CA2001790A1 - Pharmaceutical compositions and use thereof - Google Patents

Abstract

The use of fusidic acid or a derivative thereof for the manufacture of a composition for interfering with cytokines in a biological system, in particular for interfering with a cytokine such as e lymphokine, interleukin, monokine, interferon or colony-stimulating factor.

Fusidic acid and derivatives thereof are useful for preventing ef-fects of cytokines known to be pathogenetically involved in patholo-gical conditions. Said conditions may e.g. be:
Insulin-dependent diabetes mellitus (type 1), hyper- or hypo-functioning of the thyroid gland (e.g. thyroiditis), Addison's di-sease, endogenous uveitis, eye surgery such as cornea transplanta-tion, cataract operation and laser surgery, transplant rejection, graft-verses-host disease, Crohn's disease or ulcerative colitis, pernicious anemia, celiac disease, contact dermatitis, allergic/ato-pic dermatitis, pemphigus vulgaris or pemphigoid, multiple sclerosis, systemic lupus erythematosus, polymyalgia rheumatica, conditions related to vasculitis phenomena, neoplastic diseases such as multiple myeloma, septic shock caused by gram-negative bacteria, DIC, arterio-sclerosis, periodontal diseases, arthritis, osteoarthrosis or ar-thritis urica.

Description

L'7~3~3 The pre.~ent invention relates to ~ new pharmRceutical us~ of fu~idie acid and derivatives thereof, in particul~r th~ u~e for p~evention and/or treatment of diseases, the pathogenesis of which is rel~ted to the production and/or function of certain i~unoinfla~matory medi~-tors, especially cytokinec. In particular, the invention relates to the use of fusidic acid snd derivatlves thereof and to the prevention and/or trestment of diabetes ~ell~tus ~type 1) (insulin-dependcnt diabe~es ~ellitu6 ~IDDM)), thyro~d diseases, rhe~matoid di~ea~es, infl~mmAtory diseases ~f the ~ut, skin di~eases, condlt~on~ related to transplant rejection, endogeno~s uveit~s and condition-c related eo opthalmological in~ervencions.

There is increasing clinical ~nd exp~r~ment~l evidence th~t ~any autoimmune diseases develop as as result of abnormalities in the immune system, especially ~.n T lymphocyte-mediated immunity. ~Any signs and symptoms of infec~iou~ infla~matory and neoplasti~ dl-seases evolve as a result of sti~ulation of cellulsr i~munity. In addition, although immunocompetenc cells may not necess~rLly be involved at the initisl ~a~e, abno~mal re~ulation of otherwise appropriste eellular im~une reactlons may lesd to acuee and chronic diseas-s. These diseases are ~ener~lly of unknown etiolo~y ~nd in-clude cystemlc rheumatLc dlsease~ ~e.g. rheumatoid arthriti.~
orgAn-specific endocrine dlsease~ ~e.g Lnsulln-dependent diabetes 25 mellitu5 (IDDM)) and infl~mmA~ory disease~ of the gut (e.g. Crohn's dicease~ ~nd ~kln ~e.g. contact derm~titis). DLcorders of cell-me-d~ted im~unity, however, contribute co ~any other i~munolnftamm~tory and proliferatlve di6eases as will be described in further de~ail in the ~ollowing, The treatments available in relation to sald disea~es ~re usually sympeomatic or palliativt treatments, ~.e. ~ost of the dru~s pre-scribed in connection with said dlseases are directed at the ~llaying of ~ymptoms and u~ually have no curative effect O~her tresement~ are so-c~lled substieutiOn therspie~ which invol~e life-lon~ supplying to 427402~A.~Z~A31tAS/ALM~ALM/30 10.19~9 ~1 3~ 0~'ON ~ 1 '0~'01 ~ NOW~ l~Ol~NII~ ~ NN~W~llOld WO~

the patient of ~ubseances~ e.g. hormone5, ne~dPd due eo d ~duc~d/ln-~ufficlent intern~l production of ~aid ~ubstanee. Said trea~ments are often un.~atlsf~ctory and i~ply unwanted and often serious side~ef~
feces. Th~s, improved methods of treatments an~ improv~d pharmaceuti-cal co~posie~onq are needed.

Fusidic acid and certain derivatlves thereof can be isolated fro~ th~
fer~ent~tlon broth of certain strains of Fu~ldiu~ coccineum and have for a n~mber of years been known aR efficiene and phar~aceuticRlly acceptable an~ibiotics. Fusidic acid, 16-(aceeyloxy)-3,11-dihydroxy-29-ds~mara-17~20),24-dien-21-oic acid; (3~ ,16~-trihydroxy-29-no~-~,9~,134,1~-da~mar~-17(20),24 -d~en- 21-oic acid 16-acetatste), is a tetracyclic trit~rpe~oic acid ~ith a steroid-like primary structure ~f formula I: e~3 CR3 s ~12~
~COOtl " ~ OCOCHI I
,~
CH~

tH3 It is well-known that f~sidic acid inhibits baeeerial protein 5ynthe-sis and lt may be bacteriostatic and/or bactericidal. It l.e es~eclal-ly actl~e against Staphylococcus aureus, incl~ding straine ~hat are resistant to ~he penicillin~ or ~o other antibiotics. Ihe drug i~
used clinically ~o~h ln Its acid form, and as the sodium and di-ethanola~lne salts. The ~odL~ salt of fu4idic acid ha~ the actions and uses of fusidic acid, It is more soluble ~n ~ater, and is read~ly absorbed fro~ the gastro-intest~nal tract. It ls also used in t~plcal preparations, ~or severe staphylococcal infectlons ~he antiblotl~ may be slven intravenously as e.~. dlethanola~ine fusldate A description of ehe~ioAl and ~lcrobiologieal ~odifications of fusidic acld 18 gl~en in Seructure-Aetiviey Rel4t~ onship in Fusidic Acld-Typ~ Ane$-biotlc~ (45), and fusitic scid and derivacives ehereof ase described 427402E~A~02~A31/AS~ALM~LM~30.10.19B0 ~1 3~ O~'ON ~ 1 '0~ NOW~ l~Ol~lNI~ ~ NN~W~nOl~ WO~

in Pharmacopoieas and In M~rtindale, The Extra Pharmacopoe~a, 2~th edit~on (1982~, Fusidlc acid and derivatlve~ thereof are described in PCT application No. UO 86/0396~, published e~A~ned D~nish applica~lon ~o. 148390 (US
4,119,717), DK 9634g (GR ~30786), DK 99802 (AT 252446), DK 101687 (US
3,334,014), DK 10439~ (US 3230240), DK lOS9~1 (US 3,230,240~, DR
107349 (GB ~997g4), DK 116940 (US 34~9012), DK 131348 (US 3,867,413), ~K 142~52 (~5 4100276, US 4162259 and US 4315004), U.S. Pa~ent ~o 3,376,324, 3,629,3~0, 3,920,817, 4,004,004, 4,025,620, 4,060,606, 4,315,004 and U.K. Patent Nos. 2,0~3,348 and 987,042, However, none of these prior art docu~ents sre ~elated to the use of fusidic acid or functional derivAtives thereof for interfering with specific immunologicsl response mechanismc or for interfering with cytokine (lymphokines, lnterferone~ interleukins, colony-stimulating fflctor.c and/or ~onokines), nor related to m~thods for treating conditions related to disturbance~ in the cytokLne sycte~. Occssion~l mentlonln~
in the medical lieerature of specific patients, e.g. di~betics, receiving a short transitory fusidic acid medication due to ~ bac-teri~l infection is obviously irrelevant ee the teaching ~nd embodl-ments of the present invenelon.

Accort~ng to the present lnvention it h~s been found thae fusldic~id and deriva~ive-~ thereof can be u~ed for the preveneion and/or treatment of certain forms of inflammatory dise~ses or processes, epecially forms related to the i~une and/or hormone system. It is contemplated ~dS described in detail in the following de~cription of im~unologicAl m~chanisms) ~hat the aetion mechanism is via ~nte~-ference wi~h the ~ction of mediators of the im~e ~ystem, in psrti-çular cy~okine~ such as monokines snd lymphokines, i,e, that ~us~dic acid ~nterfere~ with/~uppresses the act~on of certain cytokine~ a~d thus inhibits pathological processes leading to ti~ue dama~e.

In its broade~t aspeet t~e present invention relatea to the u~e o~
fusidic acid or d derivative thereof for ~ubstantially inhibitln~ a biological effect related co a cytokine s~h as a lymphokine, inter-4Z7~02a~002/J~31/AS/ALM/ALM/30.10.19~9 ~1 3~1 Ç~ O~'ON ~,61 'O~'OI ~ NOW~ l~Ol~NIn ~ NN~W~ Oll WOl~

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leukin, monokine, interferon or colony-stlmulating fact~r ~or the prophylaxis or treatment of a condition relsted to a di~turbance of a cytok~ne system such as the lymphoki~e, interleukine, monokine, lnterferon or colony-stimulating factor syste~. The broadese ~spect 5 of the lnventlon al~o relates to a method of treating in A human a condition related to a di~turbance in a cytokine system which method comprise~ administering to the subJect an effective a~o~nt of fusldlc acid or a functional derivat~ve thereof. Thus, ths us~ accordlng to the present invention i9 noe based on the use of fusidic acid or functlonal derivatLve~ thereof ag antibiotic~.

(~ene~l lnformation on cytoklnes ~nd immunoinfla~ ory d~sedses Many Qlgn~ and symptoms of infectious, infla~matory and ~eoplastic d~sesses evolve 8s ~ result of stimulation oi cellular ~mun~y. In addition, although i~munocompetent cells may not necessarIly be involved at the initia~ stage, abnormal regulation of othe~wi~e appropriate cell~l~r ~mmune reaccion-~ may lead to acute snd chronic diseases The-~e diseases are generally of unknown etiology and in-clude systemic rheumatic diseases ~e.g. rhe~toid arehrlti~ (RA)), orgAn. speclfic endocrine di~ea~e~ (e.~. insulin-dependent di~betes mellitus (IDDM)), and infla~matory diseases of the gut te.g. Crohn's disease) and ~kin (e.g. contact der~atitis). Diso~ders of cell~edi~-ted immunity, howeve~, contribute to ~any other infla~matory and prollferati~e dl~eases ~see Table 1).

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~0~ 7~

Some diseases ~here ~aerophage~/T-lymphocyte-mediated immune reac-tlons ~re considered pathogenetioally ~mportan~.

Atopic der~Atiti~ dnd cont~ct derma~itis.
Psori~sis and psoriatic arthritls, Mycosis fungoides, S~zary syndrome and other T-lymphocyte prolifera-tive disorders Pe~phigu~ vulgarls and pemphigoid Erythema n~dosum S~lerodenna Uveiti~
Bechet'~ diQea~e Sarcoidosis Boeck S~ogren's syndromo ~heu~atoid arthritis Juvenile arthritis Reit~r~ syndrome Gout Osteoarchro~
Syste~ic lupus eryche~atosis Poly~yosltls snd myocarditi~
Pri~ary biliary cirrhosis Crohn'~ di~e~e Ulcerative colitis Multlpl~ sclerosis Apla~tlc anemi~
Idlopathlc thro~bocytopenic purpur~
M~ltlple myeloma and some B-oell lymphomas Multiple sclerosis and other demyelinating diseases Si~onds' psn-hypopituitarism Çrave~' disease and Graves' opthal~opathy Subacute thyreoditis and Hashi~oto's disease Addison's disease Insulln-dependent tiabete~ mellitu~ (type 1) 427~Q~P~ AS/ALMIALM130.10,19~9 g' 3~ g0~'O~ 'g~'g~ NOW~ l~Ol~NI n ~ w~nol~ wo~

Z(~'75 Miscell~neou~
Varlous cllnal syndromeA with vasculltis (e.g. polyarteritls nodos~, Uegener'c granulomato~i~, giant-cell arteritl~, fe~er, mal~lse, anorexia (e.g. ln acute and ~hronl~ ~nflam~atory ~nd infectious dlseases) Disse~lnated intra~ascular cosgulation (ditto) Arterlosclcrosis Shock te.g. ln g~ ne~ative ~psis) Cachexla (e.g. in canoer, chronic infectious and chronic inflammatory dices6e~
Tr~n~plant ~e~ection and graft-ver~s-host disea~e T lymphocytes govern the induction and regulatlon of cell-mediated immune reacelons, and proteins and glycoproteins produced by T ly~-phocytes (ly~phoklnes) initiate and control the im~une re~ponse(1,2). However, antlgen actlvation of T cells re~uires physicsl interdction with antigen-presenting cellc such ~Q macrophage~
and the~e cellc also produce media~or molecules crucial for T cell activation and the subsequent triggering of B lymphocytes to become antibody-producing plasma oells. These mediators ~re also responsible for the recruitment and actl~ion of many other cell types which ~uild up inflam~atory infiltrate~ i~ the diseased tissues (1-4).

The ly~phokines and the lymphocyte-act~ating metiators produced by antlgen-presenting cells are collecti~ely term~d cy~okin4s. Hence, cytokine~ are essen~l trans~itters of cell to-cell communlcatlon in both physiological and pathophys~ological infectious, immunoinflam-matory, and growth processe~. In many ca~ss ~hey also func~ion as hormone~ providing informa~ion between the im~une syste~ and other tis6ues and or~ans, Furthermore, many cytokines are produced by cells outside the imm~ne syste~ ~.g. in the skin (5) ~nd in bloot vessels), and cytokines may therefore act as ~utocrine or paracrine hormones without nece~-carily involving imm~nocompetent cells.

Cytokines ~re Active at extremely low concentrations (10-1 to 10-15 M) via bindin~ to specific receptors on a large number of t~rget cell~. Most cytokines probably act ~n ~he viclnity of the 42~4020A002/A~ ALM/30. 10,19~
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production site but, ac wentloned abo~e, so~e of the ~edtators modu-late functlon~ of cells at distant sites v$a blood find lymph cir-culation. Fig. 1 ~how~ the dynamics of protuction and function of cytokine~ during an immune ~eactlon ~n & vascularized tissue.

M~cropha~e~

Macrophage~ (M0) ~lay a central role in the defence a~ainst microblal and neoplastic disea~esl and they are i~portant in a wide ~riety of tissue repair processes ~Z).

Some M0 functions sre performed continuously, such as removal of aged erythrocytes. Other functions are performed periodically, ~nd these functions need prior activation of the M0. The cellc may be activa~ed direçtly, for instan~e by conta~t with mlcroorgsnisms or their product~, ~uch 8~ endotoxins (lipopolysaccharides), mur~myl peptides, snd other cell wall componenes. This direct means of M~ a~ti~atlon iQ
in hi~her animals improved considerably by a more indirect form of activatio~ via lymphokines (1-3). Thus, lnterferon a and -~ (IFN~ and IFN~) ~re important activators of M~, but other M0 ~ctivating lymphokines exist ~1).

Phagocytosi~ itself and, perhaps ~ore importantly, i~munecomplex- or ly~phokine-~edisted activation t~i~ger the M0 to synthes1ze and release a number of b~ologl~ally ac~1~e ~olecules, including pro~
taglandins, proteolytic enzymes, complement components, e~c. Fo~r of these factors, interleukin 1Q (IL-lQ), lnterleukin 1~ (IL-l~), inter-leukin 6 (IL-6), ~nd tumour necrosi6 factor-~ ~TNF~), have far-reach-ing biologicsl ~nd p~thophysiological significance (1-8).

Macrophage-derived polypept~de mediators The molecular biology of the best char~c~erized hu~an M0 hormones has re~ently been extensively reviewed (2,4,6-~), They ~re all member~ of a growing family of cytokines which includes the interferons and 50~e hemopo~etlc growth fsctor~. The M0 cytoklnes are also produced during condition~ of "stre~", snd ehey sre important medis~ors of fever, the ~cute-ph~se re~ction, and the ~eneration of cac~exi~ as a re~ult 427402~002/A31/AS/ALMjAl_M~30,10 19~3 ~ 3~d ~ IgO~'0N ~,61 'O'01 6g~NOW~ 1301~1NI~ ~ NN~W~IllOld WOd3 L7~

of chronic infectious, infla~matory, autoi~une, and neopl~s~lc diseases.

Production of lL~ , IL-6 ant TNFa Although IL~ , IL-6 and TNF~ are prod~ced p~i~arily by activa~ed M0, the capaclty to elabor~te the3e ~olecule~, particul~rly IL-1~/~
and IL-6, is not confined to M0 ~2). Natural killer cells ~NK cells) ~nd B lymp~ocytes ~rq already known to produce IL-l~f~, and fi~ro-blaets were the cells originally sho~n to produce IL-6 (- IFN~2), In fact, it i5 likely that all nucleated cells ~ay produce these peptide~, provided they are triggered by an Appropriate sti-mulus.

The m~ture mediator molecules sre single-chsin polypeptides consist-ing of 169 (IL~ , 153 (IL-1~ 4 (IL-6), ~nd 157 ~TNF~) amino acids. Native human IL-6 is glycosylated, and the re-s~ltinR molecular weight of this cytokine species range between 17~nd 28 kD.

The genes for TNF~ and TNF~ are both located in the class III region of the m~jor his~ocomp~tibility co~plex, i.e, the L~ region ln man.
R~cent st~dies in our laboratories show that blood mononuclear cells

2~ obtsined from ~LA-DR2 pocitlve ind~vldualc exhiblt a slg~lfic~ntly di~inished TNF~ response in vit~o (9). Thls is important, becauQe the ~LA-DR2 type is positively associsted with diseases such as multiple s~lerosis and narcolepsy and negstively associated with rheumatoid ~rthr~tls and IDDM.

Im~unological functionc of IL-lc~ ~nd IL-6 - çllni~al relevance Recepeors for IL-1~/~, IL-6 and T~F~ have been found on most of thc cell types known to respond eo the cytokLnes~ IL-1~ and IL~1~ are ehOught to bind to the same recep~or on T ant B lymphocyte~, ~nd both peptides are cap~ble of down-regulating the expression of the CD4 ~truc~ure on human T cells (10). The ~D4 structure is directly en-gaged in ~ntigen presentation, poss~bly through ~n~eraction w~th MHC
clas~ II molecules on antigen-presenting cells (Fig. 1). I~ is there-427~2~0~2/A3 l /A~/ALMIALM~30. ~ 0.1 r7~ 3~ g~'ON ~'6~ 6g/~N0 fore possible that the T ly~phocyte IL-l receptor bind~ to the m~o~
membrane Associ~ted IL-l species ~IL~ on the ~ntigen-presentin~
cell, ~nd that thl~ receptor is phy~ically or functionally rela~ed to the CD4 molecule (Fig. 1 and ref. 2).

IL-6 ha~ been ~hown to be identical with B cell-stimulating factor 2 and a factor necexs~ry for the growth cf plasm~cyto~a~ and B cell hybridomas (2, 7). IL-6 is ~lso a cofactor in T cell ~ctiv~tion, prob~bly beca~se it induces IL-2 receptors and funct~ons as B seçond s~gnal for IL-2 prod~ctlon ~2).

Motulaeion of immune re~ctions and tolerance induction It may prove clinically lmportant that interference with the IL-l-mediated "cecond si~nal" for T cell activation may lead to antigen-specIfic tolerance. Thus, triggering the T cell receptor by a proper-1~ proces~ed sntigen, whlle at ehe same ti~e prevenelng the ensulng IL-l-induced activation of these specifically re~cti~e cell~, pre~
vents the selected clone(s) fro~ responding to the ssme ~nti~en st later time, both in vitro ~ll) and in vivo (12).

Because of the central importance o I~ and IL-6 in T ~nd B
lymphocyte ~ctlvation, any treaemene that block~ ~he production or the lmmunological functions of thefie ~edi~to~ would be expected to suppress the entire cellular lmmune syste~. Thls ~ay be especially beneficisl in the mana~e~ent of patie~t~ reçeiving tran~pl8ne3.

Other functions of IL-l, IL-6 and TNF~ - cllnlcal rele~ce ~ , IL-6 and TNF~ ~re pleiotropic in thst they exert ~ultiple functions on ~ny dlfferent cell types (2~. It h~ becomo cleAr thAt IL-l~/~, T~F~, IL-6 and lymphotoxin (-TNF~) are identlcal with the factors originally desçribed AS endogenous or leukoeytic pyro~ens;
i.e,, hormone~ of leukocyt~c orlgln whlch produced fe~er ~3). It ~c no~ entirely cle~r, however, whether ~11 these cytoklne~ a~t ~n similar fashion in t~e br~in durln~ fever induçtion.

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Acu~e-ph~ce reactlon Fe~er 1~ often part of the ~cute-phase reactlon u~u~lly ~een in eon~nctlon with acute and chronic infectious and i~nolnfl~mmatory diseases, ~nd in cPncer ~2-4, 8~. IL~ and IT6, and to a les~er S extent T~F~, induce hepatocytes to synthesize a¢ute-phase proteins, including ser~m amyloid A, C-reactive protein, ~ibr~nogen, hapto-globin, complement co~ponents and clotting facto~s. The ele~ated level of fibrino~en, especlally lf ~ccomp~nied by anem~a, cause~ an increa ed erythrocyte sediment~tion rate, a commonly used clinlcal parameter of ~inflamma~ion~ Sinoe IL~ nd TNFa ere potent in-ducers of IL-6 in many cell types, including hepatocytes, lt is possible tha~ IL-6 is a se~ond ~ed~ato~ of the acute-phase re6ponse elicited by IL-1~/9 and T~F~.

Cachexia, tisse~inated intravascul~r coagulation ~DIC) and shock The above mentioned clinical picture is oftcn assoclated with di~-turbances in carbohydrate-, llp~d- and protein metabolis~ eventually resultlng in wa~ting (c~chexi~), In rare situations, clot~ing ab-norm~lltiec and shock ~ay occur (6~.

It wa6 previously thought that microbial product~, e~pecially endo-toxin fro~ the cell wall of gr~m-negative bacteria (llpopolysacchari-de (LPS)) were directly responsible for these often life-threatening symptoms, if triggered by bacterlAl infection. Thls ~s now known to be incorrect, because ~PS is a potent inducer of M0 IL-la/~, IL-6 ~nd T~F~, ant all pathophysiological proce~ses associated Yith LPS-in-duced ~hock can be reproduced by in~ection of TNF~ ~nd to a lesserextent by IL-lat~ (2,6).

TNF~ is probably slso responsible for other phenomena a~so~lated with LPS-induced disease, such as metabolic acidosls, DIC and cachexia (2,6). lt is l~kely that so~e of the~e phenomena are ~ediated through TNF~-Induced production of IL-l~/~ or IL-6, or one or se~er~l other (unknown) hormoneR. For exa~ple, TNFa is ~ potent ind~oer of IL-l~/~ in endo~helial cells, and all three cytokine~ trigger the relea~e of procoagulan~ factor(s) fro~ these cells. It i~ th~refore 4~740213~00~/A31~AS/ALM/ALM/3~.10.1!~9 S~ 3~ gO~'ON g~,61 '0~'01 6g~(NOW~ l~Ol~NI~ ~ NN~W~I101~ ~0~

~a~7~0 likely that both mediators may be involved in t~e p~thogenesls of ~ome Yascular diseases with or ~ithout accompanying clotting ab-normalltles.

~eaplastlc disea~es 5 Unregulated prod~ction of IL-6 may be pa~hogenetically ln~olvcd ln the ~anifestation of se~e~al human dlseases, including csrdiac myxoma and varlous l~mphold malignancie~ such as ~ultiple myeloma~ and v~rlou~ T and B cell lymphomas (6). It i~ interesting, that myeloma cell~ constltuti~ely produ~e IT-6 and express IL-6 ~eceptors, and th~t the prolife~ation of myelo~a cells ln vitro is inhibited by speciflc ~nelbod~e.~ to hu~an IL-6 (13).

Othe~ p~olif~ative di~eases - psoriasis IL-6 and, posslbly, T~Fh ~ay also b~ Involved in other proliferat~e disorders, Usin~ cpecific antibodies to human rTNF~ and hum~n ~IL-6, we have recently demonstrated TNF~ and IL-6 i~ skin biopsies (5, 5A~
The expression of both medi~tors was considerably augmented by W -irradiation, and relatively large amounts of IL-6 and T~Fu were found in psoriatic lesions. Interectingly, local trea~ment of the psorlatic leslons with a vitamin ~3 a~alo~ue ~e~uleed in clinical impro~ement, and biop3ie3 performed after treatment showed unsltered expre~ion of TNF~, but sn almost co~plete eli~ination of the expresslon of IL-6 in thq ~pper epidermal layer~. It ~s therefore possible ehat IL-6 ~ay be In~olved in the uncontrolled prollfer~tion of keratinocytes seen in psoriasis.

R~eum~tic d~sedses IL~ -6 and T~F~ hav~ many biologlcal effect~ which qualify them as important in the pathology leading to rheumHtic ti~eA~es (2,~). The most relevant actions of lL-l~ ~nd -~ a~e most likely their stimula~ory effects on T and B cells (no~peciflc productlon of inflammatory ly~phokines ant im~unoglobulins), M0 (e.g. MH~ class II
antigen expre~sion ~nd productlon of eico~anold~, chondrocy~es ~production of collagen type II), ~ib~obl~sts ~productlon of collagen 427~A~/Ai1/AS/ALM/A~l~.10.l~

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types I a~d II and eico~snoids), and octeoclasts ~bone re~orption).
TNFn and IL-6 share many of these effects ~ith IL-l, although IL-6 does not cau~e bone resorptio~ (Peder~en JG ~ Bendtzen K, unpublls-hed).

Se~eral cellulsr source~ of the cytokLnes are present in the ~rth-ritic ~oint, incl~ding ~0, B ly~phocytes, fibroblasts and ~ascular endPthelial cells (2,8). The demon~tration of IL-l~/~ in synovial fluids from rheumatoid and osteoarthritic ~oints further suggest~
that IL-l in partlcular may play an important role ln the p~thogene-sls of arthritis and, possibly, in osteoarthrocis (2, 14). An un-balanced reaction between immunocompe~en~ cells and accessory cellJ
such as M0, NK cells and poly~orphonuclear leukocytes ~ighe con~inu-ally induoe IL-l~/~, IL-6 ~nd other inflammatory ~olecule~, for exa~ple prosea~landin~ ~nd leukotrienes. This ml~ht contribute to the chronlc nacure of di~ea~es such as RA, The arthrlto6enic ~ctivity of IL-l~ is most clearly seen fro~ in vi w experi~ents using a rabbit arthritis model ~15~. Hu~an rIL-l~ ind~ce~
~0 and neutrophil ~cc~mul~tion 24 h after inJection in the knP~, and thi~ is a~ociated with depletion of proeeoglycan fram the articular car~ilage and an increase ln the glyco~minoglycan con~ent of the ~oint fluid. The same signs of cartllage da~age by IL-l~ ~re seen in ~oints of rabbi~s prevLou ly depleted of polymorphonuclear leu~ocytes and M~ by ~yste~lc administra~ion of nitrogen ~ust~rd. This sugges~s that IL-l~ itself ic capable of sti~ulat~ng resident cells of ~he Jolnt, such ac the chondrocytes, to c~u~e proteo~lycan depletion.
A~ain, it is possible tha~ some of the effects ascr~bed to IL-l~ may be mediated via local, IL~ Lnduced production of othe~ cytokine~, psrticulArly IL-6 ant IL-l~.

It ~s noteworthy that urate orystals ~re potent sctivators of X0 IL- l production (16). Thus, IL-l may coneribute to the manifestatlons of gout.

Insul~n-dependent diabetes mellitus (I~M) and thyroiditi~

427~ A.QQ~ 1/ASIAl M/A~M/~.10.19 ~ 39~Y ~ 180~'0N 0~,~1 '0'01 ~8/~NOW~ 13019NI~ ~ NN~W9nOlY WOY3 79(~

Recently, the predominant ~pecies of hum~n IL-l, IL~ ag shown to be a po~ent suppressor of insulin production in viero, possibly as a resulc of a direct ~nd selective cytotoxic effect on panc~eat~c ~slet ~-cells ~17). IL-l~ also retuces ln~ulln productlon, but it ~s 10 tlm~s le9~ potent than IL-l~. The effect of IL-l~ is blphasic because increased insulin productlon iQ con~istently obser~ed at low con-centrations of rIL-l~ (10 - 200 pg/~l 5*10 13 - lO ll M), ~he~eas higher concen~rations reduce both the extr~cellular and intracellular oontentc of ins~lin, probably by a direct and select~ve damag~n~
1~ effect on the ~-cells.

Althou~h TNF~ itself fails to ~ffect ~-cell function, the 4uppres~ive effect of IL-l~ is augm~n~ed si~nificantly by TNF~ ~3,8~, Ocher ~ytokinex, by them~elves or in conjunotion wl~h ILl~, fail to affect ~-~ell functLon~

It is interestlng th~t IL-6 au~ments the rele~se of insulin from ~or~al rat islet5 and that IL-lc snd IL2~ lnduce IL-6 in thè islets the~selve~ (17A). Furthermore, e~en though IL-6 {ncreases insulin prod~ction, this cytoklne c~ucec ctructural changes in ~-cells ~lmi-lar to those caused by IL-l (17A). Thus, looal IL-l-induced produc-tlon of IL-6 may underly the biphasIe insulin respon~e obser~ed with ~ar~ous conceneraeions of IL-l (17). Further~ore, IL-6, in con~unc-tion with IL-l, m~y contrioute to the immunoinflam~atory processes eventutually leadint to IDDM.

Similar effects ag those de~oribed abo~e have been obtained ~hen testing IL-l~ on h~an thyroid cell~ re~o~ed fro~ normal thyroid tissue during s~rgery of paraadenomatous ~lands (17A, 18). The ~ecre-tion of thyro~lob~lin ant cAH~ is markedly suppressed e~en by low concent~atlons of rIL-l~ (15 pg/~l - 10 12 M). The effect is a~g-mented by T~F~ ~nd, in addition, by IF~. The thyrocytes are not k~lled ~y IL-l~, but their ability to form follioles ~nd accumulate glycogen in re6ponse to thyroid stimul~ting hor~one i~ suppressed (18). Ag~in, ~ sti~ulatory effec~ on thyroglob~lln production i~
consl5tently observed at very low concentrations of IL-l~ (1.5 - 150 fgfml - 10 16 1~14 M~ (18), snd IL-1~ i~ a very potent inducer of IL-6 in thyrocytes (17A).

427~E~..0~/~31~AS/ALM~ 1.10.1~
8~ 3~ 80~'0N Ot.61 '0~'01 68/~NOW~ 1~019NI~ ~ NN~W~Ol~ WO~

~1790 The above findings and conslderat~ons indica~e a central role of M~
and perhaps ~K cell~, And ~heir product~ IL~ , TNF~ and IL-6 in a nu~ber of physiolog~cal and pdthophysiological condieions (3,8).
Prolonged ~xposure of cell~ to increAsed levels of these cyto~ines might lead to Gtruct~ral or funct~onal da~a~e to the cells (e.g., ln IDD~), for instance lf the target cells for unknown reasons are surrounded by cytokine-producing M0, NK cells and T and B ly~phocy-tes, a~ ~ the case in the early stage of IDDM deve~opment. ~ndo-~heli~l cells, fibroblasts and other cells in the connective ti~sue ~0 ~ay also contribute in these situations through an inappropriate production of cytokines ~uch as IL-l~/~ and IL-6. ~oreover, low concentrations of IL~ and IL-6 may accu~ulste in t~rget tis6ues by d~ffus~on fro~ the blood during conditions of stre6s, and the cytokine~ may therefore have important physiologic~l funcelon~ by potent1at~ng the secretion of insulin, thyroid- and, posgibly, pitul-tary- and adrenocortic~l hormones under these circum-~tances (Fig. 1).

As described above, the neg~t~ve associat~on between HLA-DR2 4TId the production of T~F~ may be i~plica~ed ln some Hl~-~s~ociated disea~e~ such as IDDM and ~A (3,8). In ehe lstter two, HLA-DR2 ls associ~ted with resistancq to disease, and thls ml~ht be expl~ined if TNF~ is involved, directly or Indirectly, in the destr~c~ion of i.clet ~-cells in IDDM snd of ~artil~e and bone ~n RA snd os~eoarthrosis.

Ther~peutie con~iderstions Considering the ~any p~tl~e pathophysiologlcal functlon9 of IL-1~/~, IL-6 and TNF~, intervention with the production or action o these hormones might be of g~eat benefit in a number of ~nfectious and immunoinfl~mmatory dlsease6 and, possibly, in certain neopla~tic di~ease~, The product~on of the ~edlators is prevenced 7~y hlgh doses of glucocorticoids, which diroctly suppress ~ene tran~cription and, if the gene$ have been tran~cribed, the ~obilization of ~R~A, at least ~n the case of IL~ and TNF~ (6,8).

The effect of IL~ on T cells in ~Lt~o i~ prevented by cyclo~porln (-Cyclo.~por~n ~) (19), by vlta~ln D3 (1725 (OH)2 D3) and a synthetlc 427~A.Oa2/AJ7 1 /AS~ALM/ALM/3C. 10.1 Y~
6~ 3~ I 80~ ' ON l ~ ~ 6 I ' 0 ' 01 6g / ~ NOW ) l~Ol~N I ~ NN~W911011 WOl~

vitamin D3 an~logue (20). Whether cyolosporin snd vitamln 33 also prevont ~o~e of ~h~ other activities of IL~ is not co~pletely clear, although their met~bolic effect on p~ncreatic ~-cell funct~on is ~lightly inhibited by both cyolosporin (21~ and vitamin D3 ~Buschard ~ ~ Bendtzen K, unpublished~.

Since cyclospo~ln has many se~ous slde-eff~cts, espec~ally during long-ter~ ~reatment (34), alternative m4ans of treating transpl~nt rejection and i~muninfla~matory disease~ are much needed, ~r~lbLtion of interleukin 1 and iT~eerieukin 6 functlons by sot~um 10 fwld~te (fusldln~

Fugldic scid ls a tetracyclic t~iterpenoic aeid wlth a steroid-llke primary st~ucture as des~ribed above. The drug is used cllnically both in i~s acid form, and a.~ the sodi~m and diethsnolamine ~alts as an antibioti~, psrt~cul~rly ao~ive agalnsc Stap~ylococcus sure~6 15 (22). It ha~ been indicated ~hae fusidic acid might have an 1nhibi-tory effect on T-lymphocytes in a mo~se model (23A), Fucldic acid inhibits in vivo and ~ vitro proteLn ~ynthesis in both prokaryotic and eukaryo~ic cells by inhibiting the attachment of tr~nsfer R~A to the 50S part of the riboso~es (23). Fus~din h~s fe~ and only trivi~l side-effect~, even d~ring lon~-term treAt~ent.

Acco~din~ to th~ presene invention it haa been ~ound that fusidin acid act as an i~munosuppressive a~ent and that its mode of action as an im~unosuppressive ~gent closely mlmicks ehat of ¢yclosporin ~ee the followin~). Hence, the potenti~ls of fu6idin ~herapy should be investlgatet in ~ll si~uations ~here cyclosporin ~reaemen~ is advooa-ted.

As ment~oned sbove, some of the key mediAtqrs of the body's response to en~lronmental faceors are a family of polypeptide hormones (cyto-kines) prod~ced by many cell types ~u~ most abundantly by ~0, The be~ characterized me~ers of the family ~re IL-l~ and IL-l~, TNF~
and the recently descr1bed IL-5 ~1,2). These cytokines part~cip~e ln man~ types of acute and c~ronic reactions lnvolving lnflammation, i~munological reaceione and tissue inJ~ry. Generally, IL-ln/~ ac-

4~7~02~A.002/~31/AS/ALM~ALM/30.10 19~
or 3~d ~ O~'ON 1~.~1 '3r'01 6g/~NOII~ l~Ol~NIn ~ NN~llOld WOd3 tlv~tes cells to cell ~rowth (e.g. flbroblasts, keraelnocytes antglial cell~) and/or increased a~tivity (e.g. pros~glandin E2 syn-the~i~ in fibroblasts, bone resorption by o~teoclaæts and activst~on of T and B lymphocytes already triggered by speci~ic antigens or ~olyclonal activators). In a few instances, however, IL-l appe~rS to inhibit cell function. For instance, IL-l is a potent suppressor of pancrestic islet ~-&ells and thyrocytes, and of melanoma cellc (2,3,8,17,24). IL~l also induees a broad spectr~m of $yctemle chan-ges, including fever, 510~ wave sleep, dnd ~ange.c in blood level~ of trace metals, ~lbu~ln and acute-phase re~ctants such ~s flbrinogen and serum amyloLd A ~2,8,25). IL-la/~ ~re therefore considered of central ~mportance in many if not all adaptive chan~es of th~ or~a-nlsm in response to varlous exogenous and, po~sibly, endogeno~s ~ti~uli.

Re~en~ly, IL~ have been recognized to share ~any activities w~th another cytokine, IL-6 (3,7). De~pite th~s, IL-6 and IL-l~/~ do not show any sequence homology, and the cytokines ac~ on different ~ocep-to~. IL-Ç has been ~hown to be identical wIth B cell-stimulating factor 2 (7~ and a fsceor ne~essa~y for the groweh o~ pla~acy~o~s ~nd B cell hybridomas (26-28).

Unre~ulated produetion of IL-la/~ and ~L-6 may be pathogeneeicAlly involved in the manIfes~ation of several human diseases a~ de.ccribed above, and the molecules have many biological effects which quallfy them ~s important ln the pathology le~ding to rheumatic dlseas~s, some endocrine disorders, and vArio~s skln dise~ses (see Table 1).

Drugs that Interfere with functions of IL~ and IL-6 are therefore of great interes~ in the manage~ent of many human disea~es.

As described in the examples, it has been observed that sot~m fusi-daee ~fusidin) inhl~Lted various IL-l~/~ functions in vitro when admini~tered in th~rapeutic and non-toxic concentrations and thus prevented the ly~phocyte cos~Lmulatory actlvLties of these humsn cytokines. The drug wa3 ~lso found to interfere wLth the hybridoma growth-promoting ~tivity of I~-6. Fu~thermore, the dru~ prevented 42740~A ~ M/ALM/3~.10.19~9 1~ 3~ 0~'ON ~ 1 'O~' 01 ~ NOW~ l301~1NI n ~ NNI~W~ I101~ WO~

~0~:~791 "non-i~munolo~ical" functions of the cytokines, including ~he toxic effect of IL-l*/~ on In~ulin prod~ctlon by pancreatic ~-cells.

The sbove described functional p etern of sc~ivity lc s~rlkingly si~ilar to that of cyclo~porin, a drug used exten3ively to prevent tr~nsplant reject~on and ln the management of seversl imm~noinflamma-tory disorders. Thus, it i& conte~plated that interference with the immunoactiv~lng signal delivered by IL~ and, po~ibly, IL 6 before and around transfer of a graft by fusidic acid tre~t~ent wlll induçe speciflc and l~stin~ paral~si~ of those host ly~phocytes otherwise programmed to respond to the graft, Such treatmen~ would spare all other clones of lymphocyte~ in the recipient, and life-long immunoguppressive ~herapy might nO longer be needed. Treat~ent with fusidic acid or derlvatlves thereof i~ con~emplated to be beneflcl~l, according to the present invention ln both the life-threatening sit~a~ons dominated by ~ nd ln other more cu~tle vasculsr di-seaees ~e,g. arteriosclerosi~, Al.~o, B dysre~ulated production and/or f~nctLon of IL-6 ~ay be a key event in the p~thogenesis of at least so~e lymphoid proliferative dL~ea~es. ~rugs that ~nterfere with the function of the cytoklne cuch ~9 fu6idic ~cld or derivAtives thereof are therefore contemplated to be cllnically useful. It is also contemplated that interference with the functions of IL~ and IL-6 with fu~idin o~ fusidic acid derivative~ ~ay be of therapeuelc i~portance in several typeA of arthritiç and de~enerative ~oint diseases.

25 U~e of fusidic acid dc~ord~g to the present ~nventlon In accordance wlth ~he present i~venelon it hss been fo~nd that fusidlc acid snd derivatives thereof ~re useful for preventing effects of cytokine~ known to be pathogenetically involved ln the previous described p~thologic~l conditions, In accordance with ehe present invention the term "fusidic scid or a deriva~ive ~-hereof" comprises any phar~ace~tically active and acceptable çompound being identiçal or structurally ~i~ilar to fusidlc scid and exhibiting relevant biological ~ctions simil~r to those of fusidic acid, includlng derivatives of fu~ltic açid, 4~ X2/~l~AS~AUM/AUM/~101 '~ 39~ S~ gO~'ON ~,61 'O~'01 ~g~NOW~ 1~019NI n ~ NN~W~llOl~ WO~

9~
1~
espeei~lly ph~rmaceutically accept~ble salts, e~ters and solva~e8 as well as con~ugat~s of fusidic acid or of the f~sidic acid deriv~tlve 6 .

Suitable salts are salts with pharmaceutically acceptable bAse~, s~ch a.c alkaline esrth ~etal salt.~ t alkali ~etal salts (especially sodl~
sslts), ~mmoniu~ .calt~ or smine salts (e~pecially the diethanol~mine sAlts).

Suitable esters are easily hydrolysable esters, e.g. i~usidic acid acetoxymethyl ester.

Suitable colva~es are e.g. hydrates, L~ particular hemihydrates, Suitsble con~gates are especially con~ugates with taurine o~ glyci-ne.

"Derivatives oi- fusldlc acid" co~prlse e.g. the following co~pounds:

3.dehydrofusidic acid;
3,11-didehydrofucld1c acid;
24,25-dihy~rofusidic acit;
17,20-24,25-tetrahydrofusidic acid;
17,~0-24,25-tet~ahydrofu~dic acid and their corresponding 3-aceeate~
~ecpecially conJ~gated wLth glycine or ta~rine);
3-0-acety1-16-ep~deacetylfusidic acid.

Also included in ~he present inven~ion is the uso of the phy~iologi-cally acceptable derLvatl~e~, includin~ f~ncti4na1 derivatives, which are disclosed, a~ well as their preparation, in the patents and the references llsted above in the section ~Baekgro~nd of the inven~lonn, e.g. the derivatives disclosed in U.S. Pa~ent No. 4,004,004. Some of these derivatives are l~sufficient as antibacterial sgents - a feAt~re which may be benefic$al in the use accordin~ to the pre~ene invention.

Of partlcular intere~t in connection with the present invention are co~pounds having the general for~ula II:

427~r''.~A311AS/ALM/~LM130.10.1989 ~ ~ 3 ~ ' O N ~ N O W ~ l ~ O l ~l N I n ~ N N ~ W ~ O l ~ W O ~ ~

F~OMPLOJ~M~N ~IJII~TOFT ~MON)~ '4~NO,~g31~4~45 PR~E 34 C~H~C~3 ~COA

R~ R~ I I

Is c~3 R~/
~ H
CHI

where~n the dashe~ lines indicate ehe presenca of eithe~ single or double bonts betwee~ ehe ato~s in question, with the proviso tha~ one of the bondc C~13-C~17) and C(17)-C(20) is a double bond, A 1Q sel~c~ced f~om the group con~L~tsng of OH, O~', and NHR", wherein R' is ~elected from ehe group consiseing of C1-C4 alkyl, aralkyl, such ~s phenyl-C1-C4-alkyl, and aryl, such a~ phe~yl or o-, n- or p-~olyl, and R~ ~ CH2COOH or C~2CH2S03~, R Is OH or 8 keto oxygen (-O), ~hereby, when R Ls OH, the C(ll) R bond is preferably Ln the n configuration with respect to the C(1g) ~ethyl group, R1 Ls hydrogen, halogen or a n~er~l ~ro~p, or R1 ls selectet from the group CnslstinB of -OR3, -~HR3 and -sR3, In which R3 is hydrogen or i9 an organic group cUch a~ acyl, in parelcular C1 C~-alkylcarbonyl, elkyl, Ln parelcular C1-C4-alkyl, aralkyl, ~27402~A.WZ/~ AS/ALM/AL!~/30. 10.19~9 F~OMPLOJ~M~N ~ ~INI~TOFT ~MON~1g~ 44 N~ g~1~4645 PRI~E 3~1 9~

such as phenyl-C1-C4-al~yl, or aryl, such as phenyl or ~-, ~- or p-tolyl, R2 is OH or ~ keto oxy~en (-O), ~hereby, when R2 is OH, the ~(3) R2 bond is preferably in the ~ configurAtion with respect to the C(l9) ~ethyl group, or R2 is hydrogen or halogen or nltril group or ~ group -OR3, -~HR3 and -SR3, in which R3 ih hydrogen or is an org~nic group such as acyl, in pa~ticular Cl-C~-~lkylcarbonyl, alkyl, in particular ~1-C4-alkyl, aralkyl, ~uch as phenyl-~1-C4-alkyl, or aryl, such AS phenyl or o , m- or p-tolyl, and ~ale~ thereof.

Examples of Cl-C4-alkyl are 8s methyl, ethyl, propyl, lsopropyl, butyl, isobutyl, sek.butyl, and tere. butyl. ~1-C4-alkyl and the other groups ment~oned ~bove may optionally be substituted where appropriatc, such a3 diQclosed in the pa~enec and other literature referred to ln the above section HBackground of the in~ention~

A~ong the compounds of the genersl formula I, preferret compounds of th0 ~ener~l for~ula I sre, e.g., compounds ~hcrein A is OH, a double bond i9 present in the posit1On ~(17)-C(20~ and in the po-q1tlon ~(24) C(25), tho hydrogen atom at C(10) is in ~hç ~ configurAtion with resyect to the C(1g) methyl g~oup, R is a h~droxy ~ro~p in the configuratlon with respeet to the C(19) methyl group, R1 is a C1-C4-alkylcarbonyloxy group such ~s an acetoxy group or ~ Cl-C4-~lkylcarbonylthio group, an arylc&rbonylth~o group such as a phenylcarbonylthio group, or a C1-C4-alkoxy group ~uch as a methoxy or eehoxy group, snd R2 is ~ nitrlle group, a Cl-C4-alkylsulfonyloxy group, or, in particular, a hydroxy gro~p in the ~ configurs~ion with respect to the C(19) methyl group, in partloula~ fusidic ac~d ~nd 3~ fusldic ~cid salt~ such as the sodium salt (fusidin).

Specific exs~ple6 of lnteresting der~vstives are the derl~te~ termed 4~02~0a21.431 ~AStALM/ALM/30. 10.1 g~

F~OM PLOU~MP~N ~ TOFT ~MO~ 5 NO. ~ 4~5 P~E ~6 V3 1177, 117~, 1303, 13~0, 13~2, 1460, 1546 and P~ 10~9 described in table 2 belo~.

~ ,,P
' 4 ~ 2 ~1~3 ~SCII~CH3I2 Substi tuen~ s Ref erences i~
Code Ser~l~tl~re Rl R2 ~3 ~4 Cpd. hlo. Tacle Pag~

VD 1177 N~ A, s ~-OH ~-OH O~OCH3 ~HC132CH2503Na XVIb III 107, lOe VD 117~ ~a A, ~ a-OH 3~-OR OC~C~3 N~2C2~a XvIa II~ 107, 10~
VD 1303 Na A, d -OH ~-OH OCH2CH3 O~a XLI lJII 120, 122 VD 1~60 Na A, d ~-OSO C~l ~t-OH OCoc~l ONa t,Xvlc XIII 139, 139 VD 1362 Na A, d a-~3 1-OH OCOCH3 Ol~a LxxvI XIV 139, l~o t~ 1460 Ya A, d Q-OH ~-Oii SC~CHl O~a ;~r.a VI 119,119 V~ 1546 ~a A, s a-OH ~-OH SCOC6H5 ONa XLb VI 113, 119 PR lO99 l~a ~ LIV X I a~, 130 Adv. Appl. Miorob~olo~y 25, 95-146 ~197g) (4 c - 2q . 25-sin~le bond, d - 24, 25-double bond 5truc~ure a~ indicated, but 24,Z5-eingle bo~d 4~ A002/~ 3/ALM/ALM/~0.10.1980 F~O~ PL~M~ UIN~TOFT (MON~ .3~ '45 NO, ~g31~6~5 PRli~ ~7 The following definitions fire e~ployed in the p~esent tex~:

"Cytokine" is a ~eneral term for ~ protelnaceous mediator relea~ed primarily but not exclu~ively by a cell popul~eion of the i~une ~y3te~ as ~ response ~o a specific stimulAting agent, e.~. a specific ant~gen o~ an alloantigen; or a nonspeclfic, polyclonal ~ctivator, e.g. an endo~oxin or other cell wall component3 of a gram-neg~tl~e bacteria;

~ly~phokine~ ls a general term for B proteinaceous ~ediator reles6ed by sensit~zed lymphocytes 8S a resp~nse to a stimulating agent, e.g.
~ 6peci~ic antigen or an alloanelgen; or by a ly~phocyte challenged by a polyclonal activator, e.g. an endotoxln or other cell wall component of a gra~-negative baceeria;

"interleukin" Is a general term for ~ proteinaceous ~ediator released primarily but not exclusively by a macrophage, T, B or ~K cell as a lS response to a stimulating agent, e.~. a specific Antigen or an al-loAntigen or by a cell ch~llenged by a polyelonsl activator, e.g~ ~n endotoxLn or other cell wall compon¢nt of a ~ram-negative bacterla;

"monoklne~ is a genersl ter~ for a protein~ceou~ mediator rele~sed by a mononucleAr ph~gocyte (e.g, a ~onocyte or B macrophage or a Kupffer cell (liver) or fi Langerhans' cell (skin)) as a response to any stl~ulat$ng agent;

"lnterferon" lc a general term for a proteinaceous sntiviral and/or ~onocyte/macrophyl activat~ne factor released by all eells a~ respon-se for a viru~ or sn interferon-induçer such ag ~ polynucleotid~; in particul~r cellc of the immune ~yste~ as a response to a specl~lc stlmulating agent, e.g. a ~peclfic antlgen or an alloantigen, or a nonspecific, polyclonal ~ct1~ator, e.g. an endotox~n or o~her cell wall co~ponents of a gra~-negative bacterla:

Rcolony-stimulatin~ façtor~ is a ~eneral term for a protelnaceou~, h~emaeopoletlc colony-stimul~ting medl~or releas~d primarily but ~ot excluslvely ~y a ~ell population of the ~mmune sy~tem as a response to ~ ~peclflc ctimulating agen~, e~ g~ a specific antigen or an al-~n~o2~oo2/A31 /AS~ /ALM/30. 10,10~4 F~OM ~OU~M~N~ TOFT ~MO~ l 19'46 NO.2~164~45 P~E 3 sa~

loantlgen; or a nonspecific, polyclonal activator, e. ~. an endotoxinor other cell wall components of ~ gram-negative bacterla, One ~spect of the lnvention is the use of fusidlc ~cid or a functlon-al derivative ~hereof for the ~anufactu~e of a phar~aceutical compo-sltion for substantially inhlbitlng a biological effect in a hum~nrelated to a cytok~ne, such 85 a lymphoklne, interleukin, ~onokine, interferon or colony-stimulating factor, for the prophylaxis o~
treatment of 8 condition related to a disturbance of a cytoklne ~ystem such as the ly~phokine, in~erleukine, ~onokine, Interferon or colony-stimulating factor system. A~ u~ed herein the term ~pha~aceu tical co~po~ition" compr~ces any composition suitable for human use.

The ln~entlon particularly ~elates to the u.~e of fusidic acid or a functional deri~ative thereof for substantially inhibitin~ a blological effect in a hu~an related to a cytokine for the prophylaxis or treatmen~ of a condltion relAted to a d~sturbance o~ a cytoklne system; ~nd/or - for subst~ntially lnhibiting a biological effect in a hu~n related to a lymphokine for the prophylaxi.~ or treat~ent of a condition related to a disturbance of 8 lymphokine system;
and~or - for ~ubctantially inhiblting a biolo~lcal effect in a hu~an related to n inte~leukin fo~ ~he prophylaxis or treatment of a condition ~elatod to ~ disturbance of ~ interleukin gystem;
and/or 25 - for substantially inhf~ftfng 8 biological effecs In ~ hum~n relatet to a monoklne for the prophyl~xis or tre~tment of B
cond~tion related to ~ distu~bance of a monokine system, and/or - for substantially fnhlbltln~ a biological effect ln a hum~nrelsted to an interferon for the prophylaxi~ o~ treatment of contltlon related tO A disturbance of a lnteri'eron ~y~te~;
and/or U7402E~002tA31/A8/~LMIALMf30.10.1~

F~OM PLOJSM~N~ TOFT ~01~ 113,~ NO.~ 3164~45 P~E ~S
'7~0 - for subQtantially inhibiting a biological effect in 8 human related to a eolony-stimulsting fac~or for the prophylaxl~ or treatment of a condition related to a dist~rbance of a colony-~ti~ul~t~ng factor system.

As used herein "substantially inhibiting~ comprlses a therapeutically relev~nt p~evention of the hsrmful biological actions of ~ald me-didtor, sald pre~ention being based on fusidic Acid or derlvatives the~eof or combinations wlth othe~ treatments/drugs e~erting a form of an~agonistic effect on the actions of th~ ~ediator in question.

Said conditions have been descrlbed above, and the ~ondltlons descr~-bed co~prise dlseases occuring in h~mans which ~lqeases demand th~ra-peutlcal intervention A9 w~ll as cond~tions in which a suppression of a cytokine sy~tem is desirable such as in connection with transplan-tstion or eye s~rgery. A~ ha~ been described in detail a~ove, clini-cally lmportant lnterleukins in thls context are in p~rticular lnter-leukin l (~ or ~), and interleukin 6.

In one of it.c bro~des~ a~peots the lnvention further relates to the u.~e of fusidic acid or i~s derivatives a5 A sub~tantially .~elective immunosuppressive drug and/or as a drug the aotion of which is via an antagonistic effect on the action of one or more cytokines.

~henever systemic treatments are employed, the adminlstration may ~e orslly, rectally and~or parenterally, the ad~nistration being depen-dent on the patlent's age and we~g~t and the particul~r condition being treated ss ~ell as the se~erity of che diseaso.

The active l~edient or combinations t~ereof ~ay be oontAined in any approprl~te amount in a co~posltion, and are generAlly ~ontalned in an ~mount of 1-95X by weight, bàsed on the total weight of the pre-paration~ The composition may be in sny appropri~e unit dosage for~.

Parent~ral admini~tration may oomprise suitable in~ect1On, ~.g.
lntraveneous, intr~muscular, intraarticular, intraooular or retroocu-4~ A31l~sJAuM/AuM13o1ol~

F~CM PLOU~M~N~ ~ v~ T~iT ~M~ .3~ 47 NO1~83164~45 P~E 41 lar inJection, infusion or implantation of e.g. sult~ble dellverydevlces, For~ulatio~s for parenteral use may be presented in unl~ dose for~s, e.g. ~mpoulec, or in ~lti-dose containers w~th an added suitable preservative. The ~omposltion may be ln orm of a solution, ~ suspen-sion, an emulsion or a delivery de~ice for i~plantation or ~y be pre~ented as A dry powder to be recon~tituted with water or ~nother suitable ~ol~ent before UBe. Ap~rt fro~ the active d~ug sub~tance the composltions may comprise suitable pharmaceutically 3cceptable carr~-er~ and/or exipients. Further~ore the co~position may, in addition,conveniently co~prise su~penting, 6tabilizlng, pH-ad~usting agents snd/or di~perslng agents.

Compositions for oral or rectal use may be formulated according to conventlonal ~har~aceutical practice and may be in the form of tab-lets, capsules, pills, powd-rs, ~mpoules, gran~lates, dragées, p~-ste~, gels, suppositories or enemas; or liquld for~ulations such as su6pensions (oily or aqueous), solutlons, elixirs, emulsions or drenche~ And the llk~.

The auxiliary additi~es of the phar~ceutical compo~ltions ~ay be any convention~l ph~rmaceutical additives and carrierc:

Binding ag~nts such as cell~los~ derivatives, starch, gelatin and polyvlnylpyrrolidine; fillcrs cuch a-~ sugar, mannitol, lactose, ~crocrystalline cellulose, potato starch, ealclu~ phosph~te; ~u~-table absorption promotorg; disintegrants such as pOtAto starch, slginic ac~d, lubrlcants such a~ ~a~nesiu~ ste~rate, stearic acid, talc, emulslfyin~ agen~s such as lecithin, sorbit~n monooleate;
wetting agents such as lecithin, polyoxyethylene esters; or buffering sgents such as acetate, citrate, phosphate, The colid compositions ~y by ~eans of specially adapted coatlng techniques be provlded wlth a coating adapted ~o protect the co~pos~-tlon from unwanted che~lcal changes prior to the release of the active compound. The coating ~ay be adapted to release the act~ve SJALM~ALM/30 .10. 19~

F~OM PLOU~MI~ TO;T ~ON)~ '4~ NO.7~1g31~4~45 P~liE 41 compo~nd in a pre~etermined p~ttern e.g. in order to achieve a con-trolled release formul~tlon.

The dosag~ of fusldic acid or its derivatives depend on the sdmini-stration method, the disea~Y, ehe ~everity of the di~ease and of the wei~ht and ~go of the patient. For compositlons adaptet for oral adm~nistration, the dosage i5 often 3.5 m8-1 g ad~lnlctered 2-6 times d~lly or for in~t~nce 0.5 g x 5 d~ily or in certaln cases 1 ~ every second or third day. Thu-c, oral ad~inistration may comprise ranges fro~ approximately l mg to 75 ~g per kg body weight per dsy or ~n appropri~te c~ses O.l mg to 20 mg per kg body weight per day. A
preferred dosage re~ime is normally 0.5 g 3 tl~es daily. In çertain csses, e.g. in the trea~ment of graft-versus-host disease the dosage is normally doubled. When fusidic acid or a derivative thereof Ls sdmin$stered rectallyt a somewhat higher amount ~uch as from about 1 my to ~bout lO0 ~g per kg body weight per day is usually preferred.

For pa~enteral administration, a dosis of about 0.1 ~g to ~bout 50 mg per kg body ~elght per day, most preferably in an a~ount from about 1 mg to about 20 mg per kg body welght per day i~ convenient, When adminlsterlng fusidic acid int~aarticularly, an amount fro~ about 0.1 ~0 ~g eo 20 mg per ~g body w-lghe per day l~ ususlly preferable. For parenteral ad~inistration, an aqueous .colution of e.g. 0.5-2X or more of the ~ct~ve i~gredlent may be employed.

Uhen iusidic scid or a derivati~e thereof is sdministered to the eye, an amount from about 0.1 mg to about 50 m~ pe~ kg bady weight per day 25 i~ usually preferre~.

In every case, the dosage should be carefully atapted so a~ to imply the specific a~tion in the cytokine system, l.e, attain~ent of op-tlmal tot~l dosa~es, dos~ge farm~ and dosage frequency. In cert~in ca~e~ it is rele~ant to admini~ter a relatively high unit dosis, a so-c~lled "boost-r dosls".

Ph~r~aceutical co~positiong for topical use such a~ co~po~ieionS
suitable for ~pplicstion to the skin aocording to ~he present in-venelon Are ~uitably creams, gels, oint~ents, lot~ons, liniments, A n'V/J~ J~/30.10.1~9 F~OM PL~ M~N~ ~ U I N~TOFT ~ MO~ , 3~ 48 NO . ~ 4~45 P~liE 4 suspension~, sol~tions. pastes, sticks, sprays, soaps, 5hAmpoos~
powders, suppositorie~ and enemac. The topical administratlon should be onto or close to the pathologlcal changes in the body, The compo-sitlons may be any sultable medicated masC adapted fo~ tirect appli-catlon or for introduction lneo the relevant orifice of the body,e,g, the rectal, urethral or vaginal orifice. The compositlons may simply be applisd d~rectly onto the diseased part, e,g, ~he skin or mucos~. Other rele~ant formulation ad~ptations for applic~t~on to the eye ~ay e.g. be eye lotlons, eye ointments, eye-drop~ o~ dru~ deli-very syste~s adapted for admlnlstration to the eye such a~ compo-s~t~on~ suitable for l~plantation admlnlstratlon. In certain ca~es it ~ay be appli~d by means of speci~l medical de~ice.~ such 85 dressings lncluding occlusive dressings, or alternaelvely plasters, p~d~, .Qponges, strip&, or oth~r forms of suitable flexible pieces of ~B-teri~l.

~ormul~tlons fo~ topical ad~lnistratlon ~y comprlse pharmaceuticsllyscceptable carrisrs and~or exlpient~ such as oint~ent bales ~e.g.
p~raff~n, polyeehylene glycols, Tween~, Span , ~eget~ble olls), suspendin~ o~ emulsii~ying agents (e.g. leclthin, ~orbitan ~onooleate, ~elatin, methyl cellulose, gum &CBcia, ~orb~tan ~onoolea~e derivati-ves), gelform~ng agents ~e.g. ~drbopol, alginates, gela~in), preser-vatlve~ (e.~. methyl o~ propyl p-hydroxybenzoates, benza~koniu~
chloride), ~ntioxidants (e,g, ~ocopherol, ascorblc acid, butylated hydroxy anisol), humectants (e.g. glyce~in, propylene glycol, urea), ~5 ant perfumes and skin protecti~e agents, Formul~tion~ fo~ topical admlnistration to the eye may comp~ise ph~maceutlcall~ lnert vehicles or be in for~ of a suit~ble carrier system, Pharmaceutically accept~ble carriers and/o~ exipients for the preparation of eyedrops lnclude for example b~ffering agents s~ch ~s ~oric acid or borate~, pH adJusting agen~s to obt~in opti~al stabili-ty or solubility of the actuve drug substAnce, ~gen~Q to ad~ust the tonicity such ~s sodl~m chlor~de or bo~ates, v~scosity altering a~ents such ~8 hy~roxypropyl cellulose, methylcellulo~e, polyvinyl-p~rrolidone, polyvinyl alcohols or polyacrylamid~, oily vehicle~ such as vehicles co~p~sing ar~chis oil, Castor ~nd mine~al oils. Eyedrops presented as ~mulsions or suspenslons ~ay f~rthen~ore co~prise stabl-427 ~~/~31/AS/~LM/ALMj30,10.1~

F~O~l P.OU~M~NN ~ TOFT ~MO~ 19' 4~ N0~ 4~45 P~E 4 liz~ng~ dl~persing~ wetting~ emulsify~ng andtor sus~ending ~gents Eye lotions and eye ointments ~dy co~prlse pharmaceutically accep-table carriers and~or exlpients such ~ used ln an eyedrop co~posi-t~on or in other relevant topical compo~ition, e. e oint~en~s, cre-am~, lot1ons and the li~e.

An ex~ple of a general ~ethod for preparing aqueous eyedroph eomprl-s~ng an ~ctive drug substance ic to dissolve the ~ompound (prefer~bly a~ the soluble sodium salt) in ~terile wster in ~ given concentr~-tion, optionally ad~ust the pH tO d suita41e value with ~ suitable buffe~ or w~th hydxochloric scid or ~odiu~ hydroxide, optionally add ~ p~eservative like phenethanol or chlorobutanol, op~ionslly add a vi~cous alterin~ exciplent like ~ethyleellulose, and ste~ilize ~he fin~l solution by e.g. autocl~vation by use of one of the ~enerally accepted cycles or by membrane filtration.

The topic~l compositlonc for use acco~din~ to the present invention co~prl~e e.g. 0.001-60~ of the ~ctive ingredient, pr~fer~bly 0.1-20X, especially 0.5-lOX or 1-5X.

According to the invention the compositions may be applied several timeC a day, e.g. 1-5 ti~es a day, depending on the condition in question and the se~e~ity thereof and furthermore on ~he ab~orption conditions on the site in question.

In e~ch case, the part~cular dosis ~ill depend on obtaining ~n appro-p~iate abæorption of the fusidic acid or de~l~at~ve thereof in ~
sufficient for~ and ~mount, In e~ch case, said ~mount and for~ should ~5 be sd~pted ~o ~s to exert 1ts specific ac~ion ln the cytokine syste~.
Hence, it ig very important e. e ~hen application onto the ~kin is performed - ~o engure that the p~tient spplies a relatively well-defined layer of the composition.

In particular, the invention relates to the use of fusidic acld or a deri~ative thereof for prophyl~xis or tre~ment of diAbe~es mellltus, in particul~r insulin-dependen~ diabete~ ~ellltu~ (type 1), in particular for the pre~ention of pro~ression of diabete6 ~ellitus (type 1), especlally ~ub~tantially im~ediately after the first 4274~2a~ ~2~1/AS/ALU/~LM/30.10 19~9 i~OM PLOll~M~N ~ ~IN~TOFT ~MON~ NO.~ 16~645 P~E
7~0 diagnostlc establishment of diabete~ ~ellitus, or for prophylaxis afte~ establi~h~ent of being ln a hl~h risk ~roup of developing dlabetes melli~us.

These uses RS well as other uses and ~ethods of the invention will preferably be noted specifically and in dètail ac explained herein in lnetructlons for use provided to the physician snd~or to the patient together with the compo~itions to be used.

During the last few years snimsl investigations have shown the capa-bility of cyclosporin (a cyclic peptide ~eeabollte of the fungi cyllndroc~pon lucidum and tr~choderma polysporum) ~o prevent diabe-te~, first of all in the spontaneou~ly diabetic BB rat.
Clinicsl studies performed on diabet~cs a~d lnvolvln~ lnve3tiga~ion of cyclo~po~in's effect on progresglon of diabetes have shown that clinlcal remicsion was obtsined by ~eans of the cyclosporln therapy;
this seemed to be due to imp~ove~ent of the functional ~-cell msss.
The re~ission ratc was greater in dlabetic pstients who ente~ed after le95 than ~ix ~eeks w~t~ symptoms and two weeks of ~nsulin therapy, than ln those who entered later. The finding~ demonstrate th~t an acute process affecting the ~-cells can be modulated by initistion of the cyclospo~ln immunosuppre~cive therapy within ~ critically brief time intervsl sfter on6et of overt disease, and th~t continued tre6t-ment can malnta~n partlal or co~plete remission th~ough a periot of at le~t one year.

A~ used in the present specification and claims ~ the term ~5 ~substsntislly immediately Af~er" co~pXi~e5 a period afte~ the first diagnostic ostablishment of the disease or a high ri~k of devYloping the di~e~e within a llmited nu~ber of months or years whLch perlod should be a~ short as possible, so as to enable intervention with the progres~ion of ir~eversible change~ in the p~hological body tissue~, sait ~nvent~on bein8 obtained by means of the use accordlng to ~he lnvention o~ fusidic scld or derivatives thereof, The admini~tration of cyclosporin Increased the remission ~ate in d~abetes mellitus (type l) of recent onset, and enhan~d ~nd preser-~ed ~-cell function th~o~gh tho flrst ~ear after diagnosis. It ~ 5 ~27402~002/~31tAS/ALMJALM/30.10.1~9 F~OM PLOU~M~IN ~ ~IN~TOFT ~MON~ .5el NO.~ 4~45 P~GE 45 lnferred that these effect3 were induced by virtue of ~he immunosup-pre~iv~ a~tion of eyclosporin. Thus the findings s~rongly support the hypothe~is th~t an immune process is involved In ~-cell damage in the hu~an disesse.

The effect of duratlon of diabetes on the early outcome sugge~ts that al~hough the immune processes affecting ~-cells may be chronic and precede dlagnosis through a long period of ti~e, the development of overt disease la associated with an acceleration of events.

From e.g. the above-described experiments it i~ inferred th~t the potenti~llty for recovery of insulin secretion is grester than for-merly believed, and that by ad~inistration of fusidlc acid or a deriv~tive thereof, the secretory capacity can be maintained ln the nor~al range through one year or more of immuno~uppression in ~any p~eients.

Thus, fusidic acid and derivatives thereof ~ay be used ~ccording to the present invention for the prevention of progression of irrever-sible pathological processes in the pancreatlc tls-~ues which other-wise le~d to diabete~ ~ellitus (type 1~. The therapy of the invention is preferably lnstituted as soon as possible, i.e. very shortly ~fter the onset of diabetes or, preferably, ~mmediately after dia~nosis of pathological proeesses in the islets of pancreas ~e.g. before clini-cal mani$estation of diabetes mellitus (type 1)). The therapy of the lnvention ~ay also ~pply to certain identified high risk g~oupc the ldentification being e.g. obtained by mean~ of a ~creenln~ program e~ploying markers relatively 5péciflc for pathologicsl chAnges ~en ln diabe~ec mellitua ~nd/or preli~inary stages the~eof, especially markers of ongoing pathological changes ln pre~icposed individuals (e.g. in HLA-DR 3/4-positive lndlviduals). A person ~s conte~plated to b- in a hlgh rlsk group of developing diabetes mellitu~ (type ~0 if hi~ risk is about lO-IO0~, such ~s a~out 50-lOOX.

In particul~r, the present invention rel~tes to the use of fusidic acid or a deriva~ive thereof for prophyl~xls or treat~ent of diabete6 mell~tus (type 1) for oral or rectal use.

42?4~ LM/3o~ ~ o~ 1 o89 F~OM PL~U~MR~ TOFT ~MON)1g~ 1~.31~ '51 ~1O,~g~1~4~45 P~I~E 4 Q

In another a~pect, the Inventlon relates to the use of fusidic acid or ~ derivativ~ thereof for the prophylaxis or treatment of condit~ons related to the function of the thyroid gl~nd, in psrticular hyper- or hypofunctlon~ng of s~id gl~nd, e.g, thyroiditl~
~acute, subficute or chronie~, includin~ Hashimoto'~ dises~e (lymphocyeic thyro~ditls; lymphoid thyroiditis~, Riedel's thyroidltis ~çhroniç fibrous thyroid~tis), de QuervAin's thyro~ditls ~sub~cute granulomatous thyroiditis), subacute lymphocytic thyroid~tls, Graves' disease, ~raves' ~ubacute thyroiditl 9 ~nd Grsves' ophthalmopathy.

For a plurallty of dise~ses e.g. the condition~ related to the func-tion of the thyroid ~land, the inflam~atory diseases of the gut, arth~itis urica, multiple sclerosi.~, B and T cell lymphoma such as ~ultlple ~yeloma treatment with fusidic scid or derivatives thereof can be beneflcial to the patlent also in a period of inapparant disease, in ~he way that the treat~ent w~th fusidic acld or deri~atl-ves thereof ean prevent or postpone a ~el~pse of the condltion 1~
question, By the t~r~ "treatment~ is therefore generally al~o meant pr~phyl~çtic treatment designated to prevent or postpone a relapse of the dl~e~ e.

In another sspect, the invention relates to the use of fusldic acid or ~ derivstive thereof for the prophylaxic or treatment of a condition related to hypofunction of the adrenal ~landc, ln part~cular Addison's dise~se or further~ore Sl~onds' panhypopituita~ism.

In ~nothe~ aspect, the invention rel~tes to the use of fusidic ~cid or a f~nctiona derivative thereof for the treatment of endogenous uvelt~s. By the term ~endogenous uveitis~ is meant e.~. nDn-infectious uveit~, such as non-infectious uveitl~ anterlor and uveitis posterior, The present invention relates in particular to the manufactu~e of a composition suitable for treat~ent of the eye, such a~ a~ eye drop composition, an eye ointment co~position, an eye }otlon composition or an in~ectable composi~ion for intraoc~la~ or retroocular in~eetion, or an oral composicion.

F~OM PLOUiM~NN ~ UIN~TOFT ~MON~1g~ 3~ '51 NO~g~1~4~45 P~l;E 47 7~0 In yee another ~spect, the invention relates eo the use of fusidic scld or a derivative thereof for the pre~ention of the infla~ation after eye surgery such as cataract operation or l~ser 5urgery. This treatment, as ~ell a~ treatment in other conditione, can in some cases be prophylactic in the ~ay the the goal of the treatment is to diminish or avoid an lnfl~mm~tion not to actually treat the condition. The present Lnvention relates in particul4r to a compo~ition ~uitable for treatment of the eye, ~uch as an eye drop composition, an eye ointment co~pogition, ~n eye lotion composltlon or an in~ectable composition for intraocular or retrooc~lar ln~éctlon, In another aspect, the invention relates eO thè use of fusidic acid or a derivative thereof for treatment of progres~ion of arthritis such as rheumatoid arthritis including ~uvenlle rheumatoit arthritis, psoriatic arthritis or Relter arthritt~ subst~ntially immediately afeer the first dla~nostie establishment of arthritis. T~e ther~py of the invention i8 prefer~bly instituted as 900n as pos~ible, i,e. very shortly after the onset of arthritis or, prefera~ly, immediately after diagnosis of p~hological processes (e.g. before clinical manlfestaelong), The therapy of the in~ention ~ay al~o apply to certain identified high risk groups the identification ~ein~ e.~.
obtained by means of a ~creening prog~a~ employ~ng markerg relatively specific for arthritic changes and~or prelim1nary ctages of artbriti~, especlally m~rker~ of ongoing pathological changes in predi~poset ind~viduals, in p~rticular indivldusls having specific HLA ti68ue types. The invention relates in particula~ to the ~nufact~re of a co~po~ition for or~l use or for use as parenteral or lntraartlcular inJectlon-~.

In another aspect, the in~ent~on relates to th~ use of fusldlc acld or a derivative thereof for the prophylaxis or ereatment of osteoa~throsls, In another aspect, the invention relates to the use of fusldlc acid or a derivatlve thereof for the prophylaxis or treatment of arthrlt~s urlcn ~ goue) .

4271~"' P~/~ SJALU/ALM/30 lO.l~

F ~I O M p L ~ T :I F T I M O ~ , 4 7 ~1 0 . ~ ~ ~ 3 1 5 l~ 3 5 ~ P R S E

In another aspect, the lnvention rQlates to the use of fuhldlc acld or a derlv~tlve thereof for proph~laxls or treAt~ent of ~ condltlon r~l~ted to transplant re;ection, such as a graft-versus-ho~t re~ct~on, or ~ny other con~ition~ related to e.g, marrow, corne~ or skin tran~-pl~ntat~on. The treatment often requires hlgher doses of fusldic acid th~n usual ~nd is often Instituted a~ a prophylactlc treatment to avoid transpl~nt reJection or graft-~ersus-hose disease, ~n parti-cular, the present 1nvention relates to a such co~position for oral 1~ or parenter~l ~dmlnIstrstion.

In ~nother aspect, the invention relates to the use of fuc~dic acid or a dsrlvatlve th~reof for the prophylaxls or trea~ment of a condition ~el~ted to cornea tran6pl~ntation, in partlcular to a composltlon ~ultable for treat~ent of the eye, such as an eye drop compo~it~on, an eye oin~ment composition, ~n eye lotion composit10n or an ln~ectable co~pas$tion for intraocular or retroocular ln~ection, or an oral co~posltion.

In yet another aspect, the in~ent~on relaee~ to the u3e of fu~idic ac$d or a derivat~ve thereof for the treatment of Crohn's dise~se or ulcerati~e colltls, especially for the pre~entlon of relap~e or progr~lon of Crohn's disease or ulcerative colitis, or for the treatment of perniceous anem~a or cellac diseas~, ln particular to the manuf~cture of Rn oral or rectal composition, or ~ eo~posl~on for parenter~l admlnistr~tlon.

In anothe~ aspeet, the inventlon relAtes to the use of fusldic acld - or A derivatlve ~hereof for prophylaxi~ or treatment of contact ~erm~tlti~ o~ allergictatopic dermatitis, or for tre~tment of pemphlgus vulgaris or pemphigoid, ln particular to the manufacture of a compo~ition for oral ~dminlstration, or for toplcal tre~tment of eontact der~aeitis, in part~cular for topic~ minictr~tion to the skln.

In ~nother ~pect, the inventi~n relates to the usY of fu5idic ~cld or ~ derl~ l~e ~oroo fc~r ~ree.~men~ c~r preventi~n uf replapse of a de~yelinating dlse~se, in particular multiple sclerosis, or for A~f~1¦AS/AUM/ALM/~.10.1~

F~ hl PLOIJG~lR~lN ~ !JI~ TI~FT l:MO~ 3 1~ '4g N~ 3~15~35~ p~lj~ -i O ~ ~ 7 3 0 treat~ent or prevention of relapse of sarcoldosic Boeck, S~og~en's syndrome, Reiter's syndrome, erythe~a nodosu~, scleroderma or Beehet' R digea6~ .

In yet anoth~r aspect, the ~nvention relates to the use of fusidic S acit or ~ deriv~tive thereaf for treatment or prevention of relapse of polymyosltl8 polymyalgia rheu~matica, myo~arditis or ~y~temic lupus erythe~atosls, in partlcular to a the manufacture of a compo~ition for oral or p~rentersl ad~ini~tration.

In another aspec~, the invention relates to th~ use of fu~idic ~cid or a derl~a~lve thereof for treatment or prevention of relapse of ~
condltion related to vasculitis phenomena, e.g. polyarteritls nodosa, U~gen~r's granuloma~o~s, or giant-cell arterltls.

In another aspect, the ~nvention r~la~es eo th~ use of fusidic ~cid or a te~vative thereof for treatment or preventlon of relapse of primary blliary cirrhosis or chronic active hepatitis.

In another aspect, the invention ~elates to the use o~ fusldic acld or a derivatlve thereof for treatment of a neoplastlc di.~e~se, such apla~tlc ane~ia or ld~opathlc thrombocytopenlc purpura, or for tr~atment or prevention of rel~pse of ~ neoplAstic disorder of the lymphold ttss~e, e.g. 8 cell lymphom~ or multiple myeloma, or a T
lymphocyte prol~ferative disorder, e.~. mycosis fungoides or S~z~ry ~Iy~d~ b, Tll~ y~ vt~ nl,c~ 1~L Lu ~ :~U~;II
composltlon for o~al or pa~enteral ad~inistration.

In ~nother aspect, the invention relates to the use of fusidic acid or a derivatlve thereof for prophylax~c or treatment of ~eptlc ~hock caused by gram-negatlve bacteria. Fusidic acid has been used for t~atment of $nfect~ons caused by gram-positive bacter~a such a~
Staphylococcus aureus, but prlor to the present inventlon it wa~ not known that fusidic acid could be u~ed for prophyl~xis or tre~tment of sepeic ~hock which is caused by gr~m-negative bacte~ia. Th~ p~esen~
invention relate~ in particular to the use of fusidic acid or ~
der~vzt~ve thereof for prophylaxiY or treatment of septic shock 4n~0c n ^ ~/A31 /AS/ALM/ALM~30.10. 1 98g F~ PlOUI~MR~ TO~T ~MO~ ,3~ '4~ 1~0.~ 15~35~ P~GE 4 9~

c~used by gra~-negative b~cterl~ for oral or psrenter~l administrat$on .

In another aspect, the invenelon relates to the use of f~sidic acid or a deriv~tive th~reof for prophylsxls or treatment of dis~eminated int~avasc~lar coa~ulQtion.

In another ~spect, the invention relate~ to the u~e of fusidlc acid or a derivat~e thereof for prophylaxi~ of ~rterlosclero~

In ~nother aspect, ehe invention relates to the ~se of fusldic acid o~ ~ d~rlv~tive th~reof for prophylaxis or treaement of a condition acute and chronic perlodont~l disea~es, in p~rticul~r periodontlt~s ~nd perlodontosls, in p~rticular by me~ns of periodontal in~ections.

In yet another aspect, the lnvention relates to the use of fusidic ~cid or ~ derlv~t~ve thereof for u~ ~8 an immuno~uppres~l~e drug, and the use ln comblnaelon with other relevant dru~s, such as - 15 eyclosporin or a derivativ~ thereof.

In a further a~pect, the lnvention rel~tes to the use of fuc~tlc acld or ~ te~ivatlve thereof to~ether with a cyclosporin (e.g.
Cyclosporln A snd G) or ~ deriv~tive thereof. Such co~bin~tion the~apy ls designed so as to reduce ehe ser~ous adverse effects of 2~ ~he cyclo~porln, In p~rticular the nephrotoxic effects, and st the s~e ti~ uti~lzlng the i~munomodulatlng effects of the cyclosporin to~ether with the effects of fusidic acid or its derlvatives, ~414~nt doc360 intorv~lc ~n~y cl . ~. b~;

- Cyclosporin A (or &nother cyclosporln of equlvalent potency):

~ Syste~ic adminlstratlon: 1-10 mg/kg body weight, - 'roplcal admln~s~raelon, 1-20 mg/~l (olntments, solut~ons, eye d~ops etc., cf. Above).

Also provided by the pres~nt invenelon ls ~ phar~aceutical co~posl~
tion co~p~isin~ fusidic acid or a de~ivative the~eof together ~Ith cyclosporin ~e,g, Cyclosporin A and G) or a derivative thereof.

427~0~F~ /A31/~/ALM/ALM/30.10.191~9 F~I~M FL~UG~lhl~ TOFT l MON ~' ~4 1~ 1, lq' 4~ NO, ~315~35~ P~I~E 5 2~ 79(~

In ~ further aspect, th~ in~ention re1ates ~o the use of fu~ldic acid or a derivatlve thereof together wlth a non-stero~d antiinfl~matory drug (~SAID) (e.g. indomeeacin snt ~cetyl salicylic ~eid) or an analogue and/or a glucocorticoid (e.g. hyd~ocortisone). S~ch combinatlon theraples ~re designed ~o a~ to reduce the serIous sdver~e effects, and At the same ti~e uti1iz~ng the effects of sald drug~ togsther with the effects of fu~ldlc acid or its derivatives.
Re1evant dosage lnterva1s ~ay e.g. be Acetyl salicyll¢ acld (or another NSAID of e~uiva1ent potency):

- Systemic ad~inl6trae~0n: 5-100 ~g/kg body weight;
- Top~ca1 adminictration: 10-100 mg/ml ~ointment~, to1utions, eye drop~ etc., cf. abo~e).

Hydroeo~tisone or ano~her glucocorticold drug of equivAlent potency;

- Sy~te~ic Adminls~r~t~on: 1-20 mg~k~ body weight;
lS - Toplcal ~dminl~tr~tion, 1~20 mgJ~1 (ointments, so1u~1~ns, eye drops etc, cf. Above).

U~7~ A ~/1~.31/A~;JALM/ALMJ30 10.1 F~O!l pll~lJGMR~ TOFT l MO~ .4~ NO,~13~15~35~ P~GE

In yet a further aspect, the invention rel~tes to use of a pharmaceutic~l composltlon comprlsing fusidic ~cid or ~ deri~tive together with ~ non-~terold antiinfl~dto~y drug (e.g. indometscin and ~cetyl 8~11cylic ~cid), and/~r glucocortico~d (e.g, hydrocortlsone).

In another aspect, the invention relates ~o the use of fuc idlc acid or a functionsl derivate thereof togethe~ ~ith FK-~06 or 8 functional derivative thereof. Such combination therapy i~ designed ~o as to reduce the serious adverse effectQ of FK-506, and a~ the same tlme utllizlng the im~unomotulating effects of the FX-506 togethe~ with the effects of fusidic ac~d or its deriv~tives.

In every case, the dossge of the additional dr~g or drugs described above should be carefully ~dapted so 8~ to i~ply ~he ~peclflc actlon in the cytoklne system, i.e. atea~nment of opti~al totsl dosages, dosage forms and dos~g~ frequency. In certain cases t 1~ relevant to administer a relatively hi~h unit dos~ge, a so-called ~booster do-8 i ~

EXAMPLE;S

IN VI'rR~ rpERIMENTs E~c~t~n, fUG~ dl~ d~riv~tivc~L, ~nd c~clo~p~rln The w~t~r soluble sodium salt~ of f~idic acid and fusid~c acidderlvative~ as w~11 as the fucithalmic eyedrops and ~h~ f~s~dln tablet~ were glft~ fro~ Leo Pharmsceutical Products (sallerup, Den-mark). Except for 2 derivative~ ~gee ~elow~, the drugs were dissol~edin 0.1 M phosphat~ buff~red saline, pH 7.4 (PBS) at a coneentration of 1 mg~ml and stored at 4~C.

The fusidic acid derivatives VD154~Na and PR108~ ~ere dissalved ~t 100 mg/ml in 98X ethanol and stored at 44C. Befor~ each experlment, FPOM PiJ~ lR~ ToFT l MON ~ ' 51~ ~10. 2~15~5q P~GE 7 7~0 the compounds were further diluted in culture medium RPMI 1640 (Ros-well Park ~emori~l Institute) wlth 25 mM hepes-buffer (~ordVacc, Stockholm, Sweden) ~upplemented with penicill~n (50~ IU~ml), strep-to~ycin (500 ~g/ml), L~glut~mine (2 mH), and 1 - lOX of a hest in-activated hum~n serum pool (~HS). The fin~l concentr~tion~ of ethanolwere always less than 0.5 o/oo ~v/v), and these concentr~tlon~ did not affect the production or funct~on of the cytokine~, Cyclosporin ~s kindly provided by S~doz ~asel, Switzerland~. The powder was dissolved ln ethanol (50 mgJml) And diluted to 20 ~gtml ln stsrile s~line. This ~tock solution was stored ~t -20C until use. A
sl~llar dllution of eth~nol was stored and used in p~rall~l experi-ments.

Huu~ L.

rIL-l~ was ~ gift fro~ T. T~uboi (Dainippon, Osaka, Japan). rIL-l~
was fro~ Cl~tron Biotechnology (P~ne Brook, ~J, USA~, Purifiet nstive hum~n IL-6 snd rIL-6 used for pancreaeic ~-cell experiments ~ere donated by J, v~n D~m~e (~n~versity of LRuven, Leuv~n, Belgium)(26) ~nd L.A. Aarden (Univ~rsley of Amsterdam, Amsterdam, Holland)(27~.
rIL-6 used for the other studies wa4 a gift from T. Hlrano (Os~k~
Unlverslty, Os3ka, Japan)(28). rTNF~ was ~ gift fro~ G.R. Adolf (Ernst Boehringer Institute, Vienn~, Au~trla). rIL-2 was f~om Boeh-ringer (Mannheim, FR~), The sctiv~tles of the cytokines were ascer-talned by bioasssys (see below). The cytokine~ were cslibrated with WHO interim standard preparatlons of the human cytokines (NBSB, ~on~on, U.K,)~ The endotoxin contents of the cytok~ne prep~rstions did not exceed 1 pg/l,OOO U ~ea~ured by a chromogenic Limulus Am~bo-cyte ~ysa~e a~say (Str~er~, R0dovre, ~enm~rk).

- Productlon of cytoklnes Hu~an ~ononuclear céll0 (K~C) were isolated from the blood of healt-hy, un~edicated adult donorc by gradlent centr~fugation of heparini zed v~nous blood on Ficoll-Hypaque (Lymphoprep, ~yegaard, Oslo, ~orway), a8 prevlously described (20,29-32), F~ PL~ TOFT l'MOI~'g'~ O,~g~ P~E g Cells, 2x106/~1, were lncubdted in culutre medium RPMI 1640 and pulged for lh at 37C vith 2 ~g/~l of E. coli-endotoxin 055:B5 (LPS) extracted by the hot-phenol-w~ter method (~alllnkrod~ Inc., St, Lou~s, M0, USA) plu~ 10 ~ l o phy~ohe~agglutlnin-P (P~A)(Difco,

- 5 De~rolt, MI, ~SA). The cells were then wsshed ~wo time~ with ~ank~' balanced sAlt solution ~HBSS) snd resuspended ln the same culture ~edium cont~n~n~. 2 ~g/ml of LPS but not PHA. In some experiments, the production of cytoklnes were carried ou~ ~n the presence of 1 ~ 1 of Indo~ethacin ~Sig~a, St. Louic~ M0, USA). After 20 h of culture, the supern~tant~ were isolsted and dialyzed for mlnim~m 2 d~ys st 4~C again~t HBS5 and, subsequently. RPMI/Hepes buffer (20), Thc cells were collected fo~ v~ablllty ~u~ t~y~an ~lue exclu~ion. In parallel expe~i~ents, 20 ~g~l of sodiu~ 3H-f~
date, specific activity g3 ~Cl/~g, (Leo Phsr~aceutical Company) was add~d ~t the lniti~tion of the cultures using 0-lOX ~v/v) NHS. After dlalysis, the sup~rnatants were tested for residual 3H fu51dlc acid by sc1ntillation couneIng.

T cell growth lndu~et by IL-2 Supernatant IL-~ activlty snd the effect of fusldin on IL-2 activity 20 ln vlero ~ere ~escured using IL-2-dependene CTLL-2 cells (American Type Cultu~e Collection, Rockville, MD, USA) cultured for 20 h at 37CC and pulsed with 3H-thy~id~ne for the lact 3 h of cultu~e ~31).
All experi~ents were c~rr~ed out ln triplicate, ~LISAs for TNF~ and IFN~

25 The presence of ~NF~ WBS ~easured by a sllght modific~tlon of a previously descrlbed ~andwlch ELISA, using high-~itered, ~onospecific rabblt antisera to human rTNF~ (30~. The ELISA utllizes the ~iotin/a-vidin system, and the detection limit i~ 1 p~/100 ~l, The assay i~
: not lnfluenced by se~um, and there is no c~oss re~ction with o~her cytoklnes, including TNFB (-ly~photoxin~. The recovery of TNF~ ~a~
Alway~ abov~ ~5X nn~ the coefficient of v~riation within dnd between ~s~ays were less than lOX.

4~4~ f~1/AS/AUU/ALM/~.10.1 F~OM p~O~ N I ~ .JINI~Tl.IFT ~PlO~ ~' g~ 13. 1~' 51 NO. ~g~15~35~ P~GE

~$7~0 IF~ was me~ured by ~ sandwlch ELISA purchased from Holland Biotech-nology (Le~den, Holland). The ELISA util~zes two different monoclonal nnt$~.odies to IF~ ~nd the blotin/antibiotin-alkaline phosphat~se ~ethot. The sensltlvity of this ~ssAy w8g 15 pg per 150 ~ nd the coefficien~ of ~ariaticn wAs less than lOZ. In the initisl experi-ment~, h~man IFN~ was tested by i~unoradlometri~ Assay (IRMA) using two monoclon~l antlh~m~n IFN~ ~ntlbodles ~Centocor, Mslvern, PA, USA). rhe sensitivlty of ehic assay is approximately O.l IU/ml.

T c~ll prollfer~tion Ind~ced ~y IL~l~J~

TotAl IL-l sctlvities in ~he supernat~nts were me~sured by 3 bio-~s~y3 u6~n~, l) murlne T cells ~thymocy~e~), 2) hu~an enriched blood T ly~phocytes (both ~ssays also detect IL-6), or 3) mou~e ly~mphoma cell8 (EL 4) (do not detect IL-6).

Th~ thymocyte co-st~mulatory ~89ay for IL-l w~ c~rried out as pre-vlously described ~29). Briefly, thymocytes from endoto~n-re~l~t~nt C3H/SSl, femal~ ~ice, 6-~ weeks of age (~o~holt~ard, Ry, Denm~rk) were grown in triplicate in ~ult~re medium supplemented wlth lOX
(v~v) N~S and 5x10 5M mercaptoethanol in 96-well~ microtiter plates (Nunc, Roskllde, ~enm~k), 106 cells/ 200 ~l~well. Two-fold dllutions of te6t material a~d 10 ~g/ml of PHA we~e added at the initi~tion of the cultures. After 4~ h, the cells ~e~e p~lced with 3H-thymldine (0.5 ~Cl/well) for 24 h, harvested on paper filters and oounted by l$quid seintillat~on.

Purified human T-lymphocyte~ were prep~ed f~om MNC as describ~d (2gA). In brlef, MNC were depletet of adherent cells by inc~bat~on for 1 h ~t ~7~C on plastic t~ssue cult~re dlshes (Becton Dicklnson, Oxnard, CA, U.S.A.). Nonadherent cells ~e~e remo~ed and passed through a nylon wool column, ~nd the resultln~ cell popula~ion con-sisted of mo~e than 80X T cells, 15X B cells, and less than lX M0.

The EL 4 assay w~s c~rried out 85 described (29). It i~ bssed on the prod~ction of IL-2 from the murine thymoma cell line (EL 4) in the precence of 1.2 x 10 7 M of th~ oalci~m lonophore, inonomyc~n ~S~g-ms), nnd IL-1~ or IL-l~. Briefly, 2 x 105 ET- 4 cell-c/200 ~l were 42~4oe~W2/A31/AS~ ALM/30.10.1~a~

FFO~l PL~IJG~ NIl ~x IJI~I~Ti~FT l MON,I~ '5~ NO,2~315335~ P~uE 1 incubated in tripllcae~ for 18 h ~ith 2- fold dilutions of test m~te~ial. After lncubation, the supernatants were collected and tested for IL 2 activity u~lng IL-2-dependen~ CTLL-2 cell~ (31), Calculations of actlvitie~ were carried out by a computerized l~ne~r regress~on analyci~ of probit-transformed data uslng seri~l 2-fold - ~upernstsnt tilution~.

~ybrido~a growth indu~ed by IL-6 IL^6 actlvity was determined by 3~-thymidine incorporatlon into IL-6-dependent mouse hybridoma eells, line B 13.29 clone ~9, essentially a~ tesc~ibet by Aarden et ~ 27). Briefly, triplicate samples each contalning 5 x 103 B9 cells were ~dded serial dilutions of cest materisl ~nd cultured for 3 dayc at 37~C. 3H-Thymid~ne wac added for the last 5 h of culture, and the cells ~ere ha~veceed and sssayed by l~quid scintillation. A tit~ation curve of a stsndard h~man rIL-6 preparation w~ carrled out in e~ch a~ssy. One ~nit~ml of IL-6 BC-elvlty was deflned as the concentratlon of the labora~ory ~tandard giving half rsxi~l 3H-thymidine lnco~poration uslng computer~zed probit analysis (1 unlt (-) 1 p~ of hum~n ~IL-6).

Flbroblast toxicity induced by T~F~

The TWF-induc~d cycotoxlclty was tested on ~-M mouse fibrobla~s as previously de~cribed (30). A t~tr~tlon curve of ~ ~tand~rd human rTNF~ prep~r~tion, prevLou~ly calibrated with ~ WHO interi~ reference prepAr~tlonl was csrried out ln each ~ssay. Calculations of HctLvlt~-el8 were carried out by a computerized linear regre-~sion analysis ~f prob~t-transfor~et data using ser$Al 3-fold ~upernatant dil~lons, Human mlxed lymphocyte react~on T~e mixed ly~phocyte cultures (~LC) were performed by adding human blood MNC, 105/100 ~l, to the s~me amoun~ of blood M~O from ~n un-related heslthy donor. The cells, 2x105 ln 200 ~l c~lture medium RPMI
1640 and 10X NHS, were then incubated at 37~C for 5 days. The ln-corporation of 3H-thymid~ne, added 16 h before ternination of the culture~, W85 dete~mined ~y liquld scintillAtion.

x~ /AS/ALM/ALM/~.10.1~W

F ~ O ~1 P l O I~T O F T l M O 11 } ' ~ I l O, ~ I~ g ~ l J ~ P R ~ E 1 1 ~Q~ 90 In6ulln production by isolated rst islet~

I~lets fro~ colla~en~se-digest~ of psncreas tl~sues, obtained from Wi~a~ rat~ (90-120 g), were lsolated and pree~ltured far 7 days, essentially as prevlously described (32), The isletc were counted, pooled and washed t~o times ln culture medlum RPMI 1640, supplemented ~ith peniclllin ~500 IU/~ treptomycin ($00 ~g/ml), amphotericin B
(2.5 ~g/~l), L-glut~m~ne ~2 ~M), 25 mmol/l Hepes buffer, 11 ~mol/l gluco6e, And O. 5~ ~HS, Islets, 25/1 ml, ~ere distributed Ht random ~n 24-well culture pla~ unc), The cultures ~ere performed in tripll-10cate. A~ter 5~7 days st 37C ln a 5X C02-humidified alr atmosphere, 0.2 ml of culture medium were removed for insulin determination by radlo~mmu~oass~y u~lng rat ~nsulin AS ~tandard.

Bone resorption ~nduced by IL-l~

Calva~leQ fro~ 5-dAy-old ~$c~, in~ected subcutaneously 2 day~ before wlth ~5Ca, were removed and cult~red as previously described (32), Ihe pariet~l bones were cut ineo 4 pieces, 2 ~ctlng a~ conerols and 2 for experlments. The isolated bone pieees were cultured ln test tu~es conta~ning 1 ~1 of ~edlu~ TC-199 (~ibco Bio-cult, Paisley, Scotland), supplemented with 5 g~l of bovine serum albumln fr~ction V and 4 m~/l of ampicillin.

The cultu~e~ were incubated at 37C in 5X CO~-hu~ldi~ied ~ir atmosp-here. After 48 h, each c~l~7arle-piece va~ removed and decalclfled for 30 mln at 90~C ln 1 ml of l~ HCl. Aliquot~, 250 ~l, from the~e salu-tions, and fro~ the ~edium, were then ss6essed by standart liquid ~5 gcintillation countln~. The relea&e of calciu~ ~ag c~lculated as a percenta~e of the total amoun~ of 4~C~ found ln the ~edium (the rele~se ot ~ Ca ~rom dead ~one tlsQue was not suotrae~e~). Lne er-fec~s were expressed a~ a ratio between th~ treated and control bone~, 427402EIA W/A:~1/AS~ALM/ALM/30.1~.19~9 F~M PL0~GMRN~ ~ IJINGTOFT ~ MOII ~ g~ 1~, 3~ ' 53 NO, ~g315~5~ P~SE 1~

CALCUI~TIO~S AND STATISTICAL E:VALUATIONS OF THE EFFECTS OF EIJSIDIN
AND FUSID~ ACID DERIVATIVES

The c~llQ were 81w~ys p~ecultured for 15 min wlth sodiu~ fusid&te (fusidin), or fucidic acid sn~logues, or wlth the solvent alone (control) before sdding th~ hum~n natur~l or reco~bLnsnt cytokine p~epar~tions to be assayed. The ~ounts oi' added cytokines ~arled $n accordance wi~h the censltivitie~ of the v~rious bioassay~, but they were always chosen to ensur~ ~uboptimal effects (~ ) 50X of ~ximum ~ctivlty) on t~eir re~pectlve tAYget cellc~ ~o test for posslble enh~nce~en~s of cytokLne functions, experi~ents were also c~ried out ~ith mlnlmally effective dmounts of sdded cytokines ((-) 10-20X of maximum ~cti~ity), In expe~iments designed to tes~ the effect of fusidin on lymphokine productlon by blood MNC, the ly~phokine-contalnLng -~upernatants were ti~lyzed to ensure remav~l of fuQidin before they we~e mea;ured for thelr cytokine contents (Qee ~bov~).

The ~nhlbitory effects of fusidin ~r fu~ldic ~cid ~nalogue~ were usually expre~ced as per cent inhibition of cytoklne ~ctivity, ~ccor-dlng to the formula;

~edian value (in the presence of f~sidin~
Y Inhlbltlon ~ x 100 med~an val~e ~ln medium alone~

P values ~re deter~ined by the Mann-Whltney rank 5um test or Wll-coxon'~ te~t for palred differences.

~XA~Le 1 Fusidin-~nduced irlhibi~fon of lnterleukin 1 productian by h~ n M0 The te~t wac performed ~s described in ~Production of cytokines~ and "T-cell prollferation induced by I~

F~OM PlOll~ lN ~ TOFT ~10~ 10~3~ '5~ NO,~ 15~5~ PI~I~E 13 A~ ~hown In Fig. 2, fusidin pro~ressively lnhibited IL-l product~on from LPS-P~A-~ctl~ated ~NC in vitr~. The vi~bility of the cells al~y~ exceeded 80X after 1 d~y in culture, ~nd the production of Another M0-deriv~d cytokine, ~NF~, wa~ co~pletely unaffected (Fig.
2), Fus~din~ tuced inhib~tfo~ of lymphoklne product~on by huma~ T Lym-pho~yees Th~ tsst wa~ performed dS described in ~roduction of Cy~oklne~ T
cell ~ro~h induced by I~-2" and ~ELISA~ for TNF~ ~nd IFN~".

As ~hown in Fig. 3, lnc~eased concentrations of f~idin progressively inhlblted the relea~e of IL-2 snd IF~ by LYS-PHA-activated M~C in vltro. Fifty ~ reduc~lon ln ehe production of these ly~phokines were achieved w~th 5-15 ~g/ml of fusidin. Under slmllar conditions, half msxi~al production of these cytokines was obtsined ~ith 15-50 ng/~l of cyclo~porln. A~ noted a~ove, the relat~vely lower concentr~tion ~ctivity level of fusitin co~pared to cyclosporin i5 not a disad~an-ta~e, considerln~ that toxic concentrations of cyclo~porine are in the range from 100 ~g~ml and above. With fusidin, on the o~her hand, there is no si~nific~nt toxicity in concentr~tLon-c up to ~bout 200 ~g~ml, ~o that the ther~peutic index of fusldln ls st least 5 times highe~ th~n that of cyclospor~n. IL~ and IFN~ are produced by the T-lymphocyt~, and these cells require IL-l a~ a co-stimulato~y cignal even in the ~a~ority of csse~ where polyclonal, noncpecific T-cell activstors are being used (see F~. 1 and ref. 1-4).

As ind~cated above, fu~idin w~s noe toxic to the cells, j~dged by tryp~n blue exclusion. The cellular vlablllty alway-c exceeded 80~.

To ~ule out the pos~lbility of csrry-over of f~sidin to the a~ ay s~ste~, ~specially import~nt in the c86e of the bioassay of IL-2, experiments ~ere carr~ed out ln the presence of radiolabelled fu~l-din. These exp~rlments confirmed that fusldin was completely ellm~n~-4~3A~2J~1/AS/ALM/A~M/~.lo.~

F~OM PL~ MR~ bTllFT ~lO~ g~ ' 5~ Nl~ 315~5~ E 1 ted from the supern~tant ~teri~l by the dialysis p~oced~re, even ~nth~ presence of lOX ~Hg ~nd with only 1 d~y of di~ly9is et 4C.

To te~t wh~ther fu~idin ae~e~ by an increased production of prostag-landin~, come of which are known to suppress the p~oductlon of cyto-kine~ (4), experinents ~ c~rrled out in the presence of indome-thscln througho~t the culture period. Indomethdcln ls a cyclooxy~e-naQe Inhibitor and therefore blocks the production of proetagl~ndins~
~owever, in 4 diff~rent experiments ~ndometh~cin did noe modify the ~uppresslon of lymphokine productlon sfforded by fusidin.

E2A~PLE 3 Fusldln- ~ntl~ced effects on cytokfne functiol2s 3.l.IL-l The test W~8 performed as described in "T cell proliferation induced by IL- ln/B ~

3.l.l. House thymocyees As ~hown in Flg. ~, fusldin inhlbited the thymocyte co-stlmulatory effect of rIL-l~ and rlL-l~ in a dose-relsted mAnne~ ~n thi~ con-ventional test for IL-l actlvlty. A 50~ reduction in IL-l~ and IL-l~
~ctiviei~s ~as aohieved at 1.5-5 ~g~l of fu~ldln, ~nder simila~ test conditlons, h~lf m~ximal IL-l response required 15-50 ng/ml of cyclo-~porin.

Since fus~d~n is bound exten~lvely ~lthough reversibly to protein, increased concen~r~tion~ of ser~m might neverthele~ interfe~e with the IL-l Inhibitory function of ehe dr~g. Experi~ents were therefore carried out ~th ~erum concentr~tions of lX, 3X and lOX (v/v), re-spectlvely, As shown in Table 3, the highest ~eru~ concentration only slightly reduced the ability of the dr~ eo interfere w~th IL-l-induced thy~ocyte actlvation.

4n4~28A002J~31JAg/ALMJ~LM/30.10.198~

F~O~ F`L~lJGM~hN ~ iJlNl~T~lFT f~MON~ g~ '55 NO,~315~5~ P~GE !5 Effect of vsrious seru~ concentrAtions on the ~biliey of fusidln to block IL-l-induced thymocyte activation Fu~idln X inhlbltion conc. ~g~ml) lX ~er~m 3X serum 10X se~u~

~9 93 ~7 g6 95 87 10 and 30 U of rIL-l~ or rIL-l~ were added to the thy~ocyte ~ay wlth or without fusidin. There was no difference between ehe two ~pecle~ of T~-l. The results are m¢ans o~ 4 different experiment~, each with the U5e of 2 different concentr~tion~ of rILrl~ and rIL~

3.1.2. Human T lymphocytes Fi~. 5 shows the effect of fusidin on the IL-l co-stimula~ory aciti-vity using enriched ~uman T lymphocyte~. Significant inhibition was obtsined at non-toxic eoncentrations of fu~ldin, eqpecially when PP~
was u~ed ~s A co-æ~l~ulator to ~lmul~te antigen-induced a~tivatlon of 2~ T cells.

3.1.3. Mo~se lymphom~ cells ~EL 4) Because thymocyte~ contaln a ç~all nu~ber of macrophages and dendrl-tlc cell~ in addition ~o T lymphocytes, ~he ~billty of f~sidin to lnhib~t IL-l~-induced activation of a mouse thy~ocyte cell l~nc ~EL
4) was next ln~est~gated. As ~hown in Fig. 6, fusldln ~l~o appeared to lnhlbie rIL~ nduced proliferation of EL 4 cells. The fact that the e~fect of the drug was much less pronounced th~n in Examples 3.1,1, ~nd 3.1,2. can ~08t likely be a~tributed to lnductlon of EL 4 cell prolIferatlon requiring co-stlmulAtion with a calclu~ ionophore, ln sdditlon to IL-l. Another po~slbllity could be that the IL-4 is t~n~formet cell llne which 18 not comp~r~ble to a u~ual cell, ~, J ',L ~ ' H '~ ' 5 5 N ~ 15 ~ ~ 5 ~ P R ~ E 1 3.2. IL-2 and TNFa Th~ test was performed a~ de~cribed in ~T cell gro~th induced by I~-2~ ~nd ~Flbroblast toxi~ity ~n~ ed by TNF~n.

In contras~ to the effect on the function of IL-l~ and IL-l~, fusidln did not modify the ablllty of hu~n IL-2 to ~ctiv~te ~ouse cytotoxic T lymphocytes ~GTLL-2) or the ability of hu~an TNF~ to k~ ou~e L-N
fibroblast (F~g. 7).

Effcce of delayed addftlon of fusidin to Il~ nduced thymocy~es 0 ThR test ~as perfor~et as described $n ~T cell proliferat~on Induced by IL~

As shown in Fig. 8, addit~on of fus~din 5 ~g/ml up eo 4~ h after lnltl~tion of thy~ocyte ~ctivation by PHA plus IL-l was progressively less effect$ve in reduclng the co-~tlmul~tory effec~ of IL-l. A
slm~lar response p~ttern Wa5 obtsined after addl~on of 50 ng/ml of cyclo~porin. Thi~ indics~e~ that fu~itin affects the early procecse~
lead~ng to T cell ac~vstion. Early start of tre~tment of immunoin-flammatory d~se~ses wlth ~usidin or derivatives thereof is therefore ~ost likely beneficidl.

E~AHPLE 5 E~fect of fusrdic acid deriv~tfve~ on IL-l-lnduced thy~ocy~c actfva-e~on The test ~as performed as descr~bed ln nT cell proli~eratlon lnduced by IL~

4274~213A002/A31/~gJ~ LM/~0.10.1~89 F~OM PLOIJI~M~llti ~ I.JI~T~FT ~MON~'g9 1~ 56 ~l0~ 15~35~ P~E 17 20U~79 Aq 6hown ln Table 4, only 3 fusidic acld deriv~tive~ were effective ln inhibitine the thy~ocyte ~o-stimu~to~y effect of I~-lu and IL~
- The ineffective derlvatives sre characterlzed by modifications ~n the sub6tituent~ ~t positlons 16 ~nt 21 of the molecule sugge~ting that the~e regions of fusidic ac~d ~r~ necess~ry fo~ the IL-l-inhlbltory effect of the drug.

TABL~ 4 Effect of fusidic acid derl~atives on the ~hymocyte co-stlmulatory funct~on of lL-l X inhlbltlon of Fu~ldic ~cid derl~ative rIL-l~ P rIL-l~ P

Fu~idSn 70 +/- 10 ~0.05 V~ 1177 21 +/-19 n~ 5 +/-9 ns VD 117~ 16 +/-13 ns ~ +J-8 n~
VD 1~0~ 2 +/-11 ns 7 +/-3 ns VD 13601 24 l/-6 C0.0522 +/-~ C~.05 VD 13621 59 +~-10 C0.0561 +/-15 c0.05 VD 14602 46 +/-13 c0.055B +/-13 c0.05 VD 1546 3 l/-9 n6 14 +/-7 n~
PR1089 -10 +~-10 ns 13 +/-5 ns Qe~ult~ sre ~ean~ +/- SEM, n-5.
1 These analogues were ldentical with fusidln at the 16 and 21 po~i-t1Ons of the ~olecule - 2 Thls analogue was also identical with fusldin at the 16 ~nd ~1 positions, except for ~ub~titution of oxygen w~eh .~u~fu~ at posltion 16, FRnM FilOl~ lRN~ INGT'FT I~MON) ~ .56 NOI~g~15~5~ PI~l~E 1 20a~L7~0 ~ 6 Fusldln-lnduced inhio~clon of th~ htlm~n two-~ay mfxed 1ymphocyte ra~c tlon (~LC) The teqt was performed ~9 de-~cribed in "Human mixed lymphocyte reac-tion", ML~ 1~ the in vitro correlate of a t~an~plant re~ectlon ln vivo .
Therefor~, if fusidin has a role in preventing graft re~ection, lt ~hould ~uppress the HLC reàction. A~ shown in Fi@. 9, fusldin indeed inhib~ted the two-way hum~n ~LC. The dose-response was almo~t 1den~i-cal to those obtained when testing the effects on IL l on ~hymocytesand PPD-actlvated hum~n T cells as well ~s IL-2 production. The 50X
~nhibitory concentrat$on of fusidin ~as 1,5-5 ~g/ml. Si~llar suppres slon of the ML~ W85 obtained ~it~ 15-50 ng/~l of cyclospor~n.

EYA~PLE 7 ~usldin-in~u~ed inhiblelon the hybridom~ growth-p~omoting effect of The test ~as performed as described ln "Hybridoma gro~th induced by IL-6~, IL-6, another M0-derived peptide med~a~or, has been reported to medlate cever~l ~unc~ions of I~-l, including ~hymocyte activation (2, 7, 6) . It was therefore of interest to ~ee whether fusidin lnter-f~r~d with the function of IL-6. ~ shown in Fig. 10, e~e growth of the B9 hybridoma cell llne wss ~nhibited by fus~din. The dose respon-4e was very si~llar to the ones obta~ned when testing ~he effect of fueldln on the IL-l-~nducèd growth of no~mal (~ouce) thymocyte~.
F$f~y ~ reduct~on in IL-6-induced growth was obtained at around 10 yg/~l of fusidin. Ag~in, there was no evldence of fu~idin-induced cytotoxicity, as demonstr~ted in the next experiments.

4271~- ~/~31/AS~ /ALM/30.10.1980 F~U PLI~IJGMI~ GTOFT (MON~ ~S 1~ .57 Nl~ 315~5~ PFI~E 1 200~790 so A~ shown in Fig . 11, the ~bility of fusidin to prevent IL- 6 - induced p~ol~feratlon of B9 hybrldoma cells wa~ not caused by a generall7ed toxic effect on the cell-~. Thus, B9 cells prelncubated ~ith 30 ~g/ml of fusidln, wlth or without rIL-6, respon~ed no~mally to a subseq~ent challenge by IL-6 afte~ washlng the cells ~ith medium and reconstitu-tion ~ithout fusldin.

F~A~PL~

F~llure to dify IL-~-fnduced ~ICtiVatiOA of bone resorptlorl The test ~a8 perfor~ed ~5 described in UBone resorption induoed by IL-l~.

Aa sho~ in T~ble 5, fusidin did not affect the incr~ased release of rAdiolAbelled Ca induced by rIL-l~. This furt~er de~onstr~tes the non~toxiclty of fusitln in biologlcal sy~tems.

Lack of effect of fus~dln on rIL la-lnduced bone ~esorption 45Ca relea~e ~X of control~

F~sldln rIL~ ean t/- SD n P

l.00 (control) lO ~/ml ~ O.g5 +/- 0 08 lO ns 10 ~g/ml 20 U/ml 1. 49 +/ 0 .121 9 C0 . 05 ~ 20 U/ml 1.35 +/ 0 lO2 B c0,05 1 and 2- no signlficant diffe~en~e between these values.

4nu~s~K~ /As/AuM/ALM~30.10.t~

';OM PLO~ MR~ JIN~TOFT ~.MO~ ~ ~q 1~ 57 ~0. ~ 15~5~ P~GE
L7gO
s~
~LE 9 P`us~ lnduced pro~e~tion of p~ncre~tic ~-cell~ agair~st ehe inhiDi-tlon/dam~g~ ~fforded by IL-l The tests were performet as descrlbed in "Insul~n production by lsolatet r~t islets~.

9.1. Fusidln Since IL~ on~ldered pathogenetic~lly i~port~nt for the tevelop-ment of IDD~, and since I~ significantly ~educe~ glucose-induced insulin p~oduct~on by pancreatic ~-cell~ Sn vlt~o, it was of interes~
1~ to test whether fusidin could re~tore nor~al ~-cell function ~
vlt~o . A~ shown in Fig. 12, e4- incubation of pancr~atlc islets isola-te~d from normal rats wlth increa~ing concentratlons of fu~idln pro-~res6ively normaliæed their glucose-induced insulln produc~ion, even ln the presence of relatively hi~h concentrseions of r~L-l~, At the highe~t concentratlon of fu~idin, ~here appe~red to be a slight red~ctlon in the protective effe~ of the drug. The ~eason i~ not cle~r bu~ ls most likely c~used by the trugs ~bility ~o lnhibit ~ngulln mR~A ~r~n~l~tion (Z3).

9.~. Fusldlc acld deriv~tives A slmllar protectlve effect ~as not ob~erved when testing a number of fusitic ~cid de~ivatives ~Flg. 13) whereas the die~hanolamlne salt (3 experiment~) gave s~milar result~ as fusidin. Thl$ lndlcates that ~odlfications of ~he basic f~sldlc acld structure except for der-lvati~e~ at the 21 position s~ch ~g the s~1t fo~mat~ OR ~re not de-sirable in connection wlth thls effect.

427~0:`''t~/A311A5/ALM/ALM/3~.10.1~B9 ' 5 ~ N 0 . ~ 1 5 ~ ~ 5 9 P ~ ~ E ~1 2~ 790 DISCUSSION RELATED ~O THE IN VI~RO EXPERIMENTS

I~Nnosuppre~ivc ~ffcat~ of fusidin The data p~esented from the ln vitro experi~ents demonstrate a dose-dependent lnhibitory effect of fusld~n on processes involved in ly~phocyte activ~tion. Inducelon of T and 3 lymphocytes requires several steps in ~dd~tlon to antigen or allogen recognition by HLA
class II positive cells such as H0 or dendritic cells: 1) production and release of IL~l and, possibly, IL-6; 2) activation of T cells by these cytokines through specific membrane receptors for IL-L and IL-

6; 3) production and release of IL-2 by T ly~phocytes; 4) acquisitlon of receptors for IL-2 on T lymphocytes, and S) internslization o~ IL-2-receptc~ compl~xes. In addition, the releace fro~ T cell~ of IF~
ls consideret important, because thls cytokine i~ a potent activator of M0 functlons, includin~ HLA class II expression (l,~).

Llke cyclosporin (19), f~sld~n appears to interfer~ with so~e of these early proces~es. First, ~elatively hlgh concent~ations of fusldln inhlblted production/secreeion of IL-l f~o~ LPS-~HA-activated hum~n blood MNC. Secondly, ~nd probably ~ore i~p~rtantly, low therA-peutl~ concentr~tlons of fusidin suppressed lymphocyte acti~at'on by IL-l and IL-6, another M~-derlved activator of T- and B ly~phocytes.
Prob~bly as ~ result of ehis, fusidin 41~o inhlblted the prod~ction by T cells of their own growth f~ctor, IL-2, leading to i~paired T
cell proliferation after antigenic, m~togeni¢ ~ well as allogeneio acti~ation. Since IFN~ is an 1~portant activator of M0 functions, including their ablll~y to secerete cyto~ines dnd expre~ ~LA class II mole~ule-c~ the drugs abilit~ eo inhibit the el~bo~at~on of IFN~
~i~ht ~on~r~but~ to T ~11 s~ reeci-~n ~n all C~se-Q, the inhibitory effects were dose-dependent, ~nd the effective concentratlon~ of fusidin appea~ed to be non-toxic to the target cellc, Furthermo~e, fusidin dld not function by ~ gener~l Antlproliferat~ve effec~, because the IL-2-lnduced prollfer~tion of CTLL-2 cells were unaffected by the drug.

~n~ x~A31/As/ALM~ALM/~1o~

FR~ OU~ JIN~TOFT l MON~'g~ '5~ ~0.~ 15q~ PR~E ~.

2~ 90 The concentrations of fusidin which were shown to ~e effe~tlve ln vl~ro seem to be clinlc~lly relevant. because ther~peutic serum concentrat~ons in the ran~e 15-100 ~g/~l ~ay be obtalned in m n ~ithout significant side-effects (33). However, fusidi~ l.c bound exten~ively to protein in vi~o, and this may contribute to a di~inis-hed cllnlcal eff~c~cy if related to the effeceive coneentrations of the drug in vitro. Th~s. however, ~oes not sppe~r to be of ma~or i~port~nce in the ca~e of fusidin, because i~creased concentrations of serum in the thymocyte assay only mar~in~lly affeceed the fusidin-lnduced immunosupp~es~ive function, Moreover, several importantclinic~l effects of fu~ldin are expected to take plsce in t~c~ues whe~e serum proteins ~re absent or present in only ~mall quantiti~s, When testlng fusidin in the mouse thymocyte co-stimulatory a~ssy, the IL-l activity ~a~ almost completely eliminated by 15 ~g~ml of ehe drug, and a SOX inhibition was achie~ed at 8 concentr~tion of 1.5-5 ~ l. At all eoncentrations, fusidin failed to decreAse the ~l~bili ty of the thy~ocytes comp~red ~th thymocytes cultured in parallel without the drug, The mechanis~ by ~hich fu~ldin Inhiblts the im~uno~timulatory f~nc-tlons of IL-l~/~ i9 unkno~n. However, the drug is known to lnhibit proteln synthesis at the translatlonal level ~23), and an 1mpaired p~ot~ln ~ynthesls may the~efore, ~t least ln part, explaln the re-sults. However, ~ co~plete lnhibition of cytokine mR~A translation cannot by itself explaln our findines. Thus, the production of T~F~
by human blood MNC activated by L~S+PHA wa~ unaffected even by 50 ~gJ~l of fusidin. Also, ehe abllity of fusidin to inhibit production of the cytoklnes, IL-2 ~nd IFN~ o~t likely a conseq~enc~ of the T~-ln/~ and the IL-6inhibitory effe~t-c of ~he drug ~ather than a direct effect on the tranclation of these ly~phokines, Hence, I~-l in particular 1~ consldered an essen~Ial 'second .~ign~l' for T ly~pho-cyte activ~tion, $ncludlng productlon of lymphokines (1-4) ehe first ~lgnal belng antigen or mitogen-induced perturbation of the T cell receptor/CD3 co~plex ~1,2,11,12).

The ~blllty of fusidin to lnhibit a ~ixed lymphocy~e reaotion ~n 35 vStro is probably also ~econdary to its lL-l-inhibltory effec~. At F~OM PLOI~ JI~ T:lFT ~MON~ 5~ ~0,2~315~5~ P~bE ~3 ~ny r~te, tho inhibltory eifece of fus$din on this in vLt~o correlate of a hu~an allo~raft reJection ~ay be import~nt, becau~e ~t suggests that the drug may b~ ~sed clinic~lly to prevent the hosts ellminatlon of allotransplants, such n kidneys, llvers, h~ares, lungs, ~kln, bone marrow, corneae, etc. Ireatment of the host with fusidin st the tl~e the tr~nsplant ~ perfor~ed, ~nd ehus interference with the 'second slgnal' of lymphocyte activstion, may al~o le~d to speclfic tolerance ~o the grafted tissue (see dbo~e).

The abllity of fu~idin to lnterfe~e with the grow~h-promotlng effects of IL-1Q/~ and IL-6 is also c elinical importance. Thus, I~-l and particularly I~-6 have been i~plicated ~n the p~thogenesLs of several lymphoproliferAtive dig~ace~ such BS multiple myeloma snd other pl~sma cell, and B- and T cell cancer~ (see above).

Antiinfl~mmntory effectQ of fu.citin IL-1 ~nd IL-6 sre known to ~ause an array of biologlcal actlvities In many cell types (1-4). It is therefore inceresting tha~ fucldin interfered ~ith some but not all the variol.ls funct~ons of IL-l that ~ere te~ted.

For example, the d~ue was ~nable to ~ffect IL-l-induced bone resorp-tion. On the oehe~ hand, the drug clearly protected psncreatio ~clet ~-cells against the inhib~toryfcytotoxic action of IL~

The apparemt inhibltory effect of fusidin on non-immunological func-t~on~ of IL-l and, pos~ibly, IL-~ is of clinicAl inte~ . Thus, ap~r~ fro~ being involved in lymphocytc ~ctl~ation and thus pres~-~ably in physiological and pathophy~iologics~ immune reactions, IL-l and IL-6 are lnvol~ed ln the development ~nd ~anlfestations of many infect~ous and i:~..unoinflam~tory diseases, Including AIDS, auto-lmmune entocrine diseases, some rheu~atic diseases, etc. (Table 1, r~f. 2,3,8). Thls, along with the ability of IL~ and IL-6 to cause fever, and of IL-l~/~ to evoke ano~exla and cachexi~, e~phasi-ze~ ~ therapeutic role of fusidic acid and deriv~ s thereof apart fro~ their usa~e a~ antibiotic~.

FROM ~LCI~ Rh~ JIN~TOFT ~MON ~ g~ 113. 3~ g315~35~ P~E

CONCLUSIONS ~OR T~E IN VITR0 EXPERIMENTS

~- ~nd B-lymphocytes, M0 and ~K cells play important roles in manls defence ~gainst m~croor~nis~s and neopl~stic diseases. On the ~her h~nd, ehere 19 ~trong e~idence for a role of these cells in the pathogenesis of cert~ln rheu~tic dlseases and im~uno~nflammatory di~ea~e~ In~ol~ing the endocrlne system, the skln, the gut, and ~any other organs. The peptid~ hormones ~cytokinee) produced ~y these cell~, par~cularly IL-l~, IL-l~, IL-6 ~nd TNF~, are potent lmmune stlmulators as well as modulators of hep~ocytes, bone cells, endot-hell~l cells, fibroblasts and synovlal tiasue ~ells, pancreatic islet~-cells, thyrocytes, and many other cells. Treatmen~ to prevent the productlon ~nd/or function of IT-l~J~, IL-6 and T~F~ ~ay prevent the de~elop~ent, ox ameliora~e the symptom$, of many immunoinflam~tory and $nfectious diseAses. Such interventlon ~ay ~160 prevent trans-plAnt r~cction eleher lf administered as ~ contln~ous, immuno~up-prP~ive treatmen~ o~ as a el~ed, short-term tre~tment to lnd~ce apecific tolerance to the grafted ti~QUe. Fu~idLn, ln therapeutl¢
relev~nt concentrations, inhibit se~erAl ~mmunological, groweh-pro-moting ~nd pro-lnflammatory functions oi IL~ and IL-6 i~ vltro, And the patterns of these respOnSes are atrikingly slmil4r to those caused by cyc~osporln. The present discovery ls therefore of impor-tance $n the syste~lc and local treatment of eeveral immunoinfla~ma-tory d~seases in humdns, whers these cytokines Bre concldered ~o be of p~thogenetlc rele~ance. These dise~ses lnclude the so-called autol~mune diseases (see Tabl~ 1), dlsea.~es sssociated with trans-plantation, including ~raft re~ection and graft-vercuc-host disease, many Infect~ous dlseases, and ~any dlsease~ charac~erized by patholo-gical csll growth, lncludln~ some neoplast~c dl~eaees, In ~he followin~, a number of ~n vlvo expe~ments are described, elther based upon evidenc~ ob~ined or ag guidelines for the experi-m~ntAl work to be per~ormed in connect~on wlth specific ~se~ of fusidic actd or deri~atlves thereof. ~ith respect ~o pharmacology, - the pharmacology of fu~d~c nc~d snd derivati~es thereof for oral, parentsral and top~c~l application appe~rs from the scientific ~nd p~te~t literature mentioned ln the section of ~Back~ro~nd of the Invention". Th1s documen~ation i5 supplemented by the following J~ ~/A311AS/ALM~ALM/~.10.1~

F~O~l ~Lol~G~l~NN ~ lJIN~TOFT l MON~1g~ 0,~315~5~ PR~E ~5 ex~ple illuctratlng penetration of the dru~ into co~part~ents of the ~ye (Example 10).

I~ ~IVO EXPERrME~TS

PHARMACOLSGICAL STUDY

~AMPL~ 10 An~lrses From Corpus Vitreu~/Sub~etL~ Fluid Th~ fusldln concentrAtion in corpus vitreum~subr~tlan~l fluid 1~
measuret ~fter 3 d~y~ o~ preceeding $yctemic therapy prio~ to oper~-tion Systemlc therspy: Fusidin eablet~ (Leo, Copenhage~, Denm~rk) 10500 ug 3 times every ~4 k~r~ h~ the~apy end~_~t Lo~r~t-~ hour~
before operatlon, Materi~l 1. corpuO vitrcwm ~n~l~o~a ~naluda 10 ~y~c ~f p~tl~nt~ who Ar~
s~b~ected to a vitreou6 body operatlon in connection ~ith bleed-15ing in the vitreous bo~y provoket by diabetes ~llltuc.

2. A~otio ope~st10n (tetach~ent of the retlna~, removal of sub-retinal fluid of 1û eyes $n connection with reein~ operstions.

PRE- CLI~IC~ SlVDIES

The following examples 11-12 are ba~ed upon ongolng studie~ and glve 20 ~uidelines for preclinic~l ctudies in ani~nal ~odels.

42~ A8/ALM/ALM/3l 1.10.1~

FF~M Pl~U~M~N~I ~ UI~TIlFT ~MON~ 1 NO~g~15~5~ P~E 2 ~UUL~ 11 In vlvo eYA~in~tion of the propllylactlc effect of f~s~dln in t~o ~n~mal models of di~betes ~ellLtus The following example ~s deQigned to examine che prophylactic effect of fu&idin ln BB-~ats snd NOD-mlce (two spont~neously dLabetic animal models for Type 1 (insulln-dep~ndent) diabetes ~ellitus).

The study includes 40-50 animals treated with fusidin and a corre-sponting nu~ber o~ control snim~ls tre~ed wlth place~o. Breeding oouples are procured ~nd the experl~ent~l anlmals are supported and breed~ The ~nimals sre treated wlth the drugs i~edlately after weanin~ And until the ~tudy ls termlnated after 200 day~. The anl~als are obse~ed daily, week-ends lncluded, ~nd once ~ ~eek the anl~ls are welghed and urlne tests are carried out to deter~in~ glucose content. P~ncreas from ~nl~ls developin~ diabetes and from sni~als at the end of the study are sub~ec~ed ~o mlcroscopic ex~minatlon to discover 1) possible infiltration of mononuclear cells in the i~letc of Lan~erh~n~ (~nsulitis), and ~) to determine ~he nu~ber of cells producing lnsulln. Furthermore, the con~ents of insulin and cytoklnec ~n con-~ecutively drswn serum samples are deter~ined in ord~r to e~sluste a pos~ible association between the develop~ent of the di-seas~ and the cytokine levels in seru~.

CXAn~l~ 12 Tbe ~ffect of fusfd~n on the ~cute-ph.ss~ response lnduced by IL-f ~nd IL-6 ln m~ce Background The acute-phase re~ct~on Is usually 8een during ficuee ~nd chronic ~fectloug and inflamms~ory di~ea~es, and ln c~ncer, and adm~nistra-tion of ~L-l, }L-6 or T~Fa reproduces thi~ r~action (8), IL-6 ~nd, to a le~ser extent, IL-l ~nd T~F~ ~nduce hepatocyees to syntho~ize acu~e 4n4~e~0oe/A31/~S/~hU/ALU/30.10,1~

FROM PL~ Ui~GT3FT ~MON )~ . 2~ 10. 2~315~35~ P~E 27 5~
phafie prote~ns, lncluding seru~ amyloid A, C-reactive proteln, flbri-nogen, haptoglobin, co~plement components and clotting factors. At the ~me tlme, the blood level of ~lbumin decrea~es, ~s doe-~ the plasma concentra~ions of Fe2~ and Zn2+, whereaa ~he level of Cu2~
- 5 increa~es. Associated ~henomena ~re fever, leukocytosis and inductlon of ~leep. The elevated level of f$brinogen, e~pec~ally if accompanled by ~ne~la, causes an increased sedi~entation rate of the blood, a co~monly used clinlcal parAmeter of 'lnflammation'.

The ~bove uentioned cllnical picture iQ ofeen sssoci~ed with di-~t~rb~nces in carbohydra~e-, lipid- snd protein met~bolis~ resulting in ~astlng (c~chexia). In rare s~tuat~ons, the acute-phase reaction may progress and lead to clottlng abnormal~tie-~, shock, and de~th, It ~as pre~ously thought tha~ microbi~l products such a~ endotoxins were directly responsible for thece sympto~s, if triggered by b~c-terial lnfectlon. Thic is now kno~n eo be incorrect, because endo-to~lns are potent inducers of M~ IL~l, IL-6 and T~F~, ant all patho-physlologlcal processes ~6sociated wlth endotoxin-induced ~hock can be rep~oduced by in~eotion of TNFa and, to e lesc~r extent, by IL-l (2)~

De~i~n Pilot study ~sing a mouse model (25).

12.1. Interleuk~n la-lnduced respon~e Fusidln, 15 ~g, and solvent alone ~control) are ~dminlstered i.v.
into S pl~s 5 (control~) female BALB/c mic~ t8-10 weeks old~. After 30 m~n, 10 y~ of human rlL-lQ ln 25 ~1 pyrogen-free isotonic saline ls gl~en i.v. After 24 h, blood i~ drawn fo~ measurements of the ~ou~e acute phaQe react~nt h~pto~lobl~.

~ending upon the~e results, the dosage of iusidln and rIL-l~ will be Altered ~n orde~ to substan~l~te the do~e-response curve of the effect of fusidin on the IL-l-induced acute-phase response in mlce, 12.2. Interleuk~n 6-induced re~pon~e 4~Y~ ~PIA31/AS/ALMJALM/~101~

F~OIl PLOJ~M~NN ~ ToFT ~MON~g~ N0~2~315~35~ P~E ~g 5~

The exper~mental approach outl~ned abo~e wlll be used, except that hu~an rIL-6, 10 ~, wlll be used.

A~ain, the dose-response curve of the effect of fusidin w~ll be establi~hed.

12.3~ Effect of fu~idin analogs on IL~ or IL6-induced acute-pha~
re~ctlons ln mlce -Sim~lar exp~r~mental ~pp~oach as abo~e, except that fusidin analogs ~111 be used instead of fusidln.

ThR following examples 13-18 concern clinical experience g~ined by certain tre~t~ents of patients w~th fusld~n and 8ive guideline~ for controlled cllnical studles ba~ed upon the evidence g~ned from these pllot stud~e~, CLINIGAL STUDIES

~h~M~L~ 13 The following Qtudies are designed to demonstr~te thc effect of f~idlc scid eyedrop treatment 88 a prophylactlc treat~ent against lnflammat~on after eye surgery.

13,1. Cl~nic~1 FY~ri~ton of t~e Antiinfl~ma~ory Effe~ of F~s~dln ~n the poseoperatlve Perlod Aft~r Ur~complc~ted OperAtion for C~-20 taract M~terial: Patlent~ ~ged over 60 opersted for cataract wLth extractlonof the lens and -~ub~equent implAntat~on of an artif~cial lens ~n the posterior chamber of the eye ~orrecponding to 20 oyes.

Exclusion: P~t~ents with eye~ with trauma, glauco~a, retinovascular bleeding or ~hrombos~s, oorioretinal lnflammstory and non-inflamma-Z7 W ~ 31 /AS/ALM/ALM130. 10 .1~

F~Otl PL0UI~MRN~ ~ UlNI~TOFT ~MON~ NO.2~315~35~ P~E

tory ~$ght-threatening diseases, di~bete~ mellitus (type I), collage-no819.

The group treated with fu~idin lncludes pa~ients corresponding to 10 ~ye~, S The control ~roup incl~des patients cor~esponding to 10 e~es tre~ted with ultralanum with chloroRmphenlcol.

Method: Randonized ther~py between the two preparations of 20 eyes in a 3 week period po~toperatively seartin~ from the fi~st postoperati~e day~

Dos~g~: Ultralanum with chloroamphenlcol eye drops 4 time~ dally.
Fuclthalmic eye drop~ 4 t~mes daily.

~v~luation para~eter8 1. Vi~ual acuity: Exa~$ned at weeks 1, 2 and 3 after the cataract OpC~at~on, 2. ~lo~lcro~copy is csr~ied Oue ae days 2, 5, 14 ~nd 21 after oper~tion. During b~o~lcroscopy, a ~ore det~lled data recording would be carried out.

Ey~ pressure is measured Bt weeks 1, 2 ~nd 3 after operation.

The e~r ~nAtion of corpus ~itreum and the corioretlnal ~tatus ls carrled out at weeks 1, 2 and 3 ~fter operat~on, and ~ubjective data ln the postoperative period are recorded ~ncluding the patients' ev~luation of the treatment with fucithalmic/ultralanum w~th &hlo~-ampfenicol, e,g, problems with dr~pp~ng of eyes, eye pain, duration of eye pain, du~ation of cight reduction, if any, after dripping of eyeç, etc.

Excluçlon during treatment: Incrcaçed lntrabulbar inflam~ation, ~sight-thre~tening), obviou~ p~nophthal~l~, non-oompliance.

~2710?''~ 11 /AS/ALM/ALMJ30.10. l 9~g FPOM PLOU~MRI~ & ~INGTOFT ~MI~N~18~ 7 N0~2~g~15~35~ P~E
Z~ 790 13.~. ClLnlc~l e~ fr~stiorl of the antiinfl~tor~ effect of fusidin ln the poscoper~t~ve p~r~od ~fter unço~plSc~ted ~aser surgery Mat~rlal Pat~ents admitted for laser ~urge~y due to e.g. loosenlng of the - 5 retin~, re~in~l biop-cy or diabetlc retinopsthy.

The group trea~ed with fusidin include~ patients corre~ponding to 10 eye~.

The con~rol group includes patients correspondin~ to 10 eye~ treated -~ith conventlonal ther~py (glucocorticoid).

Method ~An~- {7ed therapy betwoen the two pre~a~ations in a 3 week period postoper~tively st~rt~ng from the fir~t poctoperative d~y.

Dosage Fusidin tabletQ 500 m~ x 3 in 2 ~eeks starting st the day of the operation.

Evaluat~on p~ra~eter~:

1. Visual acuity: Ex~mined a~ ~eeks 1, 2 and 3 after the laser ~urger~ operstion.

2. Biomicrosco~y is carried out at d~ys 2, 5, 14 and 21 afte~
operation. During bio~icros~opy, ~ ~ore detsiled d~t~ recordin~
~lll be carried out.

The examlnatlon of corpu~ v~treu~ and the cor~oretinAl status is carried out at weeks 1, ~ ant 3 after operation, and sub~ective dst~
in the ~ostoperati~ perlod are recorded incl~d~ng the patient~' evaluation of the tre~tment with fu~ldin.

4Z7t~A~ /ALU/30.10.1989 F~O~ PLOU~lR~N ~ IJI~T~IFT I~M0~'8~ 3~ 3 ~O~g315~59 P~E ~11 14.1. Tre~ment ~ith Fwldin of ~on-infec~fous, IrPm~nolnfl~mmatory Uv~itis B~ckground Severe uveal tract infls~mstion (uveitis) is respons~ble for a large percent~ge of the visually handlcapped patients in de~elopin~ and ~v~lu~4~1 ~;vu~LL I~ L uf ~ e~ ~f u~re~ t~ ~ ~n ~c~l~ped oo~m - trle~ are classified as ldiopsthic and are pre~umed to have an under-lying d~toimmune oause, The ~reat~ent in these ca~es is malnly ba~ed on the use o ~lucocorticolds, cytotoxic drug~ or cyclospor~n. In many of the cases, however, the treatment only delsys th~ loss of ~ision side-effects (Cushin~ yndrome, snd severe bone-marrow depre~s~on ~nd nephropa~hy (cyclosporin~ may force withdrawal of therapy with resultin~ los~ of vi~lon.

~5 1~.2. Pllot ~udies In the following Tables 6 and 7 are shown the res~lt~ from two pilot ~tudl~. In the fir~t study, three patlent~ are ineluded correspond-in~ to t~eatment of flve eyes. Ihe treatment was converted from cyclospo~in to fu~idin (0.5 g 3 times d~ily given as tsblets). In the 8econd 8tudy, four p~tient~ are lnol~det ~8 eyes). The p~tlents were treated wi~h fusidin tO.5 g 3 time~ daily given as tablets) without e~rlier cyclo~porin medicat~on.

42t4028~002/A31/ASJAL~I~LMt30.10.198~

F~OM PLOUGMRNN ~ TOFT ~MON~18~ 1~.3~, 2~,134 NO.~lg~15~5~ P~GE 3 6~

Pllot study: Severe, slght-~hreatening uveltis Three pstient~ (5 eyes) - conver~lon from cyclosporin (~yA~ to fusl-d~n (Fue) Prednlsolone (mg~day) ~d Case hi~tory CyA ther~. ~before - l~st) .
~JN One eye blind 5 years 10 7.5 JK Relapse on CyA + l.S years O O
pred. 7.5 mg/day 2 x relapse on pred.pul~e Mb. Cushing M~ 2.5 ye~ 100 15 (nephropathy) TABLE 6 ~continued):

Ac~te infl.
Visual acuity f~ndal changeg Id oc (before - la~t) (before - last) Notes ~J~ ~in 6/9 6/9 +
JK dxt. 6~9 6/18 ~ - Z x vitrectomy sin. 6~36 6/36 ~ - turing Fus. t~era-py without re~p~e MN dxt. 6/6 6/6 ~ -sln. <3/60c3/60 ~ -Co~ments: All 3 patlents ~S eyes) beneiited from fusidin ther~py.
Deterloration in visual acuit.y ~ y~_~A~ ~.Au~ed by vitreal ~aze s~condary to a vitrectomy. Two vitrectocles were performed d~ing Fu~. therApy ~ithout flare-up of disease activityt ~one of the pAti-ents had side-effects of Fu~. All. 3 patient~ had severe ~id~-ef-fects c~used by th~ long-term treatment with moderate~hlgh doses of glucocortlcoid~ snd CyA.

4~4o23AX~ /AS/ALMIAU~

F~OM PLOIJl~MiNN ~ INI~TOFT ~MON ~ g~ 4 NO~ 7~8~15~35~ PI~I~E

TABL~ 7 Pilot ~eudy: Seve~e, ~lght-th~eatenlng uveitis Four patients ~ eyes) - fusldin (Fu-~ trea~ent ~no cyclosporin) Prednisolone (~g/day) Id Ca~e histo~y Fus the~before - last) ~J 7 month~ 50 7, 5/50 20 (relapce) VCK R~lap~e on pred. 7 D~onth~ 30 10 30 ~g/dAy Mt. Cushlng FVT 7 ~onth~ 60 10/60 0/30 20 (2 relapse~) OHP R~lap~è on pred.
30 m~/d y 3 months 60 10/40 25 (rel~ps~) .
20 TA~LE 7 (contlnued):

Acute Infl.
Vi s~al scuity ~undal changes 2S Id oc (beforR - last) (before last) Notes BNJ dxt, 6~60 3/60 ++ - Plus Fus. ey~-drop s sln. 6/9 ~6/9 + - Relspse at pred. 7,5 mg/day VCK dxt. 6/9 6~9 + Sub~. lmp~a~e-~in. 6/g 6/9 + - ment afte~ 2 days of E~s, FVI dxt. 6/6 6/6 ~ - 2 relapse-~ at ~in. 6/18 6J18 ~ - pred 10 ~nd O mg/day, re~p, GHP dxt. 6/9 6/9 + - Relapse ~t ~ln. 6/1~ 6/1~ + - pred 10 mg/day 427402~002/A91 J~S/ALM/AIM/30. 10.1989 F~OM PLOUGM~ IN6TOFT ~MON) g~ 3~, 2~,~5 NO,~g~15~5~ PR~E 34 ~oa~7so - Comment~: Even though the evalatlon of th~ clinic~l r6spon~e is complicaeed by conco~itant therapy wlth glucooor~icoid6, all 4 pa-tients ~ eyes) ~enefited fro~ fusidin therapy. Thus, there w~e no ~igns of acute chorioretinal inflammation, ~nd ~ patient~ were ~ble to lo~er the do~age of gl~cocorticoid. ~owever, attempts to complete-ly ~ithdraw glucocort~cold treatment resulted ln relapse in 3 patl-~ne~. Similar r~lap~e~ sre seen lf glucocortlcoid therapy is with-drawn fro~ CyA-trea~ed patlent~ with severe uveitis. ~one of the patients hat side-effect~ sttributed to fusidin.

14.3. Cl~nlcal Examinatlon of the Antlinfl~mmatory Efflcacy of Fusi-. dln in Patientc with Uv~iti4 Anterior Material~ P~tlRnts with acute onset or recurrence of uveitis an~erlor co~espondlng to 20 eyes.

Group 1 includes patients corresponding to 10 eyes t~eated with fuc~thAlmic eyed~ops 6 ti~e~ daily, ~roup ~ ls tre~ed wlth ~luc~r~l~o~ eyedr~p~ 6 t~es d~lly (~.g.
Maxldex~, from Alcon, TexaY, U,S.A.) Both groups are tre~ted wlth atropine eyedrops ~e,g. f~o~ DAK, Copen-hagen, Den~ark) ~X twlce daily.

~0 Dur~tlon of tre~tm~nt~ 3 weeks.

Cllnlcal control: Ae least once a week.

Excluslon: Uveitis as a result of ba~eerial/viral lnfections, ongoin~
sy~te~lc ther~py for collageno~i~, Aggrav~tion of ~veitis durin~
actual treatment, and non-co~plianee.

FROM PLOU~M~N~ ~ UI~I~TOiT (MON)~ '5~ N~ 4~5 PRI~E
~01790 15.3. Multlcenter st~dy of the efflcacy of fusidin in sight-threste-ning, non-infectiou~, immunoinflam~atory, posterlor uveitis Ai~s of Study To comp~re ~fficacy, ssfety and tolerability of oral fusidin with conventlonal therapy (corticosteroids) for the treatmen~ of ~lght-th~estenin~ uveltls.

Study p~rameter~ a~

- Number of patlents ~ithdra~n fro~ ~tudy medication because of contr~indlcations to continued therapy Vlsual ~cuity - Par~mete~c ~f inflam~atory ac~lvlty - Fluorescein angIography - Nu~ber of relapses - Side-effect~, safety parameters - ~lobal evaluation of efficacy and tolèrability by investigstor and p~tient, - I~munologlcal studies, includin~ measurements of blood level~ of cytokine~.

Type of Study Mult~center, open, rando~i2~d, concrollet, parallel-group study with "mA~ked" opth~lmologlse for unbiAsed recordin~ of symptoms in uveitis ~Compari-con of fusidin vs. conventlonal therapy).

4~A~/A31/AS/~M/AUM/~10l~

F~AOM PLOU~H~ N~TOFT ~MON~Y ~ Y~5~ hO,~g~1~4645 PH~E 5 Patient~

Nu~ber: Initially 2~ patient~. At this s~sge, decision wlll be m~de ~hether to contlnue.

Startin~ eriteria Actl~e unl- or bilateral ocular involve~ent.

Exclusion criteria Te~inal 6tAge of dise~se ~lth nonreve~sible degeneration of retin in which no active lesion i5 observed.

Pb~lents who are 6uffering from myosis or cataract which diseases m~de it lmpos~ible to note the posterior pole in both eyes will ~e excluded.

Ongoi~g ~nfectious disease, hepatic dysfunction, l.e elevatlon 2.5 above upper limit of nor~al vaLue of either ALAT, ASAT, bilirubln (direct and intirect) and total protein, non eompli~nce.

Genersl Stuty Outllne Patients on co~ticoste~oids and~or cytostatlc agent~ cho~ld h~ve these drugs withdrawn before definite selection and before rando~iza-tion.

In con~ecuti~e order, patients should be randomlzed ta elther fusidln (monotherapy) or conventional therapy.

Two investlgators ~hould take part in ~he sCudy. Investigator 1 should know the ~ssigned treatment, whereas the lnves~ig~tor 2 (oph-thal~ologlct) should be ~asked in this re6pect. Investigator l should evaluate s~fety par~meters, c~de-effeets and ~hould prescriSe ~edica-tion. Invectlgator 2 should evaluate only the efficacy para~eters. Heg~ould not have access to other data.

~27~B~2J~lJA~l~LM/ALu/3o lo l~

FROM PLOUI~M~ UIh~TOFT ~MON~ 8~ '53 ~10.2~ 4~q5 P~I~E 51 The a~,cl~ned therapy rhould be maintalned unless contraindicstion~, occur, Patients ~ando~ized to conventional therapy r,hould start on monothe~apy wi~h corticoseerold~. If ~fter a ~ini~ of 4 weeks, the response is inadequate, fusidin therapy should be ~n~tiated (in combinat~on with low-dose cortlcosteroids). If, sfter further 4 weekc, ~he response ls ~natequate, fusldln should be replaced with cyclorporln therapy, ~nd the patient should be ~Ithdrawn from the study.

Patient-~ ~hDuld be investigatet a~ baseline, at weeks 1, 2, month~ 1, 2, 3, 4, 6, 9, 12 and thereater a~ 6 monthly Intervals if the di-sease Is qules~ent or a~ ~ny ~i~e if acute attacks occur.

Flrst full analy~i~ of data should be performed on 6-months data of the first 2~ pattents, and thereafter at 12 ~onths intervals.

The only conco~it~nt medica~ion fo~ uveitis allowed are gluco-corticoids and cyclopley,ic eye drop~, Subcc,n~un~tival glucocortlcoid ln~ections are not ~llowed.

A) ~e~t ~edlcation Fusidin tablets 0.5 g 3 time~ d~lly B) Conventional TherApy-Group 1,5 mg p~ednirolone (e.g. from DAK, Copenhagen, Denmsrk) per kg per day for A week. Th- dose will be decreased gradu~lly to reach a ~aintenance level of 5 to 10 mg ~rednisolone per day. In case of relaps~, ehe dosa~e will be incre~sed.

Co~traindicstions for Con~lnued Therapy ~for both trea~ent groups) - Visual acuity: If afte~ msxi~u~ therapy of at least two weeks, the vLsual aeulty falls t~o lines below basel~ne at the in~ti~-tion of therapy) on two suecessive day5 in either affected eye.

~U~A~ S/ALM~MJ~lo~

F~OM PLOU~M~NII ~ UIN~TO;T ~MO~ .3~ 54 NO.~g~164~5 P~E 5 ~(~0:~790 6~
- Inflamm~tion: If, ~fter maximu~ therspy of at least two weeks, the ~nflammatory activity Ag deter~ined by a second observer, i~
worse snd the visual acuity is not improved in either eye.

- If the di~ease process progresses into the ~acula ~hich in the opinion of a second observer mi~ht lead to permanent 10s6 of vl~lon ln either eye.

~oxicity or 5ide-effecte a) 1~psired hepatic function, ~uch ~s any value 2.5 time~
that of the upper li~ of normal b) Allergic reaction~ to fusldin c) SLde-e~fects felt by the pa~ient to be of such a nature that it l~ impossible for hlm/her to conelnue.

Dlgcase actlvity Patient~ w~ll be evaluated At baseline and at weeks 1, 2 snd ~onths l, 2, 3, 4, 6, 9, 12, and thereafter at 6-mon~hly lneervals during at le~s~ l year if di~ease i~ qulscent or at any t~me if acute ~ttdck Occllr .

Conco~itant medlcatlons: Any concomLtant medication~ will be listed du~lng ~he pretreat~ent evalutlon and a~ each subseque~t evalu~tion.

Ophthalmological evaluations by ~a~ked~ ophthalmolog~st ~I) V'~ tu~l QCUity b) Inflammaelon, anterior cha~ber fl~re, vitreal haze, and opacity c) Other ocular flndlng~

d) Flurore~celn angiogr~phy (only to b~ done when medLa allowJ and 2S ~t ~onths 3, 6 and 12) 4Z7~31/AS/ALM/ALI~/30.lO,lg~

F~OM PLOU~M~N ~ UIN~TOFT ~MOII/~g~ '54 NO.~g~164~45 P~E 53 Z()01790 Laborato~y tests a) Blood: He~oglobln, he~atocrit, WBC count and different~al, platelets, sedimentation rate, total protein, total billrubln, ~lkaline phosphatase, ALAT, ASAT, potassiu~ and creatinine.

b) Qualitati~e urinalysis ~protein, sugar, ~ediment).

c) At entry to ~tudy only: Pregnancy test.

Immunologlcal Investi~atlons (optional):

Serum parameter~.

a) Seru~ protein electrophoresis b) Ig l~els c) Ser~m IFN~, IL-2, IL-2 receptor, ILlo~, TMF~

Cellular parAmeters ~) Blood lymphocyte ~nd ~onocyte analyses - total count ~nd subsets b~ H~stocompatibll~ty antigen~ - HLA-DR (only at time 0).

Autoim~une re~ponslveness~

a) Antinucle~r antibodies b~ ~he~matoid ~actor Functlon~l immune c~pacit~ec 0 ~ Baokground level~ and ~esponses to LPS and P~A measure~ by proli~erAtion ~ss~y~ and cytokine produot~on b) A~to-antlbodies for cytokines (ILl~ and TNF~) ~27U)2BA.002/A31~ ALI,I/ALMJ3~.10.1~

F~OM PLOU~M~NN & ~IN~TOFT ~MO~ 1.313. 1~'55 NOI~g~1~4~45 P~E 54 Z~O~790 Withdrawals / Drop-out6 Withdrawals are patients who for re~sons related to test medlcAtlon ~contralndca~ions a~ speclfied) stop further therapy. They should be fully analysed and should not be replaced by ne~ patients. At time of withdra~al, a complete evaluation should be performed.

Drop-out6 are patient~ lost to follow-~p or non-co~plying to pro-tocol, They should be replaced (next free patient number~.

~X~MPLE 15 Fusidin treatment of Crohn '8 dise~e Bac~round It has been establ~hed that cyolos~orin, is effective aeainst Crohn's disease (46). ~owever, the treat~en~ can only be gi~en ln ~mall doses and in short periods of time due to the risk of d~ngerous side-effect~.

Alm~ of study~ Examination of the gastrointestinal absorption of fusidin and the possible effect of fusidin in the trestment of Crohn'~ dl~ease.

Type of study: Open pilot study Patients The lnltlal ~aterial includes a total of 6 patients with Crohn' 5 dlsease.

Startlng criteria - Active Crohn' 8 t~ease where ~ed~cal ~reatment l~ ~lanned.

/AS~M/ALM/~.10.1~

F~OM PLOUuMHN~ TOFT ~ON)~g~ '55 ~0~ 4645 P~GE 55 ~:~790 - Any prednicolone treatment should not exceed 20 ~g per day and must be kept unchanged for ae lea~t 2 weeks.
- Any salazopyrin ~SASP) or 5-aminosalicylic ~cid ~5-ASA) treat-ment should algo be kept ~nchanged for at lea~t ~ weeks.

Exclusion crleeria - Patients receiving ~ny oeher treatment of Crohn's disease.
Patients having re~eLved cytostatic treae~ent with~n the lsst 2 w~ks.
- P~tients with d kno~n hypersensitivity to fusidln.
- Patlents with known, serious liver diseases.
- Patients pl~nning to beco~e pregnant or being pregnant.

General Study OutlinP

Fucidin tabletJ O.5 g 3 ti~es daily for 4 weeks. If no po~clve effect is regl~tered, the patien~ should be excluded fro~ the test.

If a positive effeç~ is registered, treat~ent ls carried on for another 4 week period. In ca~e the disea6e is aggrevated and other treat~ent ~ 9 needed, the patiene c~n at any ti~e ~e excluded fro~ the test.

Other treatment If the patlent Is receiving SASP ~sallcylazos~lfapy~idine) / 5-ASA
(5-~minoæalicylic acid) treatment by the start of the test, dosaee ~hould be kept unchanged. ~oncerning prednisolone, see below.

Dlsea~e ~ctivity The tise~se actlv~ty is ~easured by means of the ~odified "Gr~dlng Score~ a.m. (46), The effect of the treatment ~ ~ defined as a po~iti~
ve ~otal score. The reduction of the dosage of prednosolone c~n be includ~d as dn individual treatment ~. Ac ~econdsry actlvlty Ai~S, P-orosomucold ~oncentra~$on and ~Crohn's DiQe~se Activity Index"
(CDAI) ~re used.

F~OM P~Oll4M~N~ IN~TOFT ~MON~ .50 NO.~g3104045 P~E 56 2~01790 Cl$nlcal control wlth blood ~ampling should be carried out after 0, 2, 4, ~ and 12 w~ks of t~eAtment an~ 4 weeks after ended therapy and at any other ti~e depending upon cl~n~cal status.

L~borAtory tests Blood: Sediment~tlon rate, orosomucoid. hemo~lobin, hematocrit, billrubin, ALAT, ASAT, alkallne phosphatase.

Further~ore, samplec are taken ~5 ml seru~, 5 ml EDTA-pl~sma and 5 ml hep~rln plas~a) for determining ~he fus~din ~on~entration - the p~tient 18 not allowed to take his ~ornin~ dosa~e until after the blood s~mples h~ve been t~ken - and pla~ma levels of cytokines and cytoklne autoantibodies.

E~AMPL~ 16 ~us~din ~n tre~tmerlt of acute graf~-versus-host disesse ~R~ute Cv~{D).

Back~ro~lnd 15 Acute GvH~ Is ~ ~ymptom complex appearlng 8 -40 tays (median 14 days) After allogene~c bone marro~ transplantation 85 ~ consequence of the re4ction of donor T-lymphocytes against recipient antigens, especial-ly tra~splantation antigens in the HLA-syste~. This occurs ~n 70X of the p~t~ent~ with HLA-identical siblin~ donors but is more frequent~y observed ln HLA lncomp~tible family donor or HLA-identic~l unrel~red donor.

The followlng grading $s valuble interims of the pro~nosis and thera-peutlc response:

F~OM PLOU4M~N~ ~ ~ I N~TOFT ~ UON ~ g~ ' 5~ NO . ~lg~ 164~45 PR~E 57 ~r~de ~ (~llght grade): Macular or confluent exanthema. ~iarrhoe~ not more than 500 ml per 24 hour~.
Bilirubin c 25 mikromol/liter, Gr~de II ~moderate grade): Exanthema as well BS
di~rrhoea SOO-1000 ml per 24 hours And~or bil~rubin ~ 25 mikromol per ~iter Gr~de III aRd IV (heavy ~rsde): Exan~he~a snd dia~rhoea more than lO00 ml per 24 hours and/or bilirubln > 50 mikromol per llter.

The occurence of acute GvHD ln modera~e to heavy ~rade $~ co~bined with incre~ed lethality after allo~eneic bone marrow transplanta-~lon. Le~hallty is especially due to Infeotions ~s a result of delay-ed l~munologicsl reconsti~ on. Thls is caused both by the GvHD
itself and by the f~ct that these patients often receive further immunosuppr~6ive treatment with prednisolone and~or anti~thymocyte globulin.

Presently ~pplied treatment s~rategy All patlents ~re treated prophylactically against acute G~HD. This includes eyclo~porin ~day l to d~y +180), posslbly supple~en~ed with methotrexate i.v. (day l, 3, 6 and po~ibly ll), dependin~ upon th~ e~ti~ated risk of developing acute GvHD, Supplementary treatment Again~t G~HD is carried out in the fo~lowing ~tuations:

1, Gr~d~ I (slight) ~YHD which i~ ~ubjeccively very annoying, or which is not reduced after app. one week'~ observation.
2, Quickly progre~slng grade Il (moderate) G~HD.
3. Or~de III (heavy) Gv~D.

Glucocorticoids are uged, po~sibly supplemented with anti-thymocyte glob~lln. The to-cage oi' cyclo~po~in is al~o increased.

/AS/AUM/ALUJ~.10.1~

FROM PL~U~ H~ IN~TOFT ~ON~'g~ '57 NO,~ 4~45 PRGE 5 ,4~O~7~90 ~5 Aim and type of .ctudy Pilot exam~nat~on to evaluate the effect of fusidin in patient~
treated with sllogeneic bone marrow er~nsplantation whc develop acute GvH~ d.esp~te adequate prophylsxis with cyclo~por~ n ~nd, o~e~onally, 5 ~ethotrexate. If fu~itin reduce~ the development of GvHD, ~ clinical-ly lmportant reduction in glucocorticold and cyclosporin consumption and, hence, a reduction in side- effects can be obtained (e,~. infec-tions and nephropathy), Patients: lO patients receiving allogeneic bone marrow transplanes lO because of leuke~l~, aplastic anemia, or other life- threatening ~arrow dysi-~nction Exclusion critsria - Patlents under 18 yea~s of age.

- Pat~ents ~ho according to the above-~entioned crlterla ~hould be treated with glucocort~coid.

- Patients receiving 21ucocorticold and/or anti-thy~ocyte globulin before or ~t the ti~e of transplantation.

~ote:
Before fusldln trea~men~ is carried out, adequate cyclosporin pro-phylaxis should be carrled out, a, If the results of the latest cyclosporln concentration test are below therapeutic level, the cyclosporin docage should be ~n-cres6ed and the effect ~hotlld be ~waited (24 hours).
b. If the re6ults of the latest cycloQporin concentration test ls at an adeque~te level, but the patient is in the ~eanti~e be-~nnine to suffer from dia~rhoea and/or vomiting which can lead to rlor~-absorption of cyclosporin, peroral cyclospor~n must be sYbst~ted w~th in~ra~reneou-~ cyclosporln and the effect should be a~sited ( 24 hours ), 42~gP'\.Q~J~.31/AS~ALM/ALAllJ30.10.191~

F~OM PLOU~M~N~ IN~TOFT ~MON~ .3~ '57 NO,~ 164~45 PR~E 5 Gen~ral study outl~ne In patients eligablo for tre~tment w~th fusidin (see above):

- Blood s~mples are taken for measuring the cy~lo~porin concen-tratlon (valley-value).
5 - Fucldln tablets or mlxture or, ~n ~ases of moderste/severe diarrhoea, fusidin for i.v. in~ection, 12 ~gtk~ 3 times daily in the first 2 d~ys, thereaf~er 8 mg~kg 3 times daily.

If the fu~idin treat~ent is considered efficaceous, t~eatm~nt is continued for 10 days whereafter fusldin is w~thdra~n.

If pro~ression occ~rs in spLte of fusidin treaement, the drug is withdr~n and conventional therapy is instituted (~ee ~bove).

Dl6ea~e ~ctivity For esch individual pat~ent, the spread of the exanthema ~s reeorded along w~th diarrhoea volume and body temperature Laboratory test6 Blood: Sedl~entation rate, orossomucoi~, hemoglobin, b~lirubin, ALA~, ASAT, alkaline phospatase, 5 ml serum and 5 ml EDTA-plasma ars taken for m~asurements of cytoklnes and autoantibodies to cy~oklnes.

4Z7~ Q~/~l/A3/ALM/ALM/30.10.1~

F~M PlO~uM~lN ~ TOFT ~UON ~ ' 5g NO. ~71~4645 PRIiE 613 2~ 790 ~PLe 17 Fusidln ln treatment of muleiple my610m~1 Clinical effect of iusidin in early ~n~gement o~ mul~iple myeloma Aim of ~tudy 5 To gain prelimin~y experience wit~ fusidin in the ereatment of patients with multiple myeloma Speclflc eims are to etabllsh:

- ~os~e schedule of fusldln.
- Safety and tolerability of fusidin.
- Effec~s of fusidin on immunoinfla~a~ory parameters.

Depending upon the results of thL-~ pilot st~dy, a deci~ion sh~uld be ~ade ~heth~r to start a controlled study.

Type of ~tudy Open, un~ontrolled pilot st~dy.

Yatient~

10 Adults of both sexes.

The dia~nosis of Dultiple myeloma is establishet accordin~ to con-ventional cr~teria, ~.e. the classic triade. Mar~ow plasmocytosis (~10 percent~, lyric bone lesions, and a ~er~m and~or urlne 11 M
eo~ponent, or pla~moeytosis associated with a progressive incresse in the M co~ponent over time o~ if extramedull~ry mass lesions develop.

Study medlcatlon Fucidin tablets O.S g three time~ daily for at lesst 3 ~eeks 4~ /AJ~/~SJALU~UU/~.10.1~

F~OM PLOU~M~ TOFT ~MON~g~ 5~ NO,~ 4~45 PRIiE ~1 2~0~790 Inve~tig~tlon~:

At entry, the followin~ dat~ are recorded~

componen~
- marrow biopsy, radiogrs~ of the skull - ConComitBnt diseAse(8) and ~edic~tion L~borstory te5 ts ~

Blood; Haemoglobin, RBG, ~C, and dlfferential count, platelets;
Serum c~eatinine, blllrubin, alkaline phosph~tase, ALAT, ASAT, elec-trolyees, se-celciu~, IGA, IgG, I~M, M component.

Urlne: 24 h-proteinuria, glucose, M co~ponent.

Immunologleal tes tc Blood level~ of çytokines And in ~itro production of IL-6 Follo~-up inv~stigations:

The study runs for minl~u~ 3 ~eeks and maxi~u~ 3 month~, Data whlch wlll be collected ~t weekly interval~ during the fLr.~t ~ weeks, and then at 2-4 weekly interval~:

- Slde-effects which ~n be ~sc~lbed to f~lsidin, - Symptomc and s~ gn~ of ~ultiple myeloma and concomitant di-oea~e(s) .
Details of ~11 therapy, Labar~tory parameters: Creatinine, creatinine clearance, liver func-tlon te~t~, hematology. urine (24 h proteinuri~), M component.

The immunolo~cal test~ ~hould be repeated ~t le~t once monthly.

427~ LM/30. 10.1~

F~OM PL~G~ & U I N~TOFT ~ MON ~ ' 5~ NO . ~83164~45 PR~E

- Trough plasma levels of usidin (blood drawn in the ~orning before flrst dosage of fusidin~ should be performed ~t bi-weekly lntervals.

Wlthdrawal~drop-out~

~ithdrawal4 are defined ac patients who di6continue therapy either becau6e of ~ide-effect6 or in_dequste efficacy. The~e pstient~ should be fully analysed and data safety and efficacy required also after withdr~wal, Drop-outs are p&tients lost to follow-up, not co~plying to instruc-t~ons or otherwise violaeing t~e protocol. These pat~ ents should be replaeed by new patients.

EYA~LE 18 Cli~al effec~ of fusidin in early r~n~emerlt o~ rheurnatoid ar-e~2rleisJ fuvenile rherlm~toid arthri~ls, polymy~ rheurnatlca and 15 systeml e lupus erythemato~us (SLE) 18.1. Effect of fusidin in moderately ~ctive cases of sy~temic lupus erythematosus ~SLE) as ~n at~unct to glucocorticold the~apy B~ck~ro~nd Th~ ~se of glucocortlcolds has ~proved the outcome of ~ystemic lupu-~
erythema~osus ~SLE), particul~rly if accompanied by renal involve-~ent. The ~ddition of other immunos~pp~essive drugs, incl~ding cy-tostatic drug~, ha~ been advocated in ~ome cases. However, there i~
a need for ~ treAtment comple~entary to glucocorticoid~ in SLE be-cause:

a, So~e p~tients, particularly patients with sys~emic vasculiti~, do not re~pond to corticosteroid~ alone.

4~4023~X~ /AS~ALM/ALM~ 01~

F~OM PLOUull~ UIN~TOFT ~MON~ .3~ 5~ NO,~3164~45 P~GE 63 b. Many pat~ents are ln need of high dose ~10 mg prednisolone per d~y) cortlco~tqroid treatment; They ~el~p~e as soon as ehe d~ug i5 progresgively diminished or stopped.

c~ Severe ~ide-efiects of high dose glucocor~icoid therapy.

Aim of Jtudy To gain prell~lna~y experience with f~sidin in the treatment of SLE
patient~ who, de~pite prolonged therapy wlt~ low doses of gluco-corticoldc, ~ 10 mg prednicolone per day), show ~ ns of clinical ~tlvlty.

10 Speclfic aimg ~re to estsblish:

- Dosage sohedule of fusidin.
- Safety and tolerability of fus~din.
~ Ei-fects of fu~idin on i~munoinflammatory parameter~.

Open, uncontrolled pilot study.

Pstient~

10 adults of both &exes.

Inclusion criterla The SLE dlagnosi~ should fulfil four or ~ore of the Ameri~an Rheuma-tis~ A~soelation'~ criteria for e~e ol~ific~cion of SLE at the ti~e of diagnosis. Patien~s who, despite receiving c 10 ~ of prednlsolon daily for ~ 3 months, presen~ ~ith ~ubJective and~o~ objective sign~
of ongoing dlsease sctivity.

Exclw ion criteria - Patient~ receiving complemencary treatment ~e.g. cytostatic drugs) fo~ SLE.

42r~A,Q~J~31J~ tALM~0.10.1985 FROU PLOll~MR~ GTOFT ~MON`1g~ 1~.3~ NO~ 316~45 P~E ~4 P~tlen~s who sre not ~reated with glucocortlcoids.
- Pregnancy and patients in child-be~ring age not practising effoctive birth control.

Study ~edication - Fucidln tablets 0.5 g three tlmes daily for st least 3 weeks.
- ~rednisclone (e.g. ~rom DAK, Copenhagen, Den~ark). rhe tosA~e eho~ld be the ~esn tally do~dge ~s 10 ~g per day) given ~n the prevlous three months. Thi~ do~ge must be kept unalte~ed throughout the study.

Inve~eigations At entry the follo~ing data should be recorded:

- Hl~to~y of SL~ ~nd ~ub~ective And obJective ~gns of SLE.
- Concomitant diseaQè~s~ ant m-dication.

Laboratory test6:

Blood: H~e~oglobin, RBC, WBC, and dif~erentlal c~l.nt, platelets;
serum ~reatinine, biliru~ln, ~lkaline phosphatase, AIAT, ASAT, elec-trolytes Urine: 24-h-proteln~ria, glucose.

I~unolog~cal blood tests:

20 - Antinucle~r snd ~nti-DNA antibodies.
- IgG, IgA, Ig~.
- Coombs'test, rheumatoid factor.
- J lymphocyte level (membrane immunoglobulin).
- T lymphocyte level ~nd subpopulaelon~ (CD4, CD~).
Levels of cytokines and in vitro production of IL-2, LT (lympho-toxine (T~F~) and IFN7.
- Complement C3 ~nd C5a.

42?4C2~W/~ J~LM/ALM/30.10.1989 FROM PLOU~M~ & ~ TOFT ~MON~ 13 NOI~g~1~4~45 P~I~E ~5 ~2 Follow-up inve~tig~tions:

The ~udy runs for ~inimu~ 3 weeks and maximum 3 months. D~ts wh~ch will be collected at weekly intervals du~ing the first 3 ~eek~, and then ~t 2-4 weekly intervals - Side-effects which can be acribed eo fusidin.
- Sy~peomJ and signs of gLE and concomitant dise~se(s).
Details of all th~rapy.

~abor~tory paramet~rs: Creatinine, creat~nine clearance, liver func-tion te~ts, hem~tolo~y, ur1ne (24-h- proteinuria).

The immunological eests will be repeaeed at le~st once monthly.

- ~rou~h pla~as levels of fusidin (blood dr~wn ln the morning before firct dosage of fusidin) will be performed at bi-weekly lntervals.

Wlthdrawals~drop-outs lS Withdr~wals are defined as p~tients ~ho discontinue ther~py either becauee of side-effect~ or inadequate efficacy. These patients will b~ fully analyeed and data s~fety ~nd efflcacy required slso after withdr~

Drop-out~ are patients lost to follow-up, not complying to instruc-20 tlonL or otherwice viola~ing the protocol. Th~- patiens ~ill be replaced by new pAt~en~s.

18.2: Effect of fu~idin as an immunolnflammatory ~odulator in rheu~a-toid arthriti~ given l~mediately ~fter diagnosis ~ackground Rheum~toid arthritis Is a co~mon disesse of unknown cause. rhe ~n~
flammatory processe6 in rheu~told a~thritis include ~ncreased pro-duction of synovlal fluid, activation ~nd prolife~ation of cell~ in 4Z7~ LM/ALM/30.10.1~89 F~OM PLOU~MR~ih ~ T~FT ~ MON ~ , 2~ . ~1 110, ~ 45 P~E ~6 ~he syno~al ~embrane, destruct~on of articular cartilsge ~nd bone, and repAi~ processe5 resulting in f~brosic, eetoplc c~lcif~cation an~
metaplastic bone formation.

Poly~orphonuclea~ leukocytes snd macrophages ~M0) ~lay A critical role in all these proce~see ~35, 36). Immunocompetent cells are ~lso lnvolved, and ln ~eve~e cases the inflamed ~ynovial ti~sue may re-~emble a lymphoid organ ~ith germinal centers of B lymphocy~eg s~r-rounded by T lymphocyte~, Generally, a relatively large proportion of the infiltra~ing T ~ells besr membrane markers characteri~tic for actlvated T cells, and such cell~ are often found in the blood as well (3S), The presence of plasma cells and immune complexes in the 6ynov~al ti~ue and the frequent finding of ~utoan~ibodies, parti-c~larly anti Fc-I~G rheumatold factors, and hypergamma~lobullnemi~
also suggests tha~ antibody-mediated reactions are involved ~n the di6esGe procecses. Howc~er, the6e humoral reaceions may reflect that the celluldr infiltrate in the ~yno~i~l tiss~e is dominated by ac-tivated T cells of the helper phenotype, and these çell~ may trigger B cells to produce antibodies ~n an uncontro~led manne~. Many lnves-t~gators believe that in rheuma~oid a~thritis, the host recponds to an exogenous antigen, or an endo~enous antigen rendered im~no~enic in an aberrant manner, to generate an Inapprcpriate cellular as well a~ hu~oral im~une re~ponse.

IL-l and, po~sibly, TL-6 ~nd TNF~ seem to play a central role In t~e processes leading to tis-~ue d~age ~n rheumatoid arthriti~ and rela-ted rhe~matic diseases (~), Ihus, ln~ectlon of IL-l and TNF~ in knee Joint~ of n~rmal rabbits causes similar bio~he~ical changes as those seen in e~rly rheu~atoid arthritis (37~, and blood levels of IL-l ~nd IL-6 clo~ely parallelc .cub~ectlv~ and ob~ective di~ease aeti~ity in pa~ient~ with rh-umatoid ~rthrici~ (3~).

The ~dence tha~ cyclo~porin i~ effective in rheu~atoid ~rthritis, both ~hen experimentally induced (39) and in clinical cases (40-43), further ~upport the notion that IL-l and/or IL-6 ~ay trigger the ln~ti~ unoinflammatory proces~es which eventually cause~ ~oint and bone de~tr~ctlon. Unfortunately, significant ~dver6e ffects of ~ LMt~LM/30 101~119 FROM PLOU~MR~N ~ ~IN4TOFT ~M~N~'g~ 1~.3~, 2~ 1 NO,~g~164~5 P~GE 6 Z~ 790 ~4 cyelosporln have been noted, and alternative therapies are therefore ~dvocat-d.

Aim of B tudy To galn pr~llmlnary experience wlth fusldin in the tre~ement of p~tients with newly diagnosed rheumatoid arthritis.

Specific aim6 are to es~abli~h the dos~ge schetule, safety and to-lerability of fu61din and effect~ of the drug on immunoinflam~atory parameters in rheumaeoid arthritis.

Open, uncontrolled pilot study.

P~tients lO adult~ of both sexes Incluslon cr$teria The diagnosis should i`ulfil four or more of the American Rheumatis~
Association's criterla for the clAs~ification of rheumato~d ~rthritls at the time o~ dia~nos~ s . Patients ~u8t not ha~e recelved medication other than non-steroidal aneiinflam~atory drugs (NSAI~) (in particu-l~r glucocorticoids, golt salts, penicillamine, cytost&tic drugs), and the duration of sctive disea4e ~hould not exceed 12 months.

Exclusion cr$terla - Patlents receiving treatment oeher than NSAID.
- PregnAncy and p~tients $n ohild-bearing ~ge not practisLng effective blrth control.

Study medication - Fucldin, tablet~ 0,5 g three times daily for at lea-4t 2 months, Investigation~

A ~ .31/AS/ALM/~.LM/30.10.1~8~

F~OM PLOlll~M~h ~ i)IN~ToFT ~MON ~ NO. ~g~ 45 P~E

At entry the following data will be recorded - ~istory of disease ~nd sub~ectiv~ and ob~ective ~i~ns of rheu~a-toid arthritis.
- Coneomitant di~ease(s) and medicat~on.

L~boratory te~t~

Blood: Haemoglobin, RBC, WBC, And tifferential count, platelets;
serum creatinlne, blllrubin, alkaline pho~phatase, A~AT, ASAT, elec-trolyte~.

Urine: proteln, glucos~.

Immunological ~lood tests' An~ln~cl~ar and anti-DNA sntibodies.
- I~G, I~A, IgM.
- Rheumatoid factor.
- B lymphoc~te level (membrane i~munoglobulin).
- T lymphocyte level and cubpopulations (CD4, CD8~
- Levels of cytokines and in vitro production of IL-2, ~T ~nt IFN~, - Complement C3 and C5a.

Follow-up ~vesti~ations~

The study runs for ~lnimum 2 months and maximu~ 6 ~onths. Data which will be collected at bi weekly intervals durin~ the first 2 months, and then ~t 4 we-kly inte~vals:

Sid~-effect6 which can be ~scribed to fucidin.
- SymptomS and el~ns of rheumatoid arthritis ~nd conco~itant dlsea~e(~).
- Detalls of ther~py.

42~442B~002/A31 J~ LM/30.10. l F~OM PLOU~M~N~ ~ ~IN~TOFT ~MO~g~ 1~.3~. 2~ 10.~08~1~4~45 P~E

Laboratory parameters: Creatin~ne, liver function tests, hem~tology, u~ine ~protein, ~lucose), The l~unological tests should be repeated at least once every second ~onth.

- Trough plasma levels of fusidin ~blood drawn in the morning before first dos~ge of fu~ldin) should be perforned ~t bi-we~kly intervals during the first two months and then at four weeks intervals.

Withdrawals/drop-out~

Withdrawals are defined a~ patlents who dlscontlnue therapy either beca~se of side-effectc or inadequ~te effic&cy. These patien~s should be fully analysed and dstA on .~afety and efficacy are required alco ~fter ~ithdrAwAl.

Drop-out~ ~re p~tlents lost to follow-up, not complylng to instruc-tLo~s o~ otherwi~e viol~ting the protocol. rhese patlenes sh~uld be replaced by new pat~ents.

18.3. Other connecti~e tlssue dlseases:

Since the term rhe~matold a~thritis applies to a syndro~e wLthout known cause, it is diffLcult to accerta~n whether the pathogenes1s of this connectLve t~sue disease differs ~arkedly from related and often ~verlapping clLnLcal syndromes. Thus, other connective tissue disease~, s~ch as ~uvenile rheuma~old arthrltis, psorlatic srthritis, resctive arthrl~ls (Reiter's syndrome~, poly~yalgla rhe~at~ca, systemic sclero~i~, and s~rcoidosls ~ay be pathogenetically related.
Thae si~ila~ disease mechanis~s may be operstive in several or all of ehese diseases is stren~thened by the fact that patients wieh dLf-ferent connective tissue di~esses benefit fro~ therapy with the T
cell-selective im~unos~ppres~ive drug cyclosporin ~44). The tr~at-ment of these d~seases, however, is often un~a~isfac~ory, and almo~t alwayc accompanied by side-effects.

4n~o~/A3l/AsJALMJAL~/3o~lo~

F~OM PLoll~M~lN ~ IJ~NI~TOFT ~MON~'g~ 113,~0. ~1~1'0~ N0~20~ 4~5 P~E 70 8~
The cl~ni~dl studies in connection with these diseases should be performed snalogously eo ehe protocols outlined abo~e.

FR~M PLOUS~NN i l)IN~TI:lFT (MON~'g~ 3~ 110,~31~4~45 P~E 71 Fig~ 1~ Cellular in~eraotions and cytoklne product~on during anelgen p~esen~ation to T- ~nt B lymphocytes, The cytokines produced by both ~mmune and non-~mmuno cells ~ay enter the b~ood ~cream and act as S hormones which affect the functions of di~tant tissues. Modified from (3).

~0~ mAcropha~e. Th: T helper lymphocyte. B: B ly~phocy~e. F: fibro-blast, ~X: nat~ural ki~ler cell. ~R: MHC clasA II molecule. IL-1 IF:
lnterle~kin 1-inducing factor.

Fig. 2~ Eff~ct of fu~idin on IL-1 and TNFa productlon, The percentage of inhiblclon 1~ ~h~ J ~ f~ct~ on of the f~ 1 t~t ~b~ tl dt~
of fu~din. * : P~0,01, n-12 ~IL-1) and n~6 (TNF~).

Fig. 3: Effect of fusidin on IL-2 and IFN~ production. ~he percenta~e of tnhibition is s~own as a function of the final test concentration of fusidin. * , PcO.05, n-6.

Pig. 4: Effect of fusidin on I~-1 (mouse thymocytes). The percentage of inhibi~ion i~ ~ho~n a~ a funcelon of the final te~t concent~ation of fus~dln. * : P<0.01, n-8.

Fig. ~: Effec~ of f~sidln on IL-1 (human ly~phocytes). The percentage of inhibition is shown as a f~nctlon of the f$nal test concentration of fusidin. * : Pc0.05, n-5. PPD: purified proteln derivative of tuberculin (~ntigen). P~A~ phytohe~sgglutinin ~nonspecific, poly-elon81 T lymphocyte activator).

Flg. 6~ Eff~ct of fusidin on IL 1 (mou~e thymocyte cell line, EL 4~.
The percentage of inhibition ~s sho~n 85 8 function of the finRl te~t concentration of fusldin. * P<0.05, n-7, Flg. 7: Effect of fusidin to inhibit IL-~ and TNF~ activities. The percentag~ of inhibition is ~hown ~s ~ function of the f~n~l test concentr~tion of fusld~n. n-4 (IL-2) and n 6 (IFN~, r-sp-ctively.

4~4~A~/A31/AS/~UMJALM~.10.1~

FROM PLOll~MhlN~ IN~TOFT (MO~ ~ g~ 1~, 3~ 4 NO, ~ 4~45 P~E 7 Fig. ~: Effect of delayed addition of S ~g~ml of fu~ldln to PHA-ac-tlvated murine thymocytes.

Fig. 9~ Effect of fusidin on the t~o-way mixed lymphocyte reaction.
The percentage of inhibition i~ sha~n as a function of the final te~
concentration of fu~idin. * : PC0.01, n-10.

Fi~. 10: Effect of fu~idin on 1~-6 (B9 cells), The perc~ntage of Inhibit~on is shown as a function of the final test concentration of fu~idin. Human rIL-6 ~as added to the test cells at a concentration of l U/ml . * : PC0 . 02, n-7 .

Fig. 11: Reversibil-ty of fu~idin ind~ced lnhibltion of IL-6 ~ctivity ~B9 sssay). The tes~ cells were treated as indicated. The ~ells were ereated with 18 U/ml of rIL-6 wlth/wlthout 30 ~g/~l of fu~ld~n, as indicatéd (PRE). After 2 days, thè cells were wa~hed three ti~es ~nd cultured further for 2 days with/without 30 ~g/ml of fu~idin, A8 Indicated ~POST), The assay for IL-6 ~ctivity wa.~ perfor~ed d~ring ehe last 2 days of culture in the presence of the lndicated levels of rIL-6, Fi~, 12, IL-l~ $ndu~ed inhi~icion of insulln secret~on (S of control:
In~ulin secretion in cultures kept in medium ~lone without IL-l).
Protec~lon afforded by fucidin. The rat ~ancreatic islets of Lan-gerhanc were precultured for 6 d~ys. The iele~s we~e then washed and added fresh medium, conta~nlng 11 mmol/l of glucose, and rIL-l~ +/-fusldLn at the indlcated concentration~.

Fig. 13: In~b$1ity of f~sidin analog~ to protect sgain~t IL-l~ ~n-duced ~-cell d mage. The culture conditions were the sa~e as in Flg.
12, except that only one conce~tr~tlon of the fusidin 8nalog8 ~ere te~ted.

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~C~l ~LOU~M~N~ ~ UINGTOFT ~MON~g~ 1~,31~ 7 NOI~g~1~4~45 P~E 7g ss 41. Fo~e 0, B~erkhoel F, S~lvesen CF, Be~g KJ, Rugs~ad HE, Sdel~d G, Mellbye OJ, Kæ5Q E. An open, controlled, randoml~ed comparlson of cyclo-~porin ~nd azathlopr~ne ~n the treatment of rhe~m~to~t ar-thrltis~ a prelimin~y report. Arthr~Rheumatis~ 1987;30:B8 92, 42. ~oug~to~ ~, Awada H, Amor B. Cyclosporln in rheumatoid srthr~ tis:
a double bllnt, plaoebo controlled Ctudy in 52 patLents.
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Claims (75)

1. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for substantially inhibiting a biological effect in a human related to a cytokine for prophylaxis or treatment of a condition related to disturbances of a cytokine system, such as for the manufacture of a composition 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 pro-phylaxis after establishment of being in a high risk group of de-veloping diabetes mellitus (type 1), or for the manufacture of a composition for the treatment of endogenous uveitis, or for the manufacture of a composition for the prevention of the in-flammation after eye surgery such as cataract operation or laser surgery, or for the manufacture of a composition for treatment of progression of arthritis such as rheumatoid arthritis, psoriatic arthritis or Reiter arthritis substantially immediately after the first diagnostic estab-lishment of arthritis, or for the manufacture of a composition for prophylaxis or treatment of arthritis urica, or for the manufacture of a composition for treatment of osteoarthrosis, or for the manufacture of a composition for prophylaxis or treatment of a condition related to transplant rejection, such as for the manufac-ture of a composition for prophylaxis or treatment of a condition related to cornea transplantation, or for the manufacture of a composition for prophylaxis or treatment of a condition related to a graft-versus-host reaction, or for the manufacture of a composition 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 for the manufacture of a composition for the treatment of perniceous anemia or celiac disease, or for the manufacture of 8 composition for prophylaxis or treatment of contact dermatitis, or for the manufacture of a composition for prophylaxis or treatment of allergic/atopic dermatitis , or for the manufacture of a composition for treatment of pemphigus vulgaris or pemphigoid, or for the manufacture of a composition for treatment of a condition related to the function of the thyroid gland, in particular hyper- or hypofunctioning of said gland, e.g. thyroiditis (acute, subacute or chronic), including Hashimoto's disease (lymphocytic thyroiditis;
lymphoid thyroiditis), Riedel's thyroiditis (chronic fibrous thyroi-ditis), de Quervain's thyroiditis (subacute granulomatous thyroidi-tis), subacute lymphocytic thyroiditis, Graves' disease, Graves'sub-acute thyroiditis or Graves' ophthalmopathy, or for the manufacture of a composition for treatment of Simmonds' panhypopituitarism or for the manufacture of a composition for treatment of a condition related to hypofunction of the adrenal glands, in particular Addi-son's disease, or for the manufacture of a composition for treatment or prevention of replapse of a demyelinating disease, in particular multiple sclero-sis, or for the manufacture of a composition for treatment of prevention of relapse of sarcoidosis Boeck. Sjögren's syndrome, Reiter's syndrome, erythema nodosum, scleroderma or Bechet's disease, or for the manufacture of a composition for treatment or prevention of relapse of polymyositis, polymyalgia rheumatics, myocarditis or systemic lupus erythematosis, or for the manufacture of a composition for treatment or prevention of relapse of a condition related to vasculitis phenomena, e.g. polyar-teritis nodosa, Wegener's granulomatosis, or giant-cell arteritis, or for the manufacture of a composition for treatment or prevention of relapse of primary biliary cirrhosis or chronic active hepatitis, or for the manufacture of a composition for treatment of aplastic anemia or idiopathic thrombocytopenic purpura, or for the manufacture of a composition for treatment or prevention of relapse of a neoplastic disorder of the lymphoid tissue, e.g. B cell lymphoma or multiple myeloma, or for the manufacture of a composition for treatment or prevention of relapse of a T lymphocyte proliferative disorder, e.g. mycosis fun-goides or Sézary syndrome, or for the manufacture of a composition for prophylaxis or treatment of septic shock caused by gram-negative bacteria, or for the manufacture of a composition for prophylaxis or treatment of disseminated intravascular coagulation, or for the manufacture of a composition for prophylaxis of arterio-sclerosis, or for the manufacture of a composition for prophylaxis or treatment of a condition acute and chronic periodontal diseases, in particular periodontitis and periodontosis.

2. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for prophylaxis or treatment of diabetes melli-tus, 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 diag-nostic establishment of diabetes mellitus, or for prophylaxis after establishment of being in a high risk group of developing diabetes mellitus (type 1).

3. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for prophylaxis or treatment of progression of arthritis such as rheumatoid arthritis, psoriatic arthritis or Reiter arthritis substantially immediately after the first diagnostic establishment of arthritis.

4. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for prophylaxis or treatment of septic shock caused by gram-negative bacteria.

5, The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for treatment of Crohn's disease or ulcerative colitis, especially for the prevention of relapse or progression of Crohn's disease or ulcerative colitis.

6. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for prophylaxis or treatment of a condition related to transplant rejection.

7. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for prophylaxis or treatment of a condition related to a graft-versus-host reaction.

8. The use according to claim 6 of fusidic acid or a derivative thereof for the manufacture of a composition for prophylaxis or treatment of a condition related to cornea transplantation.

9. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for the treatment of endogenous uveitis.

10. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for the prevention of the inflammation after eye surgery such as cataract operation or laser surgery.

11. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for prophylaxis or treatment of contact der-matitis.

12. The use of fusidic acid or a functional derivate thereof for the manufacture of a composition for treatment or prevention of relapse of systemic lupus erythematosis.

13. The use of fusidic acid or a functional derivate thereof for the manufacture of a composition for treatment or prevention of relapse of a neoplastic disorder of the lymphoid tissue such as B cell lymp-homa or multiple myeloma.

14. The use of fusidic acid or a derivative thereof for use as an immunosuppressive drug.

15. The use according to any of claims 1 - 14 wherein the composition is a composition suitable for oral administration.

16. The use according to any of claims 1 - 14 wherein the composition is a composition suitable for rectal administration.

17. The use according to any of claims 1 - 14 wherein the composition is a composition suitable for parenteral administration.

18. The use according to any of claims 1 - 14 wherein the composition is a composition suitable for intraarticular administration.

19. The use according to any of claims 1 - 14 wherein the composition is a composition suitable for application to the skin,

20. The use according to any of claims 1-14 wherein the composition is a composition suitable for application to the eye.

21. The use according to any of claims 1-14 wherein the composition is a composition suitable for implantation administration.

22. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for the treatment of contact dermatitis, in particular for the manufacture of a composition for oral administra-tion.

23. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for the topical treatment of contact dermati-tis, in particular for the manufacture of a composition for topical administration to the skin.

24. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for treatment of Crohn's disease or colitis ulcerosa, in particular for the manufacture of an oral or rectal composition, or a composition for parenteral administration.

25. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for treatment of arthritis substantially imme-diately after the first diagnostic establishment of arthritis, in particular for the manufacture of a composition for oral use or for use as parenteral or intraarticular injections.

26. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for treatment of endogenous uveitis, in parti-cular for the manufacture of a composition suitable for treatment of the eye, such as an eye drop composition, an eye ointment composi-tion, an eye lotion composition or an injectable composition for intraocular or retroocular injection, or an oral composition.

27. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for the prevention of inflammation after eye surgery, in particular an oral composition, or 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 injection.

28. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for prophylaxis or treatment of a condition related to transplant rejection, in particular for the manufacture of composition for oral or parenteral administration.

29, The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for prophylaxis or treatment of a condition related to a graft-versus-host reaction, in particular for the manu-facture of a composition for oral or parenteral administration.

30. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for prophylaxis or treatment of diabetes melli-tus (type 1), in particular for the manufacture of a composition for oral or rectal use.

31. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for prophylaxis or treatment of septic shock caused by gram-negative bacteria, in particular for the manufacture of a composition for oral or parenteral administration,

32, The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for treatment or prevention of relapse of systemic lupus erythematosis, in particular for the manufacture of composition for oral or parenteral administration.

33. The use of fusidic acid or a derivative thereof for the manufac-ture of a composition for treatment or prevention of relapse of a neoplastic disorder of the lymphoid tissue such as B cell lymphoma or multiple myeloma, in particular for the manufacture of a composition for oral or parenteral administration.

34. The use according to any of the preceding claims wherein the fusidic acid or a derivative is used together with a ciclosporin (Cyclosporin A) or a derivative thereof or together with FK-506.

35. A method for substantially inhibiting a biological effect in a human related to a cytokine for prophylaxis or treatment of a condition related to disturbances of a cytokine system, such as 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 (type 1), or for the treatment of endogenous uveitis, or for the prevention of the inflammation after eye surgery such as cataract operation or laser surgery, or for treatment of progression of arthritis such as rheumatoid arth-ritis, psoriatic arthritis or Reiter arthritis substantially immedia-tely after the first diagnostic establishment of arthritis, or for prophylaxis or treatment of arthritis urica, or for treatment of osteoarthrosis, or for prophylaxis or treatment of B condition related to transplant rejection, such as for prophylaxis or treatment of a condition rela-ted to cornea transplantation, or for prophylaxis or treatment of a condition related to a graft-ver-sus-host reaction, or for the treatment of Crohn's disease or ulcerative colitis, especial-ly for the prevention of relapse or progression of Crohn's disease or ulcerative colitis for the treatment of perniceous anemia or celiac disease, or for prophylaxis or treatment of contact dermatitis, or for prophylaxis or treatment of allergic/atopic dermatitis, or for treatment of pemphigus vulgaris or pemphigoid, or for treatment of a condition related to the function of the thyroid gland, in particular hyper- or hypofunctioning of said gland, e.g.
thyroiditis (acute, subacute or chronic), including Hashimoto's disease (lymphocytic thyroiditis; lymphoid thyroiditis), Riedel's thyroiditis (chronic fibrous thyroiditis), de Quervain's thyroiditis (subacute granulomatous thyroiditis, subacute lymphocytic thyroidi-tis, Graves' disease, Graves' subacute thyroiditis or Graves' oph-thalmopathy, or for treatment of Simmonds' panhypopituitarism or for treatment of a condition related to hypofunction of the adrenal glands, in particular Addison's disease, or for treatment or prevention of replapse of a demyelinating disease, in particular multiple sclerosis, or for treatment or prevention of relapse of sarcoidosis Boeck, Sjö-gren's syndrome, Reiter's syndrome, erythema nodosum, scleroderma or Bechet's disease, or for treatment or prevention of relapse of polymyositis, polymyalgia rheumatica, myocarditis or systemic lupus erythematosis, or for treatment or prevention of relapse of a condition related to vasculitis phenomena, e.g. polyarteritis nodosa, Wegener's granuloma-tosis, or giant-cell arteritis, or for treatment or prevention of relapse of primary biliary cirrhosis or chronic active hepatitis, or for treatment of aplastic anemia or idiopathic thrombocytopenic purpura, or for treatment or prevention of relapse of a neoplastic disorder of the lymphoid tissue, e.g. B cell lymphoma or multiple myeloma, or for treatment or prevention of relapse of a T lymphocyte proliferati-ve disorder, e.g. mycosis fungoides or Sézary syndrome, or for prophylaxis or treatment of septic shock caused by gram-negative bacteria, or for prophylaxis or treatment of disseminated intravascular coagula-tion, or for prophylaxis of arteriosclerosis, or for prophylaxis or treatment of a condition acute and chronic perio-dontal diseases, in particular periodontitis and periodontosis, the method comprising administering to a human in need thereof an effective amount of fusidic acid or a derivative thereof,

36, A method of treating diabetes mellitus, in particular insulin-dependent diabetes mellitus (type 1) which method comprises admini-stering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

37, A method of preventing a progression of diabetes mellitus, in particular insulin-dependent diabetes mellitus (type 1) which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

38. A method according to claim 37 wherein the effective amount of fusidic acid or a derivative thereof is administered substantially immediately after the first diagnostic establishment of diabetes mellitus.

39. A method of prophylactic treatment of diabetes mellitus (type 1) after establishment of being in a high risk group of developing diabetes mellitus (type 1).

40. A method of treatment or prevention of the inflammation after eye surgery, which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

41. A method of treating endogeneous uveitis, which method comprises administering to a human in need of such treatment an effective amount of fusidic acid of a derivative thereof.

42. A method of treating arthritis substantially immediately after the first diagnostic establishment of arthritis which method compri-ses administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

43. A method of treating arthritis urica which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

44. A method of treating osteoarthrosis which method comprises ad-ministering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

45. A method of treating a condition related to transplant rejection which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative there-of.

46. A method of treating a condition related to a graft-versus-host reaction which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

47. A method of treating a condition cornea transplantation, which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

68. A method of treating or preventing a relapse of Crohn's disease or ulcerative colitis which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

49. A method of treating pernicious anemia or celiac disease which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

50. A method of treating contact dermatitis which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

51. A method of treating allergic/atopic dermatitis which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof,

52. A method of treating pemphigus vulgaris or pemphigoid which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

53. A method of treating a condition which is related to the function of the thyroid gland, in particular hyper- or hypofunctioning of said gland, e.g. thyroiditis (acute, subacute or chronic), including Hashimoto's disease (lymphocytic thyroiditis; lymphoid thyroiditis), Riedel's thyroiditis (chronic fibrous thyroiditis), de Quervain's thyroiditis (subacute granulomatous thyroiditis), subacute lymphocy-tic thyroiditis, Graves' disease, Graves' subacute thyroiditis, Gra-ves' ophthalmopathy or Simmond's panhypopituitarism which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

54. A method of treating a condition which is related to the hypo-function of the adrenal glands, in particular Addison's disease, which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative there-of.

55. A method of treating or preventing relapse of a demyelinating disease, in particular multiple sclerosis, which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

56. A method of treating or preventing relapse of sarcoidosis Boeck, Sjögren's syndrome, Reiter's syndrome, erythema nodosum, scleroderma or Bechet's disease which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

57. A method of treating or preventing relapse of polymyositis, polymyalgia rheumatica, myocarditis or systemic lupus erythematosis which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative there-of.

58. A method of treating or preventing a relapse of a condition related to vasculitis phenomena, e.g. polyarteritis nodosa, Wegener's granulomatosis, or giant-cell arteritis which method comprises ad-ministering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

59. A method of treating or preventing a relapse of primary biliary cirrhosis which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

60. A method of treating aplastic anemia or idiopathic thrombocyto-penic purpura which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

61. A method of treating or preventing a relapse of a neoplastic disorder of the lymphoid tissue, e.g. B-cell lymphoma or multiple myeloma, which method comprises administering to a human in need of such treatment an effective amount of fusidic acid of a derivative thereof.

62. A method according to claim 62 wherein disorder is multiple myeloma.

63. A method of treating or preventing a relapse of a T-lymphocyte proliferative disorder, e.g. mycosis fungoides or Sézary syndrome which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative there of.

64. A method of treating or preventing a condition of septic shock caused by gram-negative bacteria, which method comprises administer-ing to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

65. A method of treating disseminated intravascular coagulation which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

66. A method of prophylactic treatment of arteriosclerosis which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative thereof.

67. A method of treating a condition related to acute and chronic periodontal diseases, in particular periodontitis and periodontosis, which method comprises administering to a human in need of such treatment an effective amount of fusidic acid or a derivative there-of.

68. A method according to any of claims 36-68 wherein the fusidic acid is administered orally, preferably in an amount from about 1 mg to about 75 mg per kg body weight per day.

69. A method according to any of claims 36-68 wherein the fusidic acid is administered rectally, preferably in an amount from about 1 mg to about 100 mg per kg body weight per day.

70. A method according to any of claims 36-68 wherein the fusidic acid is administered topically to the skin in an effective amount.

71. A method according to any of claims 36-68 wherein the fusidic acid is administered to the eye, preferably in an amount from about 0.1 mg to about 50 mg per kg body weight per day.

72. A method according to any of claims 36-68 wherein the fusidic acid is administered parenterally, preferably in an amount from 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.

73. A method according to any of claims 36-68 wherein the fusidic acid is administered intraarticularly, preferably in an amount from about 0,1 mg to 20 mg per kg body weight per day.

74. The use according to any of claims 1-35 wherein the derivative of fusidic acid is selected from the group consisting of dehydrofusidic acid, 3,11-didehydrofusidic acid; 24,25-dihydrofusidic acid; 17,20-24,25-tetrahydrofusidic acid; 17,20-24,25-tetrahydrofusidic acid and their correspcnting 3 acetates (especially conjugated with glycine or taurine): and 3-0-acetyl-16-epideacetylfusidic acid.

75. A method according to any of claims 36-68 wherein the derivative of fusidic acid is selected from the group consisting of dehydrofusi-dic acid: 3,11-didehydrofusidic acid; 24,25-dihydrofusidic acid;
17,20-24,25-tetrahydrofusidic acid; 17,20-24,25-tetrahydrofusidic acid and their corresponding 3-acetates (especially conjugated with glycine or taurine); and 3-0-acetyl-16-epideacetylfusidic acid.