fusidic acid or a derivative thereof for the treatment of Crohn's disease or ulcerative colitis, especially for the prevention of relapse or progression of Crohn's disease or ulcerative colitis, or for the treatment of perniceous anemia or celiac disease, in particular to the manufacture of an oral or rectal composition, or a composition for parenteral administration.
• the invention relates to the use of fusidic acid or a derivative thereof for prophylaxis or treatment of contact dermatitis or allergic/atopic dermatitis, or for treatment of pemphigus vulgaris or pemphigoid, in particular to the manufacture of a composition for oral administration, or for topical treatment of contact dermatitis, in particular for topical administration to the skin.
• the invention relates to the use of fusidic acid or a derivative thereof for treatment or prevention of relapse of a demyelinating disease, in particular multiple sclerosis, or for treatment or prevention of relapse of sarcoidosis Boeck, Sjogren's syndrome, Reiter's syndrome, erythema nodosum, scleroderma or
• the invention relates to the use of fusidic acid or a derivative thereof for treatment or prevention of relapse of polymyositis polymyalgia rheumatica, myocarditis or systemic lupus erythematosis, in particular to a the manufacture of a composition for oral or parenteral administration.
• the invention relates to the use of fusidic acid or a derivative thereof for treatment or prevention of relapse of a condition related to vasculitis phenomena, e.g. polyarteritis nodosa, Wegener's granulomatosis, or giant-cell arteritis.
• a condition related to vasculitis phenomena e.g. polyarteritis nodosa, Wegener's granulomatosis, or giant-cell arteritis.
• the invention relates to the use of fusidic acid or a derivative thereof for treatment or prevention of relapse of primary biliary cirrhosis or chronic active hepatitis.
• the invention relates to the use of fusidic acid or a derivative thereof for treatment of a neoplastic disease, such as
• aplastic anemia or idiopathic thrombocytopenic purpura or for treatment or prevention of relapse of a neoplastic disorder of the lymphold tissue, e.g. B cell lymphoma or multiple myeloma, or a T lymphocyte proliferative disorder, e.g. mycosis fungoides or Sezary syndrome.
• the present invention relates in particular to a such composition for oral or parenteral administration.
• the invention relates to the use of fusidic acid or a derivative thereof for prophylaxis or treatment of septic shock caused by gram-negative bacteria.
• Fusidic acid has been used for treatment of infections caused by gram-positive bacteria such as Staphylococcus aureus, but prior to the present invention it was not known that fusidic acid could be used for prophylaxis or treatment of septic shock which is caused by gram-negative bacteria.
• the present invention relates in particular to the use of fusidic acid or a derivative thereof for prophylaxis or treatment of septic shock caused by gram-negative bacteria for oral or parenteral
• the invention relates to the use of fusidic acid or a derivative thereof for prophylaxis or treatment of disseminated intravascular coagulation.
• the invention relates to the use of fusidic acid or a derivative thereof for prophylaxis of arteriosclerosis.
• the invention relates to the use of fusidic acid or a derivative thereof for prophylaxis or treatment of a condition acute and chronic periodontal diseases, in particular periodontitis and periodontosis, in particular by means of periodontal injections.
• the invention relates to the use of fusidic acid or a derivative thereof for use as an immunosuppressive drug, and the use in combination with other relevant drugs, such as cyclosporin or a derivative thereof.
• the invention relates to the use of fusidic acid or a derivative thereof together with a cyclosporin (e.g.
• Cyclosporin A and G or a derivative thereof.
• Such combination therapy is designed so as to reduce the serious adverse effects of the cyclosporin, in particular the nephrotoxic effects, and at the same time utilizing the immunomodulating effects of the cyclosporin together with the effects of fusidic acid or its derivatives.
• Relevant dosage intervals may e.g. be:
• Cyclosporin A (or another cyclosporin of equivalent potency): - Systemic administration: 1-10 mg/kg body weight;
• Topical administration 1-20 mg/ml (ointments, solutions, eye drops etc., cf. above).
• a pharmaceutical composition comprising fusidic acid or a derivative thereof together with a cyclosporin (e.g. Cyclosporin A and G) or a derivative thereof.
• a cyclosporin e.g. Cyclosporin A and G
• the invention relates to the use of fusidic acid or a derivative thereof together with a non-steroid antiinflammatory drug (NSAID) (e.g. indometacin and acetyl salicylic acid) or an analogue and/or a glucocorticoid (e.g. hydrocortisone).
• NSAID non-steroid antiinflammatory drug
• Such combination therapies are designed so as to reduce the serious adverse effects, and at the same time utilizing the effects of said drugs together with the effects of fusidic acid or its derivatives.
• Relevant dosage intervals may e.g. be:
• Acetyl salicylic acid (or another NSAID of equivalent potency): - Systemic administration: 5-100 mg/kg body weight;
• Topical administration 10-100 mg/ml (ointments, solutions, eye drops etc., cf. above).
• Hydrocortisone or another glucocorticoid drug of equivalent potency - Systemic administration: 1-20 mg/kg body weight;
• Topical administration 1-20 mg/ml (ointments, solutions, eye drops etc, cf. above).
• the invention relates to use of a
• composition comprising fusidic acid or a derivative together with a non-steroid antiinflammatory drug (e.g. indometacin and acetyl salicylic acid), and/or glucocorticoid (e.g.
• a non-steroid antiinflammatory drug e.g. indometacin and acetyl salicylic acid
• glucocorticoid e.g.
• the invention relates to the use of fusidic acid or a functional derivate thereof together with FK-506 or a functional derivative thereof.
• Such combination therapy is designed so as to reduce the serious adverse effects of FK-506, and at the same time utilizing the immunomodulating effects of the FK-506 together with the effects of fusidic acid or its derivatives.
• the dosage of the additional drug or drugs described above should be carefully adapted so as to imply the specific action in the cytokine system, i.e. attainment of optimal total dosages, dosage forms and dosage frequency. In certain cases it is relevant to administer a relatively high unit dosage, a so-called "booster dosis".
• the fusidic acid derivatives VDl546Na and PR1089Na were dissolved at 100 mg/ml in 98% ethanol and stored at 4°C. Before each experiment, the compounds were further diluted in culture medium RPMI 1640 (Ros- well Park Memorial Institute) with 25 mM hepes-buffer (NordVacc, Sweden) supplemented with penicillin (500 IU/ml), streptomycin (500 ⁇ g/ml), L-glutamine (2 mM) , and 1 - 10% of a heat-in- activated human serum pool (NHS). The final concentrations of ethanol were always less than 0.5 o/oo (v/v), and these concentrations did not affect the production or function of the cytokines.
• Cyclosporin was kindly provided by Sandoz (Basel, Switzerland). The powder was dissolved in ethanol (50 mg/ml) and diluted to 20 ⁇ g/ml in sterile saline. This stock solution was stored at -20°C until use. A similar dilution of ethanol was stored and used in parallel experiments.
• rIL-1 ⁇ Human cytokines rIL-1 ⁇ was a gift from T. Tsuboi (Dainippon, Osaka, Japan). rIL-1 ⁇ was from Cistron Biotechnology (Pine Brook, NJ, USA). Purified native human IL-6 and rIL-6 used for pancreatic ⁇ -cell experiments were donated by J. van Damme (University of Leuven, Leuven, Belgium) (26) and L.A. Aarden (University of Amsterdam, Amsterdam, Holland) (27). rIL-6 used for the other studies was a gift from T. Hirano (Osaka University, Osaka, Japan) (28). rTNF ⁇ was a gift from G.R. Adolf
• rIL-2 was from Boehringer (Mannheim, FRG).
• the activities of the cytokines were ascertained by bioassays (see below).
• the cytokines were calibrated with WHO interim standard preparations of the human cytokines (NBSB, London, U.K.).
• the endotoxin contents of the cytokine preparations did not exceed 1 pg/1,000 U measured by a chromogenic Limulus Amebocyte Lysate assay (Struers, R ⁇ dovre, Denmark).
• MNC Human mononuclear cells
• cytokines were carried out in the presence of 1 ⁇ g/ml of indomethacin (Sigma, St. Louis, MO, USA). After 20 h of culture, the supernatants were isolated and dialyzed for minimum 2 days at 4°C against HBSS and, subsequently, RPMI/Hepes buffer (20).
• the cells were collected for viability studies using trypan blue exclusion.
• 20 ⁇ g/ml of sodium-11 ⁇ - 3 H-fusi- date specific activity 93 ⁇ Ci/mg, (Leo Pharmaceutical Company) was added at the initiation of the cultures using 0-10% (v/v) NHS.
• the supernatants were tested for residual 3 H fusidic acid by scintillation counting.
• TNF ⁇ and IFN ⁇ The presence of TNF ⁇ was measured by a slight modification of a previously described sandwich ELISA, using high-titered, monospecific rabbit antisera to human rTNF ⁇ (30).
• the ELISA utilizes the biotin/avidin system, and the detection limit is 1 pg/100 ⁇ l.
• the recovery of TNF ⁇ was always above 85% and the coefficient of variation within and between assays were less than 10%.
• IFN ⁇ was measured by a sandwich ELISA purchased from Holland Biotechnology (Leiden, Holland).
• the ELISA utilizes two different monoclonal antibodies to IFN7 and the biotin/antibiotin-alkaline phosphatase method.
• the sensitivity of this assay was 15 pg per 150 ⁇ l, and the coefficient of variation was less than 10%.
• human IFN ⁇ was tested by Immunoradiometric assay (IRMA) using two monoclonal antihuman IFN7 antibodies (Centocor, Malvern, PA, USA). The sensitivity of this assay is approximately 0.1 IU/ml.
• T cell proliferation induced by TL-1 ⁇ / ⁇ Total IL-1 activities in the supernatants were measured by 3 bioassays using 1) murine T cells (thymocytes), 2) human enriched blood T lymphocytes (both assays also detect IL-6), or 3) mouse lymphoma cells (EL 4) (do not detect IL-6).
• thymocyte co-stimulatory assay for IL-1 was carried out as previously described (29). Briefly, thymocytes from endotoxin-resistant C3H/SSI, female mice, 6-8 weeks of age (Bomholtgaard, Ry, Denmark) were grown in triplicate In culture medium supplemented with 10% (v/v) NHS and 5x10 -5 M mercaptoethanol in 96-wells microtiter plates (Nunc, Roskilde, Denmark), 10 6 cells/ 200 ⁇ l/well. Two-fold dilutions of test material and 10 ⁇ g/ml of PHA were added at the initiation of the cultures. After 48 h, the cells were pulsed with 3 H-thymidine (0.5 ⁇ Ci/well) for 24 h, harvested on paper filters and counted by liquid scintillation.
• 3 H-thymidine 0.5 ⁇ Ci/well
• Purified human T-lymphocytes were prepared from MNC as described (29A).
• MNC were depleted of adherent cells by Incubation for 1 h at 37°C on plastic tissue culture dishes (Becton Dickinson, Oxnard, CA, U.S.A.). Nonadherent cells were removed and passed through a nylon wool column, and the resulting cell population consisted of more than 80% T cells, 15% B cells, and less than 1% MO.
• the EL 4 assay was carried out as described (29).
• IL-2 is based on the production of IL-2 from the murine thymoma cell line (EL 4) in the presence of 1.2 x 10 -7 M of the calcium ionophore, inonomycin (Sigma), and IL-l ⁇ or TL-1 ⁇ . Briefly, 2 x 10 5 EL 4 cells/200 ⁇ l werer incubated in triplicate for 18 h with 2- fold dilutions of test material. After incubation, the supernatants were collected and tested for IL-2 activity using IL-2-dependent CTLL-2 cells (31).
• TNF ⁇ Fibroblast toxicity induced by TNF ⁇
• the TNF-induced cytotoxicity was tested on L-M mouse fibroblasts as previously described (30).
• the mixed lymphocyte cultures were performed by adding human blood MNC, 10 5 /100 ⁇ l, to the same amount of blood MNC from an unrelated healthy donor.
• the cells 2x10 5 in 200 ⁇ l culture medium RPMI 1640 and 10% NHS, were then incubated at 37°C for 5 days.
• Islets from collagenase-digests of pancreas tissues obtained from Wistar rats (90-120 g), were isolated and precultured for 7 days, essentially as previously described (32). The islets were counted, pooled and washed two times in culture medium RPMI 1640, supplemented with penicillin (500 IU/ml) streptomycin (500 ⁇ g/ml), amphotericin B (2.5 ⁇ g/ml), L-glutamine (2 mM), 25 mmol/1 Hepes buffer, 11 mmol/1 glucose, and 0.5% NHS. Islets, 25/1 ml, were distributed at random in 24-well culture plates (Nunc). The cultures were performed in triplicate. After 5-7 days at 37oC in a 5% CO 2 -humidified air atmosphere, 0.2 ml of culture medium were removed for insulin determination by a radioimmunoassay using rat insulin as standard.
• the parietal bones were cut into 4 pieces, 2 acting as controls and 2 for experiments.
• the isolated bone pieces were cultured in test tubes containing 1 ml of medium TC-199 (Gibco Bio-cult, Paisley, Scotland), supplemented with 5 g/1 of bovine serum albumin fraction V and 40 mg/1 of ampicillin.
• the cells were always precultured for 15 min with sodium fusidate (fusidin), or fusidic acid analogues, or with the solvent alone (control) before adding the human natural or recombinant cytokine preparations to be assayed.
• the amounts of added cytokines varied in accordance with the sensitivities of the various bioassays, but they were always chosen to ensure suboptimal effects ((-) 50% of maximum activity) on their respective target cells.
• lymphokine-containing supernatants were dialyzed to ensure removal of fusidin before they were measured for their cytokine contents (see above).
• P values are determined by the Mann-Whitney rank sum test or Wilcoxon's test for paired differences.
• the test was performed as described in "Production of Cytokines", “T cell growth induced by IL-2” and “ELISAs for TNF ⁇ and IFN ⁇ ” .
• fusidin on the other hand, there is no significant toxicity in concentrations up to about 200 ⁇ g/ml, so that the therapeutic index of fusidin is at least 5 times higher than that of cyclosporin.
• IL-2 and IFN7 are produced by the T- lymphocytes, and these cells require IL-1 as a co-stimulatory signal even in the majority of cases where polyclonal, nonspecific T-cell activators are being used (see Fig. 1 and ref. 1-4).
• fusidin was not toxic to the cells, judged by trypan blue exclusion. The cellular viability always exceeded 80%.
• fusidin inhibited the thymocyte co-stimulatory effect of rIL-1 ⁇ and rIL-1 ⁇ in a dose-related manner in this conventional test for IL-1 activity.
• a 50% reduction in IL-1 ⁇ and TL-1 ⁇ activities was achieved at 1.5-5 ⁇ g/ml of fusidin.
• half maximal IL-1 response required 15-50 ng/ml of cyclosporin.
• Fig. 5 shows the effect of fusidin on the IL-1 co-stimulatory acitivity using enriched human T lymphocytes. Significant inhibition was obtained at non-toxic concentrations of fusidin, especially when PPD was used as a co-stimulator to simulate antigen-induced activation of T cells.
• thymocytes contain a small number of macrophages and dendritic cells in addition to T lymphocytes
• fusidin also appeared to inhibit rIL-1 ⁇ -induced proliferation of EL 4 cells.
• the fact that the effect of the drug was much less pronounced than in Examples 3.1.1. and 3.1.2. can most likely be attributed to induction of EL 4 cell proliferation requiring co-stimulation with a calcium ionophore, in addition to IL-1.
• the EL-4 is a transformed cell line which is not comparable to a usual cell.
• test was performed as described in "T cell growth induced by IL-2" and "Fibroblast toxicity induced by TNF ⁇ ".
• fusidin did not modify the ability of human IL-2 to activate mouse cytotoxic T lymphocytes (CTLL-2) or the ability of human TNF ⁇ to kill mouse L-M fibroblast (Fig. 7).
• MLC is the in vitro correlate of a transplant rejection in vivo .
• fusidin has a role in preventing graft rejection, it should suppress the MLC reaction.
• fusidin indeed inhibited the two-way human MLC.
• the dose-response was almost identical to those obtained when testing the effects on IL-1 on thymocytes and PPD-activated human T cells as well as IL-2 production.
• the 50% inhibitory concentration of fusidin was 1.5-5 ⁇ g/ml. Similar suppression of the MLC was obtained with 15-50 ng/ml of cyclosporin.
• IL-6 another M ⁇ -derived peptide mediator, has been reported to mediate several functions of IL-1, including thymocyte activation
• fusidin did not affect the increased release of radiolabelled Ca induced by rIL-1 ⁇ . This further demonstrates the non-toxicity of fusidin in biological systems.
• T and B lymphocytes require several steps in addition to antigen or allogen recognition by HLA class II positive cells such as M0 or dendritic cells: 1) production and release of IL-1 and, possibly, IL-6; 2) activation of T cells hy these cytokines through specific membrane receptors for IL-1 and IL- 6; 3) production and release of IL-2 by T lymphocytes; 4) acquisition of receptors for IL-2 on T lymphocytes, and 5) internalization of IL- 2-receptor complexes.
• HLA class II positive cells such as M0 or dendritic cells
• fusidin also inhibited the production by T cells of their own growth factor, IL-2, leading to impaired T cell proliferation after antigenic, mitogenic as well as allogeneic activation.
• IFN7 is an important activator of M0 functions, including their ability to secerete cytokines and express HLA class II molecules, the drugs ability to inhibit the elaboration of IFN ⁇ might contribute to T cell suppression.
• fusidin did not function by a general antiproliferative effect, because the IL-2-induced proliferation of CTLL-2 cells were unaffected by the drug.
• concentrations of fusidin which were shown to be effective in vitro seem to be clinically relevant, because therapeutic serum concentrations in the range 15-100 ⁇ g/ml may be obtained in man without significant side-effects (33).
• fusidin is bound extensively to protein in vivo , and this may contribute to a diminished clinical efficacy if related to the effective concentrations of the drug in vitro .
• fusidin does not appear to be of major importance in the case of fusidin, because increased concentrations of serum in the thymocyte assay only marginally affected the fusidin- induced immunosuppressive function. Moreover, several important clinical effects of fusidin are expected to take place in tissues where serum proteins are absent or present in only small quantities.
• IL-1 in particular is considered an essential 'second signal' for T lymphocyte activation, including production of lymphokines (1-4) the first signal being antigen or mitogen-induced perturbation of the T cell receptor/CD3 complex (1,2,11,12).
• fusidin to inhibit a mixed lymphocyte reaction in vitro is probably also secondary to its IL-1-inhibitory effect.
• the inhibitory effect of fusidin on this in vitro correlate of a human allograft rejection may be important, because it suggests that the drug may be used clinically to prevent the hosts elimination of allotransplants, such as kidneys, livers, hearts, lungs, skin, bone marrow, corneae, etc.
• Treatment of the host with fusidin at the time the transplant is performed, and thus interference with the 'second signal' of lymphocyte activation, may also lead to specific tolerance to the grafted tissue (see above).
• IL-1 and particularly IL-6 have been implicated in the pathogenesis of several lymphoproliferative diseases, such as multiple myeloma and other plasma cell, and B- and T cell cancers (see above).
• fusidin IL-1 and IL-6 Antiinflammatory effects of fusidin IL-1 and IL-6 are known to cause an array of biological activities in many cell types (1-4). It is therefore interesting that fusidin interfered with some but not all the various functions of IL-1 that were tested.
• the drug was unable to affect IL-1-induced bone resorption.
• the drug clearly protected pancreatic islet /3-cells against the inhibitory/cytotoxic action of IL-1 ⁇ .
• IL-1 and IL-6 are involved in the development and manifestations of many infectious and immunoinflammatory diseases, including AIDS, auto- immune endocrine diseases, some rheumatic diseases, etc. (Table 1, ref. 2,3,8).
• AIDS auto- immune endocrine diseases
• rheumatic diseases etc.
• This along with the ability of TL-1 ⁇ / ⁇ and IL-6 to cause fever, and of TL-1 ⁇ / ⁇ to evoke anorexia and cachexia, emphasizes a therapeutic role of fusidic acid and deriyatives thereof apart from their usage as antibiotics.
• T- and B-lymphocytes M ⁇ and NK cells play important roles in man's defence against microorganisms and neoplastic diseases.
• cytokines peptide hormones produced by these cells, particularly IL-1 ⁇ , IL-1 ⁇ , IL-6 and TNF ⁇ , are potent immune stimulators as well as modulators of hepatocytes, bone cells, endothelial cells, fibroblasts and synovial tissue cells, pancreatic islet ⁇ -cells, thyrocytes, and many other cells.
• Treatment to prevent the production and/or function of IL-1 ⁇ / ⁇ , IL-6 and TNF ⁇ may prevent the development, or ameliorate the symptoms, of many immunoinflammatory and infectious diseases.
• Such intervention may also prevent transplant rejection either if administered as a continuous, immunosuppressive treatment or as a timed, short-term treatment to induce specific tolerance to the grafted tissue.
• Fusidin in therapeutic relevant concentrations, inhibit several immunological, growth-promoting and pro-inflammatory functions of TL-1 ⁇ / ⁇ and IL-6 in vitro , and the patterns of these responses are strikingly similar to those caused by cyclosporin.
• the present discovery is therefore of importance in the systemic and local treatment of several immunoinflammatory diseases in humans, where these cytokines are considered to be of pathogenetic relevance.
• These diseases include the so-called autoimmune diseases (see Table 1), diseases associated with transplantation, including graft rejection and graft-versus-host disease, many infectious diseases, and many diseases characterized by pathological cell growth, including some neoplastic diseases.
• the fusidin concentration in corpus vitreum/subretinal fluid is measured after 3 days of preceeding systemic therapy prior to operation.
• Systemic therapy Fusidin tablets (Leo, Copenhagen, Denmark) 500 mg 3 times every 24 hours . The therapy ends at least 6 hours before operation.
• Corpus vltreum analyses include 10 eyes of patients who are
• Amotio operation detachment of the retina
• removal of subretinal fluid of 10 eyes in connection with retina operations removal of subretinal fluid of 10 eyes in connection with retina operations.
• the following example is designed to examine the prophylactic effect of fusidin in BB-rats and NOD-mice (two spontaneously diabetic animal models for Type 1 (insulin-dependent) diabetes mellitus).
• the study includes 40-50 animals treated with fusidin and a corresponding number of control animals treated with placebo. Breeding couples are procured and the experimental animals are supported and breed. The animals are treated with the drugs immediately after weaning and until the study is terminated after 200 days. The animals are observed daily, week-ends included, and once a week the animals are weighed and urine tests are carried out to determine glucose content. Pancreas from animals developing diabetes and from animals at the end of the study are subjected to microscopic examination to discover 1) possible infiltration of mononuclear cells in the islets of Langerhans (insulitis), and 2) to determine the number of cells producing insulin. Furthermore, the contents of insulin and cytokines in consecutively drawn serum samples are determined in order to evaluate a possible association between the development of the disease and the cytokine levels in serum.
• the acute-phase reaction is usually seen during acute and chronic infectious and inflammatory diseases, and in cancer, and administration of IL-1, IL-6 or TNF ⁇ reproduces this reaction (8).
• IL-6 and, to a lesser extent, IL-1 and TNF ⁇ induce hepatocytes to synthesize acute phase proteins, including serum amyloid A, C-reactive protein, fibrinogen, haptoglobin, complement components and clotting factors.
• the blood level of albumin decreases, as does the plasma concentrations of Fe 2+ and Zn 2+ , whereas the level of Cu 2+ increases.
• Associated phenomena are fever, leukocytosis and induction of sleep.
• the elevated level of fibrinogen especially if accompanied by anemia, causes an increased sedimentation rate of the blood, a commonly used clinical parameter of 'inflammation'.
• Fusidin, 15 mg, and solvent alone (control) are administered i.v. into 5 plus 5 (controls) female BALB/c mice (8-10 weeks old). After 30 min, 10 ⁇ g of human rIL-1 ⁇ in 25 ⁇ l pyrogen-free isotonic saline is given i.v. After 24 h, blood is drawn for measurements of the mouse acute phase reactant haptoglobin.
• the dosage of fusidin and rIL-1 ⁇ will be altered in order to substantiate the dose-response curve of the effect of fusidin on the IL-1-induced acute-phase response in mice.
• Interleukin 6-induced responsse The experimental approach outlined above will be used, except that human rIL-6, 10 ⁇ g, will be used.
• EXAMPLE 13 The following studies are designed to demonstrate the effect of fusidic acid eyedrop treatment as a prophylactic treatment against inflammation after eye surgery.
• Exclusion Patients with eyes with trauma, glaucoma, retinovascular bleeding or thrombosis, corioretinal inflammatory and non-inflamma tory sight-threatening diseases, diabetes mellitus (type I), collagenosis.
• the group treated with fusidin includes patients corresponding to 10 eyes.
• the control group includes patients corresponding to 10 eyes treated with ultralanum with chloroamphenicol.
• Eye pressure is measured at weeks 1, 2 and 3 after operation.
• the examination of corpus vitreum and the corioretinal status is carried out at weeks 1, 2 and 3 after operation, and subjective data In the postoperative period are recorded including the patients' evaluation of the treatment with fucithalmic/ultralanum with chlor- ampfenicol, e.g. problems with dripping of eyes, eye pain, duration of eye pain, duration of sight reduction, if any, after dripping of eyes, etc.
• the group treated with fusidin includes patients corresponding to 10 eyes.
• the control group includes patients corresponding to 10 eyes treated with conventional therapy (glucocorticoid).
• Method Randomized therapy between the two preparations in a 3 week period postoperatively starting from the first postoperative day.
• Evaluation parameters 1. Visual acuity: Examined at weeks 1, 2 and 3 after the laser surgery operation.
• Biomicroscopy is carried out at days 2, 5, 14 and 21 after
• Severe uveal tract inflammation is responsible for a large percentage of the visually handicapped patients in developing and developed countries. Most of the cases of uveitis in developed countries are classified as idiopathic and are presumed to have an underlying autoimmune cause.
• the treatment in these cases is mainly based on the use of glucocorticoids, cytotoxic drugs or cyclosporin. In many of the cases, however, the treatment only delays the loss of vision side-effects (Cushing's syndrome, and severe bone-marrow depression and nephropathy (cyclosporin) may force withdrawal of therapy with resulting loss of vision.
• Pilot studies In the following Tables 6 and 7 are shown the results from two pilot studies. In the first study, three patients are included corresponding to treatment of five eyes. The treatment was converted from cyclosporin to fusidin (0.5 g 3 times daily given as tablets). In the second study, four patients are included (8 eyes). The patients were treated with fusidin (0.5 g 3 times daily given as tablets) without earlier cyclosporin medication.
• Group 1 includes patients corresponding to 10 eyes treated with fucithalmic eyedrops 6 times daily.
• Group 2 is treated with glucocorticoid eyedrops 6 times daily (e.g. Maxidex ® , from Alcon, Texas , U.S.A.)
• Both groups are treated with atropine eyedrops (e.g. from DAK, Copenhagen, Denmark) 1% twice daily. Duration of treatment: 3 weeks.
• Clinical control At least once a week.
• Study parameters are: - Number of patients withdrawn from study medication because of contraindications to continued therapy - Visual acuity - Parameters of inflammatory activity - Fluorescein angiography - Number of relapses - Side-effects, safety parameters - Global evaluation of efficacy and tolerability by investigator and patient. - Immunological studies, including measurements of blood levels of cytokines.
• Investigator 1 should know the assigned treatment, whereas the investigator 2 (ophthalmologist) should be masked in this respect.
• Investigator 1 should evaluate safety parameters, side-effects and should prescribe medication.
• Investigator 2 should evaluate only the efficacy parameters. He should not have access to other data.
• the assigned therapy should be maintained unless contraindications occur. Patients randomized to conventional therapy should start on monotherapy with corticosteroids. If after a minimum of 4 weeks, the response is inadequate, fusidin therapy should be initiated (in combination with low-dose corticosteroids). If, after further 4 weeks, the response is inadequate, fusidin should be replaced with cyclosporin therapy, and the patient should be withdrawn from the study.
• Patients should be investigated at baseline, at weeks 1, 2, months 1, 2, 3, 4, 6, 9 , 12 and thereafter at 6 monthly intervals if the disease is quiescent or at any time if acute attacks occur.
• glucocorticoids The only concomitant medication for uveitis allowed are glucocorticoids and cycloplegic eye drops. Subconjunctival glucocorticoid injections are not allowed.
• Contraindications for Continued Therapy (for both treatment groups): - Visual acuity: If after maximum therapy of at least two weeks, the visual acuity falls two lines below baseline at the initiation of therapy) on two successive days in either affected eye. - Inflammation: If, after maximum therapy of at least two weeks, the inflammatory activity as determined by a second observer, is worse and the visual acuity is not improved in either eye. - If the disease process progresses into the macula which in the opinion of a second observer might lead to permanent loss of vision in either eye. - Toxicity or side-effects a) Impaired hepatic function, such as any value 2.5 times
• Patients will be evaluated at baseline and at weeks 1, 2 and months 1, 2, 3, 4, 6, 9, 12, and thereafter at 6-monthly intervals during at least 1 year if disease is quiscent or at any time if acute attack occur.
• Concomitant medications Any concomitant medications will be listed during the pretreatment evalution and at each subsequent evaluation. Ophthalmological evaluations by "masked" ophthalmologist: a) Visual acuity b) Inflammation, anterior chamber flare, vitreal haze, and opacity c) Other ocular findings d) Flurorescein angiography (only to be done when media allows and at months 3, 6 and 12) Laboratory tests a) Blood: Hemoglobin, hematocrit, WBC count and differential,
• Serum parameters a) Serum protein electrophoresis b) Ig levels c) Serum IFN7, IL-2, IL-2 receptor, TL1 ⁇ / ⁇ , TNF ⁇
• Cellular parameters a) Blood lymphocyte and monocyte analyses - total count and subsets b) Histocompatibllity antigens - HLA-DR (only at time 0).
• Withdrawals are patients who for reasons related to test medication (contraindications as specified) stop further therapy. They should be fully analysed and should not be replaced by new patients. At time of Withdrawal, a complete evaluation should be performed.
• Drop-outs are patients lost to follow-up or non-complying to protocol. They should be replaced (next free patient number).
• the initial material includes a total of 6 patients with Crohn's disease.
• the disease activity is measured by means of the modified "Grading Score" a.m. (46).
• the effect of the treatment is defined as a positive total score.
• the reduction of the dosage of prednosolone can be included as an individual treatment aim.
• P-orosomucoid concentration and "Crohn's Disease Activity Index” (CDAI) are used.
• Clinical control with blood sampling should be carried out after 0, 2, 4, 8 and 12 weeks of treatment and 4 weeks after ended therapy and at any other time depending upon clinical status.
• samples are taken (5 ml serum, 5 ml EDTA-plasma and 5 ml heparin plasma) for determining the fusidin concentration - the patient is not allowed to take his morning dosage until after the blood samples have been taken - and plasma levels of cytokines and cytokine autoantibodies.
• Acute GvHD is a symptom complex appearing 8-40 days (median 14 days) after allogeneic bone marrow transplantation as a consequence of the reaction of donor T-lymphocytes against recipient antigens, especially transplantation antigens in the HLA-system. This occurs in 70% of the patients with HIA-identical sibling donors but is more frequently observed in HLA incompatible family donor or HIA-identical unrelated donor.
• Grade I serum grade: Macular or confluent exanthema. Diarrhoea not more than 500 ml per 24 hours.
• diarrhoea more than 1000 ml per 24 hours and/or
• bilirubin > 50 mikromol per liter.
• the occurence of acute GvHD in moderate to heavy grade is combined with increased lethality after allogeneic bone marrow transplantation. Lethality is especially due to infections as a result of delayed immunological reconstitution. This is caused both by the GvHD itself and by the fact that these patients often receive further immunosuppressive treatment with prednisolone and/or anti-thymocyte globulin.
• All patients are treated prophylactically against acute GvHD. This includes cyclosporin (day -1 to day +180), possibly supplemented with methotrexate i.v. (day 1, 3, 6 and possibly 11), depending upon the estimated risk of developing acute GvHD.
• Glucocorticoids are used, possibly supplemented with anti-thymocyte globulin. The dosage of cyclosporin is also increased. Aim and type of study
• Exclusion criteria Patients under 18 years of age. - Patients who according to the above-mentioned criteria should be treated with glucocorticoid. - Patients receiving glucocorticoid and/or anti-thymocyte globulin before or at the time of transplantation.
• cyclosporin prophylaxis should be carried out: a. If the results of the latest cyclosporin concentration test are below therapeutic level, the cyclosporin dosage should be increased and the effect should be awaited (24 hours).
• diarrhoea, fusidin for i.v. injection 12 mg/kg 3 times daily in the first 2 days, thereafter 8 mg/kg 3 times daily.
• fusidin treatment is considered therapeutically, treatment is continued for 10 days whereafter fusidin is withdrawn. If progression occurs in spite of fusidin treatment, the drug is withdrwan and conventional therapy is instituted (see above).
• Blood Sedimentation rate, orossomucoid, hemoglobin, bilirubin, ALAT, ASAT, alkaline phospatase. 5 ml serum and 5 ml EDTA-plasma are taken for measurements of cytokines and autoantibodies to cytokines.
• Marrow plasmocytosis (>10 percent), lytic bone lesions, and a serum and/or urine M component, or plasmocytosis associated with a progressive increase in the M component over time or if extramedullary mass lesions develop.
• Blood Haemoglobin, RBC, WBC, and differential count, platelets; serum creatinine, bilirubin, alkaline phosphatase, ALAT, ASAT, electrolytes, se-calcium, IGA, IgG, IgM, M component.
• Urine 24-h-proteinuria, glucose, M component.
• the immunological tests should be repeated at least once monthly. - True plasma levels of fusidin (blood drawn in the morning before first dosage of fusidin) should be performed at bi-weekly intervals.
• Withdrawals/drop-outs Withdrawals are defined as patients who discontinue therapy either because of side-effects or inadequate efficacy. These patients should be fully analysed and data safety and efficacy required also after withdrawal.
• glucocorticoids has improved the outcome of systemic lupus erythematosus (SLE), particularly if accompanied by renal involvement.
• SLE systemic lupus erythematosus
• cytostatic drugs including cytostatic drugs
• a treatment complementary to glucocorticoids in SLE because: a. Some patients, particularly patients with systemic vasculitis, do not respond to corticosteroids alone. b. Many patients are in need of high dose (>10 mg prednisolone per day) corticosteroid treatment: They relapse as soon as the drug is progressively diminished or stopped.
• the SLE diagnosis should fulfil four or more of the American Rheumatism Association's criteria for the classification of SLE at the time of diagnosis. Patients who, despite receiving ⁇ 10 mg of prednisolon daily for ⁇ .3 months, present with subjective and/or objective signs of ongoing disease activity.
• Exclusion criteria Patients receiving complementary treatment (e.g. cytostatic drugs) for SLE. - Patients who are not treated with glucocorticoids.
• Urine 24-h-proteinuria, glucose.
• Immunological blood tests - Antinuclear and anti-DNA antibodies.
• IL-2 IL-2
• LT lymphotoxine (TNF ⁇ )
• IFN7 IFN7
• Withdrawals/drop-outs Withdrawals are defined as patients who discontinue therapy either because fo side-effects or inadequate efficacy. These patients will be fully analysed and data safety and efficacy required also after withdrawal.
• Rheumatoid arthritis is a common disease of unknown cause.
• the inflammatory processes in rheumatoid arthritis include increased production of synovial fluid, activation and proliferation of cells in the synovial membrane, destruction of articular cartilage and bone, and repair processes resulting in fibrosis, ectopic calcification and metaplastic bone formation.
• Polymorphonuclear leukocytes and macrophages play a critical role in all these processes (35, 36). Immunocompetent cells are also involved, and in severe cases the inflamed synovial tissue may resemble a lymphoid organ with germinal centers of B lymphocytes surrounded by T lymphocytes. Generally, a relatively large proportion of the infiltrating T cells bear membrane markers characteristic for activated T cells, and such cells are often found in the blood as well (35). The presence of plasma cells and immune complexes in the synovial tissue and the frequent finding of autoantibodies, particularly anti Fc-IgG rheumatoid factors, and hypergammaglobulinemia also suggests that antibody-mediated reactions are involved in the disease processes.
• IL-1 and, possibly, IL-6 and TNF ⁇ seem to play a central role in the processes leading to tissue damage in rheumatoid arthritis and related rheumatic diseases (8).
• injection of IL-1 and TNF ⁇ in knee joints of normal rabbits causes similar biochemical changes as those seen in early rheumatoid arthritis (37), and blood levels of IL-1 and IL-6 closely parallels subjective and objective disease activity in patients with rheumatoid arthritis (38).
• the evidence that cyclosporin is effective in rheumatoid arthritis, both when experimentally induced (39) and in clinical cases (40-43), further support the notion that IL-1 and/or IL-6 may trigger the initial immunoinflammatory processes which eventually causes joint and bone destruction.
• significant adverse effects of cyclosporin have been noted, and alternative therapies are therefore advocated.
• NSAID non-steroidal antiinflammatory drugs
• glucocorticoids In particular glucocorticoids, gold salts, penicillamine, cytostatic drugs
• duration of active disease should not exceed 12 months.
• Urine protein, glucose.
• Immunological blood tests - Antinuclear and anti-DNA antibodies.
• the immunological tests should be repeated at least once every second month. - Trough plasma levels of fusidin (blood drawn in the morning before first dosage of fusidin) should be performed at bi-weakly intervals during the first two months and then at four weeks intervals.
• connective tissue diseases such as juvenile rheumatoid arthritis, psoriatic arthritis, reactive arthritis (Reiter' s syndrome), polymyalgia rheumatica, systemic sclerosis, and sarcoidosis may be pathogenetically related. That similar disease mechanisms may be operative in several or all of these diseases is strengthened by the fact that patients with different connective tissue diseases benefit from therapy with the T cell-selective immunosuppressive drug cyclosporin (44). The treatment of these diseases, however, is often unsatisfactory, and almost always accompanied by side-effects. The clinical studies in connection with these diseases should be performed analogoosly to the protocols outlined above.
• Fig. 1 Cellular interactions and cytokine production during antigen presentation to T- and B lymphocytes.
• the cytokines produced by both immune and non-immune cells may enter the blood stream and act as hormones which affect the functions of distant tissues. Modified from (3).
• M ⁇ macrophage.
• Th T helper lymphocyte.
• B B lymphocyte.
• F fibroblast.
• NK natural killer cell.
• DR MHC class II molecule.
• IL-1 IF interleukin 1-inducing factor.
• PPD purified protein derivative of tuberculin (antigen).
• PHA phytohemagglutinin (nonspecific, polyclonal T lymphocyte activator).
• Fig. 8 Effect of delayed addition of 5 ⁇ g/ml of fusidin to PHA-activated murine thymocytes.
• Fig. 11 Reversibility of fusidin-induced inhibition of IL-6 activity (B9 assay). The test cells were treated as indicated. The cells were treated with 18 U/ml of rIL-6 with/without 30 ⁇ g/ml of fusidin, as indicated (PRE). After 2 days, the cells were washed three times and cultured further for 2 days with/without 30 ⁇ g/ml of fusidin, as indicated (POST). The assay for IL-6 activity was performed during the last 2 days of culture in the presence of the indicated levels of rIL-6.
• Fig. 12 IL-1 ⁇ induced inhibition of insulin secretion (% of control: Insulin secretion in cultures kept in medium alone without IL-1). Protection afforded by fusidin.
• the rat pancreatic islets of Langerhans were precultured for 6 days. The islets were then washed and added fresh medium, containing 11 mmol/1 of glucose, and rIL-1 ⁇ +/- fusidin at the indicated concentrations.
• Fig. 13 Inability of fusidin analogs to protect against IL-1 ⁇ induced ⁇ -cell damage.
• the culture conditions were the same as in Fig. 12, except that only one concentration of the fusidin analogs were tested.
• Oxholm A Oxholm P, Staberg B, Bendtzen K. Interleukin-6 in the epidermis of patients with psoriasis before and during PUVA treatment, Acta Derm Venereol 1989; 69: 195-199.
• Muller K, Svenson M, Bendtzen K. l ⁇ ,25-dihydroxyvitamin D3 and a novel vitamin D analogue MC 903 are potent inhibitors of human interleukin 1 in vitro . Immunol Lett 1988; 17: 361-366.

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