Note: Descriptions are shown in the official language in which they were submitted.
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).
4271~''~ ~5~A31/AS/ALM/ALM/30.10.19~9
6; ~ S~ O~'ON S,61 '0'01 6~ ~NOW~ 1~019NIn ~ NN~W~llOl~ WO~.
~0~ 7~
TABLE 1
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~
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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
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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.
42740~002/~31f~S/ALM~ALM/34.10.19~0
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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
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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(~
13
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
14
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
16
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
17
"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
21
V3 1177, 117~, 1303, 13~0, 13~2, 1460, 1546 and P~ 10~9 described in
table 2 belo~.
TAPILE 2
~ ,,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~
23
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
24
- 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
790
26
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
73
27
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
L79
28
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
29
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
79
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
31
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
32
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
33
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
34
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 ¬her 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(~
36
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
L790
37
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
20 MATERIALS AN3 METHODS
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
38
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
39
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
41
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
- 42
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~
43
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
790
44
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),
ESAMPLE 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
790
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
790
46
TABLE 3
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
790
47
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).
ESAMPLE 4
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
48
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$on of fusidin ~as 1,5-5 ~g/ml. Si~llar suppres
slon of the ML~ W85 obtained ~it~ 15-50 ng/~l of cyclospor~n.
EYA~PLE 7
~usldin-in~u~ed inhiblelon the hybridom~ growth-p~omoting effect of
IL-6
The test ~as performed as described ln "Hybridoma gro~th induced by
IL-6~,
IL-6, another M0-derived peptide med~a~or, has been reported to
medlate cever~l ~unc~ions of I~-l, including ~hymocyte activation
(2, 7, 6) . It was therefore of interest to ~ee whether fusidin lnter-
f~r~d with the function of IL-6. ~ shown in Fig. 10, e~e growth of
the B9 hybridoma cell llne wss ~nhibited by fus~din. The dose respon-
4e was very si~llar to the ones obta~ned when testing ~he effect of
fueldln on the IL-l-~nducèd growth of no~mal (~ouce) thymocyte~.
F$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.
TABLE 5
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
52
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, $ncludlng productlon of lymphokines (1-4) ehe first
~lgnal belng antigen or mitogen-induced perturbation of the T cell
receptor/CD3 co~plex ~1,2,11,12).
The ~blllty of fusidin to lnhibit a ~ixed lymphocy~e reaotion ~n
35 vStro is probably also ~econdary to its lL-l-inhibltory effec~. At
F~OM PLOI~ JI~ T:lFT ~MON~ 5~ ~0,2~315~5~ P~bE ~3
790
54
~ny r~te, tho inhibltory eifece of fus$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
790
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
56
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
57
~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
790
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
790
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
61
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
790
62
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
1790
6~
TA3LE 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
790
64
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
66
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
0
67
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
790
68
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
71
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
72
- 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~$on and ~Crohn's DiQe~se Activity Index"
(CDAI) ~re used.
F~OM P~Oll4M~N~ IN~TOFT ~MON~ .50 NO.~g3104045 P~E 56
2~01790
73
Cl$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
74
~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
790
76
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
77
~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
78
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
2001790
79
- 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
790
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
790
81
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
790
~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
790
83
~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
790
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
790
86
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
790
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
790
88
LEGENDS T0 FIGURES
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
L790
B9
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$1ity of f~sidin analog~ to protect sgain~t IL-l~ ~n-
duced ~-cell d mage. The culture conditions were the sa~e as in Flg.
12, except that only one conce~tr~tlon of the fusidin 8nalog8 ~ere
te~ted.
42~402~002/~31J~S/ALM/ALh~30.10.15~9
FROM PLOU~M~N~I ~ UIN~TOFT ~MON~'g~ 4 NO,~ 164~4$ P~E 7
790
REFERENCES
1, Bendtzen K. Lymphokines in inflamm~elon, InflA~ation Basic ~echa-
nisms ~Issue InJuring Princ~ple~ and Cl~nical Mod~ls (P Venge ~ A
Lindbom et6 ) 1~8S: Almqvi~t L Wiksell Internat~onal. Stockholm
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