Note: Descriptions are shown in the official language in which they were submitted.
~39~5~
73605-14
B~CKGROUND OF THE INVENTION
~ he present invention describes drugs for the
treatment of a variety of diseasas and medical conditions.
The common elem~nt of the diseases and medical conditions
that axe suitable ~or treatment according to the methods
described her~in is an involvement o~ interleukin-1. This
invention describes methods for the treatment of
interleukin-l mediated diseases and medical conditions.
Cytokines are extracellular proteins which modify
the behavior o~ cells, particularly those cells that are in
the immediate area of cytokin2 synthesi~ and release. one
of the most potent inflammatory cytokines yet discovered and
a cytokine which is thought to be a key mediator in many
diseases and medical conditions is interleukin-l (IL-1).
Interleukin-l, which is manufactured, though not
exclusively, by cells of the macrophagaJmonocyte lineage,
may be produced in two forms, IL-l alpha (Il-la) and IL 1
beta (IL-lb).
A disease or madical condition is considered to be
a "interleukin-l mediated disease" i~ the spontaneous or
experimental disease or ~edic~l condition is as~ociated with
elevated level~ of IL-l in bodily fluid~ or tissua or if
cell~ or tis~ues taken fro~ the body produce elevated levels
of IL 1 in culture. In ~any cases, such interleukin~1
mediated diseasss are also recoyniz2d by he following
additional two conditions~ pathological findings
associated wlth th~ diseasQ or medical condition can be
mimicked experimentally in animal~ by tha admini~tration of
IL-l; and (~ the pathology induced in experimental animal
models of the clisease or medical condition can be inhibited
or abolished by treatment with agents which inhibit the
action of IL-1~ In most "Interleukin-1 mediated diseases'~
~3~
at least two of the three conditions re. met, and in many
"interleuXin-1 mediated diseases" all three conditions are
met. A list of diseases or medi.cal conditions which are
interleukin~l mediated includes, but i5 no~ limi~ed to, the
following:
1) Arthritis
2) Inflammatory Bowel Disease
3) Septic 9,hock
4) Ischemic: injury
5) Reperfusion injury
6) Osteoporosis
7~ Asthma
8) Insulin diabetes
9) ~yelogenous and other leukemias
10) Psoriasis
11) Cachexia/anorexia
Arthritis is a chronic joint ~isease which
afflicts and disable~, to varying degrees, millions of
people worldwide. The disease is typically characterized at
the microscopic lev~l by the inflammation o~ synovial tissue
and by a progressive degradation of the molecular components
constituting the ~oint cartilage and bone. Continued
inflammation and erosion of the joint frequently lead to
con iderable pain, swellin~, and loss of ~unction.
~ hile the etiology of arthritis is poorly
understoodt considerable information has recently been
gained regarding the molecular aspects of inflammation.
This research has lead to the identi~ication o~ certain
cytokines, which are believed to figure prominently in the
mediation of in~lammation. The involYement o~ interleukin-l
in arthritis has been implicated by two distinct lines of
evidence. First! increased levels o~ interleukin-1 and of
the mRNA encoding it have been ~ound in the synovial tissue
and nuid of arthritic join~s. Re~erencas of interest
include G. Buchan e~ ~1., Third Annual ~eneral Meeting of
-2=
~ ~ 3 ~
the ~ritish Society for Rheumatology, London, England,
November 19-?1, 1988, PR. J. Rhieumatol 25 (Supplemental 2)
1986; A. Fontana et al., Rheumatology Int., 2, pp. 49~53
(1982); and G. Duff et al, Mono:kines and Oth~r Non-
Lymphocytic Cytokines, M. Powanda et al., editors, pp. 387
392, 1988 Alan R. Liss, Inc.
Second, the administration o~ interleukin~1 to
healthy joint tissue has been shown on numerous sccasions to
result in the erosion of cartilage and bone. In one
experiment, intraarticular injections oP I~ 1 into rabbits
were ~hown to cause cartilage destruction ln vivo as
described by E. Pettipher ~_3~., Proc. Natl. Acad. Sci.
USA, Vol. 83, pp. 8749-8753, ~ovember, 1986. In other
s udies, IL-l was shown to cause the degradation of both
cartilage and bone in tissue explants. Relevant references
include J. SaXlatavala ~ lo~ Development of Diseases of
Cartilage and Bone Matrix, Alan R. Liss, Inc.~ pp. 291~298,
and P. Stashenko et al., The American Association of
I~munologists, Vol. 138, pp. 1464-1468, No. 5, March 1,
1987.
One generally accep~ed theory used to explain the
causal link between IL-l and inflammation is that IL-l
sti~ulates various cell types, such as ~ibroblasts and
chondrocytes, to produce and secrete proinflammatory or
degradative compounds, such as prostaglandin E2 and
collagenase Consequently, the present inventors postulated
that substanc that inter~ere with the aetivity o~
interleukin-l would appear to make excellent candidates ~or
use in the treatment o~ inflammatory diseases like
arthritis.
Inflammatory bowel disease ("IBD") is a term used
to describe both acute and chronic in~lamma ory conditions
of the tissue of th~ intestinal tract and encompasses two
generally distinct maladies known as Ulcerative Colitis and
Crohn's disease. Ulcerative Colitis is a mucosal ulceration
-3-
of the colon. Crohn's disease, which is also referred to as
Ileitis, Ileocolitis and Colitis, is a transmural
inflammation that can be found throughout the yeneral
intestinal tract.
IBD is characterized by various histological
features including transmural acute and chronic
granulomatous inflammation with ulceration, crypt abbesses
and marked fibrosis. Not all of these indications will be
found in all IBD cases. Spontaneous reactivation,
extraintestinal inflammation and anemia are o~ten associated
with IBD. Large joint arthritis is commonly found in
pati2nts suffering from Crohn's disease.
A5 is found in the molecular processes of the
in~lammation associated with arthritis, research has found
that various cytokines appear to mediate aspects of IBD. In
particular, IL-l has been implicated as a mediating material
in IBD. Again, two distinct lines of evidence lead to this
conclusion.
Increased levels of IL-l have been found in
aff2cted areas of intestines fro~ patients with I8D.
Tissues from pati~nts with active Ulcerative Colitis showed
IL-1 levels about 15 times the level found in control
samples. TissuQs with active Crohn's disease showed IL-l
levels about 6 times that of ths control, and tissues with
inac~ive Crohn's diseasa were about 3 times that o~ the
control tissue sa~ples. Sartor çt ~1. Gastroenterology, 94,
Pg. A399)(Abstract o~ Paper). See also 5a sangi et al.
Clin. Exp. Immunol., 67, Pp. 594 605 ~1987); Rachmilewitz et
al. Gastroenterology, 97, Pg. 326 (19B9) (the bioassay used
th~rein to determine IL-1 concentration levels is known to
also unselectively detect lL-2, IL-4, IL-6 and IL-7~.
Th2 role of IL-l in IBD ~a~ also been implicated
by studies that have shown that the perfusion of rabbit
colons with IL-1 induces the production of prosta~landin and
thromboxane. Comminelli ~ 31- Gastroenterology, 97, Pp.
-4-
~ ~ r~ ~ ~,L ~ ~
1400-1405 (1989). This is consistent w:ith the hypotheSis
~escribed above, that IL-l is linked to the in~lammation of
tissues due to its stimulation of the production of
proinflamm~tory or degradativa compounds. Thus, it is
likely that systemic and local IL-l production initiates or
contributes to the infla~matory response in IBD, and plays
an active role in the pathogenesis of the disease. The
systemic production of IL-l may also be responsible, in
part, for the extraintestinal inflammation associated with
Crohn's disease.
These re~ults have led the inventors hereo~ to
propose that substances that would interfere with the
activity of interleukin-1 could be ef~ective compounds for
the treatment of IBD.
Septic shock is a condition as~ociated with
massive bacterial invasion. It is commonly believed that
the shock is brought on, at least in part, by the presence
o~ bacterial toxins (e.g., liyopolysaccharides). Septic
shock is a relatively common causa of mortality in the
hospital setting. At present there are few treatment
Qpti.ons for patients sufferi.ng from septic shocX~ a~d the
treatments available are generally supportive in nature.
5eptic shock is characteri2ed by various symptoms
including a drop in ~ean art~rial blocd pressure tMAP), a
decrease in cardiac output, tachycardia, tachypnea,
lacticacidemia and leukopenia. Yarious ~ytokines, including
interleukin-l, hava been implicated in the mediation of
septic shock, although the specific etiolo~y of the disease
is not ~ully understood.
That IL-l may have a role in the mediation of
septic shock has been suggested by two lines o~ evidence.
one study has been conducted wherein the blood serum of
children suffering from gram-negative septicemia was
analyzed for IL-l concentration. Thi~ study showed that
elevated levP~ls of IL-1 were found in 21% o~ the patients
--5--
2 ~
~xamin~d. In addition, it was shown that IL-l serum levels
were significantly higher in patients who died than in the
survivors. Girardin ~t_al. New England J. of M~dicine 319,
pp~ 397-400 (1988). See alsQ, Cannon et al. Critical Care
Medicine, p. S58 (Abstract) April, 1989 (increased I~-l
levels in patients suffering ~rom sepsis ~yndrome).
It has also been shown that human IL-l induces
shock-like state in rabbits. A single bolus injection o~
human IL-lb brought about hypotension and several
hemodynamic and hematological parameters characteristic o~
septic shock. For example, the M~P of IL-1 injected ra~bits
decrea~ed by a minimum of 19.1%. Okusawa et al., J. Clin.
Invest., 81, pp. 1162-1171 (198~).
These result~ hav~ led the inventors hereof to
propose that substanc~s which interfere with the activity of
interleuXin-l could be effective compounds for the treatment
of septic shock.
Ischemic injury may occur to a tissue or organ
whenever that tissue or organ is depriv~d of its normal
blood flow. Further damage may occur wh~n the flow of
oxyg~nated blood is restored to that tissue. The extent and
reversibility of th~ damage imparted depends, partly, on the
severity o~ the original insult. It is possible, however,
to mitigate the ~xtent o~ tissue damage resulting from
reperfusion by a variety of th~rapeutic interventions.
Simpson PJ ~t al. In: ~alliw~ll B. (ed) Ox~q~L_adicals an~
Tlssu~ Intury, Brook ~odge Symp - Upjohn (1988).
Reperfusion injury is a well documented sequela to
ische~ic episodes in thQ h~art, gut, kidney, liver and other
organs. S~mpson PJ ~ . In: Halliwell B~ (ed) Oxyaen
Rad~als and Tissu~ Iniuxy, Brook Lodge S~mp - Upjohn
(1988); H~rman B. et ~1. FASEB J. 2:1460151 (1988); McDougal
WS. Th~ J. o~ Urology, 1~0:1325-1330 (1988); Finn WF. Kidney
Int., 7:171-182 (1990); Schrier XW. Klin Wochenschr, 66:800-
807 (lg88); and Winchel RJ. Transplantation 48:393-3g6
-6-
(1989). The exact pathogenesis of reperfusion injury may
vary depending on the tissue aPfected. In the heart, for
instance, reperfusion injury is accompanied by a dramatic
influx of neutrophils, and these cell5 are thought to play a
major role affecting the reperfu.sion damage. Lucchesi BR
et al. Ann Rev Pharmacol Toxicol 26:201-224 (1988). Renal
ischemia and reperfusion injury, on the other hand, appear
to involve an increase in tubula,r cell membrane
permeability, increa~ed levPls of intracellular calcium,
altersd mitochondrial re~pira~or~ function, and the
generation of ~ree radicals. In the kidney, the role of
extravasating neutrophils in affecting the reperfusion
injury is less certain. McDougal WS. The J. Urology,
140:1325 1330 ~19883; Finn WF. Xidney Int., 37:171-182
~1990); Schrier RW. Rlin ~ochenschr, 66:800~807 (1988); and
Winchel RJ. Transplantation 48:393-3~6 (1989).
Despite the differences in csllular participa~ion
during ischemia and reperfusion inju~y, there may he
similarities in the und~rlying mechanism. Interleukin-1 is
recognized as an early stage mediator of organ injury, and
may be generated by re~ident or newly infiltrated
in1ammatory cells giving rise to organ specific tissue
pathology. In such an in~tance, the ability to inhibit the
biological activity of IL-l would represent a novel
therapeutic intervention aimed at limiting the extent of
tissue damage.
This information has led the inventors hereof to
propo~e that substances which interfere with the activity of
interleukin-l could be e~ective compounds for the
minimization of ischemia and reperfu~ion injury.
To date, an ef~ective, yet ~elective, inhibitor of
IL-l has not been available in su~ficient quantities or
purity to prove that IL 1 is a target for pharmaceutical
intervention in the treatment o~ arthriti~ D, septic
shock, ischemia injury or reperfusion injury and for use as
--7--
73605-1
a therapeutic agent in the treatment of inflammation.
Despite the prior art, the present inventors have iden-
tified a class of compounds, referred to herein as interleukin-l
inhibitors, that prevent and treat interleukin-l mediated diseases.
Moreover the present therapeutic interventions may be practiced
without unacceptable compromise of normal physiological processes
(e.g. immuno competency) which are essential to the patient'swell
being.
In currently pending United States Patent Application
Serial No. 07/506,522, filed April 6, 1990 specifically incor-
porated herein by reference, a preferred class of naturally
occurring, proteinaceous interleukin-l inhibitors and a method for
manufacturing a substantial quantity of the same with a high
degree of purity are described. In particular, the aforementioned
application describes in detail three such interleukin-l inhibitors
which are interleukin-l receptor antagonists (IL-lra's), namely,
IL-lialpha (IL-lia), IL-libeta IL-liB, and IL-lix. These IL-lra's
will be referred to as IL-lraa, IL-lrab and IL-lrax, respectively,
in this application.
SUM~lARY OF THE INVENTION
The present invention describes a medicine (or a
therapeutic agent) for the prevention or treatment of interleukin-
1 mediated diseases of patientsin need thereof.
An object of the present invention is to provide a
therapeutic agent for treating interleukin-l mediated diseases,
such as interleukin-l mediated arthritis, interleukin-l mediated
2~39~
73605-14
inflammatory bowel disease ("IBD"), interleukin-l mediated septic
shock, interleukin-l mediated ischemia injury and interleukin-l
mediated reperfusion injury.
Additional objects and advantages of this invention
will be set forth in part in the description which follows and in
part will be obvious from the description or may be learned from
practice of the invention. The objects and advantages may be
realized and attained by means of the instrumentalities and com-
binations particularly pointed out in the appended claims.
To achieve the objects and in accordance with the
purposes of the present invention, medicines are disclosed for
treating interleukin-l mediated diseases, including interleukin-l
mediated arthritis, interleukin-l mediated inflammatory bowel
disease, interleukin-l mediated septic shock, interleukin-l
mediated ischemia injury and interleukin-l mediated reperfusion
injury. These medicines comprise a therapeutically effective
amount of an interleukin-l inhibitor in admixture with a pharma-
ceutically acceptable carrier. The medicines are practically
almost always placed in packages for commercial use. The packages
may carry indications or instructions that the medicines can be
used for preventing or treating such interleukin-l mediated
diseases.
Preferred interleukin-l inhibitors of the present
invention are proteins and, more particularly, are naturally-
occurring proteins. The naturally-occurring proteins are preferred
because they pose cL relatively low risk of producing unforeseen
":
-
73605-14
side effects in patients treated therewith.
A preferred class of interleukin-l inhibitors are
the human proteins which naturally serve as interleukin-l recep-
tor antagonists (IL-lra's). Preferably, those IL-lra's which
are preferred in the practice of the present invention are
selected from the group consisting of IL-lraa, IL-lraB, IL-lrax,
or the N-terminal methionyl derivatives of IL-lrax. Also pre-
ferred are proteins which have been modified for example by the
addition of polyethylene glycol (PEG) or any other repeat
polymer to increase their circulating half-life and/or to decrease
their immunogenicity.
While the production of IL-lra may be achieved by
extraction from naturally available sources, such as by isolation
from the conditioned medium of cultured human
- 9a -
:,:
~3~ $~
monocytes, a preferred method of IL-lra production is by
recombinant DNA technology. Recombinant DNA technology is
prsferred in part because it is capable of producing
comparativPly high~r amounts of IL~lra at greater purities.
It is to be understood that both the foregoing
general description and the ~ollowing detailed description
are exemplary and explanatory only and are not restrictive
of the invantion as claimed.
BRIEF DESCRIPTION pF T~E DRAWINGS
FIG. 1 depicts the release of glycosaminoglycans
(GA~) from bovine nasal cartilage in rssponse to increasing
amounts of IL-lB;
FIG. 2 depicts the inhibitory ef~ect of increasing
amounts of IL-lra on the IL-lB-induced release of ÇAG from
bovine nasal car~ilage;
FIGS. 3 and 4 depict the inhibitory effect of IL-
lra on the pathogenesis of type II collagen~induced
arthritis in mice:
FIG. 5 depicts the inhibitory eff2ct of IL-lra on
SCW reactivation of SCW-induced arthritis in rats.
FIG. 6 depicts the effects of IL-lra treatment on
various indications of interleukin-l mediated IBD,
FIG. 7 depicts the e~fects of IL-lra on PG-APS
reactiYation of joint in~lammation in conjl~nction with
indomethacin.
FIG. 8 depicts the effects o~ IL-lra on survival
rate in rabbits with Pndotoxin~induced shock.
ETAILED DESCRIPTION OF_PREFERRED EMBODIMENTS
Re~erence will now be made in detail to the
presPntly preferred embodiments of the invention, which,
together with ~he following examples, serYe to explain the
principles of the invention.
As noted aboYe, the present invention relates to
--10--
2~3~
73605-1
medicines for treating in-terleukin-l mediated di.s~ases
including in~erleukin-1 mediated arthritis, interleukin-l
mediated inflammatory bowel disease, interleukin-l mediated
septic shock, interleukin-l mediated ischemia injury, and
interleukin-l mediated reperfusion injury in patients
sufferinq therefrom. This medicine co~prises
a therapeutically effective amount of an
interleukin-l inhibitor. In one Qmbodimsnt, th0 preferred
interleukin-l inhibitors of the present invention are
naturally-occurring proteins that serve as IL~1 receptor
antagonists (IL-lra's).
A disease or medical condition i~ considered to be
a 1l interleukin-l mediated disease" if the spontaneous or
experimental disease or medical condition is associated with
elevated levels of IL-l in bodily fluid3 or tissue or if
cellc or tissues taken from the body produca elevated levels
of IL-l in culture. In many cases, such in erleukin-l
mediated diseases are also recognized by the following
additional two conditions: (1) pathological findings
associated with the diseas~ or m~dical condition can b~
mimicked experimentally in animal 5 by the administration oP
IL-1: and (2) th~ pathology inducQd in experimental animal
modsls of the diseasQ or medical condition can be inhibited
or abolished by treatment with agents which inhibit the
action o~ IL-l. In mo3t "Interleukin-1 ~ediated diseases"
at least two of the three condition~ are met, and in many
"interleukin-l mediated diseases" all thre~ conditions are
met. A list of disea-e~ or medical conditions which are
int~rleukin-l mediated includa~, but is not limited to, the
following:
1) Arthritis
2) Inflammatory Bowel ~isease
3) Septic 5hock
4) Ischemic injury
5) Reparfu~ion injury
2~4~g
73605-1
6) Osteoporosis
7) A thma
8) Insulin cliabetes
9) Myelogenous and other leukemias
10) Psoriasis;
11) Cachexia/anorexia
The naturally-occurrinq protein6 are preferred in
part because th~y pose a comparat:ively low risk of produoing
un~oreseen and undesirabl9 physiological sid~ efgects in
patients treated therewith.
For purpose~ oX ths specification and claims, a
protein is deemed to be I'natural:Ly-occurring" if it or a
sub~tantially equivalent protein can b~ found to exist
normally in healthy humans. "Naturally occurring" proteins
may b~ obtainQd by r~combinant DNA methods as well a~ by
isola.ion fro~ cells which 3rdinarily produce them.
"Naturally-occurring" encompasse~ prot~in~ that contain an
N terminal methionyl group as a consequenc~ of expression in
. Coli.
"Substantially equivalent" as used throu~hout the
specification and claim~ is defined to mean possessing a
VQry high degree of a~ino acid residue homology (See
qene~ally ~. Dayho~ s.og Protei~ Seq~ence and
~t~UC~U~, Vol. 5, p. 124 (1972), Na~ional Biochemical
Research Foundation, Wa~hington, D.C., SpQ ifically
incorporated herein by re~rence~ as w~ll as possessing
comparable ~iological a¢tivity.
~ articularly pre~erred IL-lra's o~ ~he present
invention arQ th~ naturally-ocGurring proteins that exist n
YiV~ a~ regulator~ o~ interleukin-l that have previously
been described in a currcntly pending Un~ted States patent
application. This application is U.~. Patent Application
SPrial No. 07~266,531, ~ d November 3, 198B, by Hannum e~
-, which i5 entitled "Interleukin-l Inhibitors."
73~0~ 3 ~ ~ 3~
Three preferred forms of IL-lra, each being
derived from th~ same DNA coding sequence, were disclosed
and described in the aforementioned Hannum et al.
application. The first of these, IL-lraa, was characterizsd
as behaving as a 22-23 kD molecule on SDS-PAGE with an
approximate isoelectric point of 4.8, eluting from a Mono Q
FPLC column at around 52 mM NaC1 in Tris buffer, p~ 7.6.
The second, IL-lraB, was charact~rized as behaving as a 22-
23 kD protein, p.I=4.8, eluting from a ~ono Q column at 60
mM NaC1. The third, IL-lrax, was characterized as behaving
as a 20 kD protein, eluting from a Mono Q column at 48 mM
NaCl. All thrPe o~ the Hannum ~t al. interleukin 1
inhibitors wer~ shown to possess similar functional and
immunological activities. The present inventlon also
includ2s modified IL-lra's. In one e~bodiment, the IL-lra
is modified by attachment of one or more polyethylene glycol
(PEG) or other repeating polymeric moietie~. In another
embodiment, the IL-lra contain~ an N-terminal methionyl
group as a consequence of expression in E~ coli.
Methods for producing the Hannum et al. inhibitor~
are also disclosed in th~ above mentioned application. One
disclosed method consisted of isolating the inhibitors ~rom
human monocyt~s (where thPy ars naturally produced). A
second disclosed method involved isolating the gene
responsible for coding the inhibitors, cloning the gene in
suitable vector~ and cell types, expressing the g~ne to
producQ tha inhibitors and harvesting the inhibitors. The
latter ~ethod, which is exemplary o~ recombinant DNA m~thods
in gen ral, is a preferred method o~ tha present inYention.
Recombinant DNA method~ are preferr~d in part becausP they
~re capable o~ achieving comparatively higher amounts at
greater purities.
Additional interleukin 1 inhibitors includa
compounds capable o~ specifically preventing activ~tion of
-13
2~3~
cellular receptors to IL-1, Such compounds include IL-1
binding prote.ins such as soluble r~ceptors and monoclonal
antibodies. Such compounds also include receptor
antagonists and monoclonal antibodi~s to the receptors.
A second class of IL-lra's include the compounds
and proteins which ~lock in vivc! synthesis and/or
extracellular release of IL-l. Such compounds include
agents which af ct transcription of IL-l genes or
processing IL-1 preproteins. Under certain conditions, the
IL-lra will block IL-l induced IL-1 production.
Preferably, the above de~cribed IL lrals are
produced by the aforementioned method in "substantially
pure" form. By "substantially pure" it is meant that th~
inhibitor, in an unmodified foxm, has a comparatively high
specific activity, preferably in the range of approximately
150,000-500,000 receptor units/mg as defined by Hannum et
al. in Nature 34~: 336-340 (1990) and Eisenberg et al. in
Nature 343~ 341-346 (1390), both of which are specifically
incorporated herein by reference. It is to be recogniz~d,
how~ver, that derivatives of IL-lra may have different
specific activities. In a preferred embodiment of the
present invention, a therapeutic co~position comprising at
least one o~ IL lraa, IL-lraB, and IL-lrax is administered
in an effective amount to patients suf~ering ~rom
interleukin-1 mediated diseases.
Because it is possible that the inhibitory
function of the preferred inhibitors is imparted by one or
mor~ discrete and separable portions, it is al50 envisioned
that the method of the pre~ent invention could be practiced
by ad~inistering a therapeutic composition whose active
ingredient consists o~ that portion (or those portions) o~
an inhibitor which controls (or control) interleukin-1
inhibition.
The therapeutic composition of the present
invention is ]preferably administered parenterally by
-14-
~. .
~3~
injection, although other effectiVe administratlon forms,
such as intraarticular înjection, inhalant mists, orally
active formulations, or supposit:orias, are also envisioned.
One preferred carrier is physio]Logical saline solution, but
it is contemplated that other pharmaceutically acceptable
carriers may also be used. In one preferred embodiment it
is envisioned ~hat the carrier s3nd the IL-lra constitute a
physiologically-compatible, slow-release formulation. The
primary solvent in such a carrier may be either aqueous or
non-agueous in nature. In addition, the carrier may contain
other pharmacologically-acceptable excipients for modifying
or maintaining the pH, osmolarity, viscosity, clarity,
color, sterility, stability, rate of dissolution, or odor of
the formulation. Similarly, the carrier may contain still
oth~r pharmacologically-acceptable excipients ~or modifying
or maintaining the stabili~y, rate of dissolution, release,
or absorp~ion of the IL-lraO Such excipients are those
substances usually and customarily employed to ~ormulate
dosages for parenteral administration in either unit dose or
multi-dose form.
once the therapeutic composition has been
~ormulated, it may b~ stored in sterile vials as a soluti~n,
suspension, gel, emulsion, solid, or dehydrated or
lyophilized powder. Such formulations may be stored either
in a ready to U58 form or requiring reconstitution
immediately prior to administration. The preferred 5 orage
of such ~ormulations is at temperature~ at least as low as
4 C and preferably at 70 C. It is also preferred that such
~ormulations containing Il~lra ar~ stored and administered
at or near physiological pH. It is presently believed that
storage and administration in a formulation at a high pH
~i.P. greater than 8~ or at a low pH (i.e. less than 5) i5
undesirable.
Preferably, the manner of administering the
formulations containing IL-lra is via an intraarticular,
-15-
~3~
subcutaneous or intramuscular route. Pr~ferably, the manner
of administering the formulations containing IL-lra is via
intra-articular, subcutaneous, intramuscular or intravenous
injection, suppositories, enema, inhaled aerosol, or oral or
topical routes. To achieve and maintain the desired dose of
IL-lra, repeated subcutaneous or intramuscular .inj ections
may be administered. Both of these methods are intended to
create a preselected concentration range of IL-lra in the
pa~ient's blood stream. It is believed that the maintenance
of circulating concentrations of IL-lra of less than OoOl ng
per ml of plasma may not be an effective composition while
the prolonged maintenanca of circulating levels in excess of
100 ug per ml may have undesirable side effacts.
A pre~erred dosage range ~or the treatment of
interleukin-l mediated arthritis is between 1 and 100 ng/ml.
Accordingly, it is preferred that, initially, doses are
administered to bring the circulating levels o~ IL-lra above
10 ng per ml of plasma and that, thereafter, doses are
administered at a suitable frequency to keep the circulating
level o~ IL-lra at or above approximately lO ng per ml of
plasma. The fre~uency o~ dosing will depend on
pharmacokinetic parameters sf the IL-lra in the formulation
us~d.
A preferred dosage rang for the treatment o~
interleukin-l mediated IBD is between ~bout .5-50 mg pex kg
of patient weight administered between about 1 and lO times
per day. In a more preferrad embodiment the dosage is
between about l-10 mg per kg of patient weight administered
between about 3 and 5 times per day. ~he frequency of
dosing will depend on ph rmacokinetic parameters of the IL-
lra in the formulation us~d.
A preferred dosage ranga for the treatment o~
interleukin-l mediated septic shock i5 between about 1.0-200
mg per kg par day of patient body weight per 24 hours
administered in equal doses ~etween about 4-15 times per 24
~16-
2~39~
hours. In a more pre~erred embodiment the dosage is between
about 10-120 mg per kg per day of patient body weight
administered in equal doses eve:ry 2 hours. In the most
preferred embodiment 100 mg per kg o~ patient body weight
per 24 hours is equally administered every 2 hours. The
frequency of dosing will depend on pharmacokinetic
parameters of the Il-lra in the formulation used.
In an additional pref~erred mode ~or the treatment
of interleukin-1 mediated septic shock, an initial bolus
injection of Il-lra is a~ministered ~ollowed by a continuous
infusion o~ IL-lra until circulating IL-1 levels are no
longer elevated. The goal of the treatment is to maintain
serum IL-lra levels between 2-20 ug per ml ~or this period.
In a preferred emhodi~ent of this mode, an initial bolus of
between about 10-20 mg per kg of patient body weight of IL-
lra is administered followed by the continuous
administration of IL-lra of between about 5-20 ug per kg of
patient body weigh~ per minute until circulating IL-l levels
are no longer elevated, SBrUm IL-1b level~ may be
ascertained by co~mercially available i~munoassay t st kits.
The initiation of treatment ~or IL-l me~iated septic shock
should be ~egun, under either mode of treatment, as soon as
possible after septicemia or the chance of septicemia is
diagnosed. For example, tr~atment ~ay be begun immediately
following surgery or an accident or any other event that may
carry the risk of inltiating ~eptic shock.
A preerred do~age range for the treatmPnt of
interleukin-l m~diated i~shemia and reperfu~ion injury is
between abou 1-50 mg per kg of patient weight administered
hourly. In a prefarred e~bodiment an initial bolus of about
15-50 mg per kg o~ lra i9 administered, ollowed by
hourly injections of about 5-20 mg per kg. The frequency of
dosing will depend on pharmacokinetic parameters of the IL-
lra in the formulation used.
It is also contemplated that certain formulations
17-
9 ~
containing IL-lra are to be admini~tered orally.
Preferably, IL-lra which is adm.Lnistered in this fashion is
encapsulated. The encapsulated IL-lra may be formulated
with or without those carriers customarily used in the
compounding of solid dosage fo~ns. Preferably, the capsul
is dPsigned so that the active portion of the formulation is
released at that point in the gastro-intestinal tract when
bioavailability i5 maximized and pre-systemic degradation is
minimized. Additional excipients may ~e included to
facilitate absorption of th~ IT.-lra. Diluents, ~lavorings,
low melting point waxes, vegetable oils, lubricants,
suspending agents~ tablet disintegrating agents, and binders
may also be employ~d.
When used for the treatment of interleukin-l
mediated IBD, the administration o~ IL-lra may also be
accomplished in a suitably ~ormulated enema.
Regardless of the manner of administration, the
specific dose is calculated according to the approximate
body weight or surface area of the patient. Further
refinement of the calculations necessary to deter~ine the
appropriate do~age for treatment involving each of the above
mentioned formulations is routinely made by those of
ordinary skill in the art and i5 within khe ambit of tasks
routinely performed by them without undue experimentation,
especially in light of ~he dosage info~mation and assays
disclo~ed herein. These dosages may be ascertained through
use of the established assays for dete~mining dosages
utilized in conjunction with appropriate dose-response data~
It should be noted that the IL-lra formulations
described herein may be used for veterinary as well as human
applic~tions and that the term "patient" should not be
construed in a limiting manner. In the case of veterinary
applications, the dosage ranges should be the same as
speci~ied abovs.
It is understood that the application of teachings
-18-
2 ~ 3 ~
of the present invention to a specific problem or
Pnvironment will be within the capabilities of one having
ordinary skill in the art in light of the teachings
contained herein. Examples of representative uses of the
present invention appear in the following examples.
In Examples 1-3, IL-lra's are shown to prevent
and/or neutralize, either totally or in part, the ePfects of
IL-1 in known arthritis models such as those described by
Wilder, R.L., in "Experimental Animal Models of Chronic
Arthritis," Goodacre, J.A. and W.C. Dick (ed.),
Immunopathogenic Mechanisms of Arthritis, Kluwer Academic
Publishers; Dordrecht, Netherlands; Boston, ~ass. (1988),
specifically incorporated herein by reference. As is noted
in the examples, in each model the IL-lra tested showed
beneficial results.
In Examples 4-S~ IL-lra's are shown to prevent
and/or neutralize, either totally or in part, the effects of
IL-l in known IBD models such as thosD described by Zipser
e~ al. Gastroenterology~ 92, pp. 33-39 (1987), specifically
incorporated herein by this re~erence; and Sartor e~ al.
Gastroenterology, 89, pp. 587-95 tl985), specifically
incorpora~ed herein by ~-his referenr~. As is notPd in the
examples, in each model the IL-lra tested showed beneficial
results.
In Example 5, IL-lra is shown to have beneficial
ef~ects on extraintestin~l inflammation associated with the
Sartor IBD model. In Example 6, intestinal inflammation
resulting ~rom the administration of an
NSAID--indomethacin-~is also neutralized by treatment with
IL-lra.
In Example 7, IL-lra's ar~ shown to prevent and/or
neutralize, in part, th~ effects of IL-l in a known septic
shock model. Rabbits are given intravenous injections QP
endotoxins--believed to be a principal cause of septic
shock--to incluce septic shock. The mortality rate of groups
--19--
~ 0 3 ~
of rabbits given varying amounts of IL-lra were examined.
As can be seen in the Example, treatment with
Il-lra showad beneficial results.
In Example 8, IL-lra's are shown to neutralize, in
part, the ef~ects of IL-l in ischemia and reperfusion injury
experiments. Dogs are subjected to regional myocardial
ischemia for two hours and then reperfused for four hours.
The size of infarct as a perc~ntage of ventricular mass and
the in~arct size as a percent of mass at risk were measured.
Treatment with Il-lra showed beneficial result~.
X~MPL~ l: DemonstratiQn of_the E~eçts of Human
InteF~eukin~l Inhi~itQx_i~_Cultured Bovine
Nasal Cartilaae 2xplant.
Numerous in vitro and in~ivo methods have been
used to study the progression of arthritis. One in vitro
model which has proven to be especially usaful in this
regard is cultured cartilaginous tissue explant. In fact,
this model has been used in the past to demonstrate that IL-
1 is a powerful mediator of cartilage destruction and,
tharefore, a propitiouC target or intervention in arthritic
joint erosion. (See qen2rall~ G. Buchan ~t_al., Third
Annual General ~eeting of the British Society for
~heumatology, London, England, Novamber 19-21, 1988, PR. J.
Rheu~atol 25 (Supplement 2) 1986; Fontana ~_31-, ~heumatol
Int (1982) 2:49-53; J. Saklatvala ~ ., D velopment of
Diseases of Cartilage and Bone Matrix, Alan R. Liss, Inc.,
pp. 291-298; P. Stashenko e~ al., The American Association
of ImmunolQgists, Vol. 138, ppO 1464-1468, No. 5, March 1,
19B7; G. Dodge et al., J. Clin. Invest., 83:647-~61; J.
Sandy et al., Journal of Orthopedic Research 4:263-272, J.
Saklatavala et al., The Control of Tissue Damage, Glauert,
ed., Elsevier Science Publishers, pp. 97-108; I. Cambell e~
~l., Biochem. J. 237:117-122; J. Tyler, ~iochem. J.,
225:493-507, and J. Eastgate et al., Sixth International
-20-
2~39Ari~
Lymphokine Workshop, 7(3):338, all of which are specifically
incorporated her~in by reference.)
The cartilage explant model essentially as
described by Steinberg et al., }3iochem. J. 180:403-412,
incorporated herein by referPnce, was used in this Example
to demonstrate the mitigating e~fect of IL-lra on IL-l
mediated cartilage breakdown. ~Yhile bovine nasal septum was
used here as the source of cart:ilaginous tissue, articular
cartilage of the type described in J. Tyler ~_3~., Br. J.
Theumatol. 24 (Supplement 1):150-155, incorporated herein by
re~erence, could also be used.
Preparatio~_~ Cartilaae.
Bovine nasal septum was removed from freshly
slaughtered yearling steers and plac~d on ice. The tissue
was thPn scrubbed with Povidone/Iodine prep solution (1-
ethanol-2~pyrolidinone homopolymer with iodin~, obtained
from Medline Industries, Mundelein, Ill). The mucosa and
perichondrium were then removed. The remaining
cartilaginous septum was then immersed in a 5% (v/v)
solution of Povldone/Iodine for one hour at room
temperature.
The following procedures were then performed
aseptically in a laminar flow hood. The septa were
repeatedly rinsed with Gay's Balanced S~lt Solution (GIBC0
Laboratories, Grand Island, NY). The cartilage sheet was
then placed on a sterile sur~ace, and uniSorm 8 mm plugs
were removed using a standard coxk borer. Approximately 1-2
mm of the top and bottom surfaces were removed using a razor
blade. The plugs were then held in the Gey's Balanced Salt
Solution~ Plugs taken fro~ different steers were kept
separately.
Each plug was then sectioned into several 0.8 mm
disks. The cutting devicP used was an aluminum block o~ the
type described by Steinberg e~_al.~ Biochem. J. 180:403-412.
The disks produced were consistently be ween 40 and 50 mg
-21-
wet weight. The dis~s were kept in culture in Delbecco's
Modified Eagle Medium plus 10% Fetal ca:l~ serum, plus
Penicillin, Streptomycin, and Neomycin (all reagents ~rom
GIBCO), hereinafter re~err~d to simply as ~Imedium.~ The
cultures were maintained in a 37 C incubator with 5% C02.
Representative disks :Erom each steer were then
tested for their ability to respond to IL-lB as indicated by
the release of glycosaminoglycans (GAG) into the culture
medium. (glycosaminoglycans are released ~rom a cell once
~he cell matrix has been degraded.) The presence o~ GAG was
detected using 1, 9-dimethylenethylene blue as described by
Ferndale et al., Connective Tissue Research, 9:247-248,
incorporated herein by reference. Disks that responded to 5
ng/ml of IL-lB by increasing output of GAG two fold or
greater as compared to an unstimulated basal rate were
selected for use in th~ following experiments. These disks
are hereinafter referred to as "IL-lB responsive disks."
IL-l_Dose Res~nse.
This preliminary experiment was performed to
determine whether a dose response curve 0xists to increasing
amounts of IL-lB.
First, several of the Il-lB responsive dis~s were
sactioned into quarter slices. (The remainder was set aside
for later experiments.) Because re~ponses to IL-1
frequently vary from animal to animal, disk to disk, and
slice to slice, the steps o~ this e~periment were designed
so that each slice serv2d as its own control.
Second, each slice was incubated in one well of a
48 well tissue culture clu~ter tCostar, Cambridge, MA) with
a constant volume of the previously described medium. After
48 hours, the amount o~ GAG present in the supernatant of
each culture was measured. This amount was then normalized
for each culture in terms o~ ug GAG per mg wPt weight of
tissue. In this manner, a basal rate of GAG release in the
absence of II.-l was established for each slice.
-22-
~39~
Third, the supernatants from all the cultures were
discarded and replaced with fresh medium containing
differing amounts of IL-lB. The IL-lB was produced in-house
(J. Childs, notebook 935, pages 49-52) and after
characterization, was utilized in all experiments calling
for its use. After a 48 hour incubation with IL-lB, the
supernatants from the cultures were recovered, and the
amount of GAG present in each was measuredO These amounts
were normalized for each culture as above. The ~asal rates
were then subtracted ~rom the IL-lB inducad rates. The
results are depicted in Fig. 1. ~The results are also
expressed in tabular form in Table 1.) As Fig. 1 clearly
indicates, the r~lease of GAG from th~ cartilaginous tissue
is dependent on the amount of IL-lB administered.
Because 5 ng/ml o~ IL-lB caused an easily
measurable increase in GAG releas~ during the 48 hour period
of culturing, this concentration was used in the following
experiment.
Effects of rlL-lra on IL-l Induced GAG Release.
Several of the remainin~ IL-~B responsive disks
were n~xt sectioned into quartar slices. As above, each
slice was used at its own control.
Each slice wa5 then incubated with a constant
volume of the previously-described medium for 48 hours in a
48 well tissue culture cluster. A basal rate of GAG release
was determined for each slice. Next, the supernatants ~rom
the cultures were discarded and rP.placed with fresh medi~m
containing 5 ng/ml o~ IL-lB and difPering amounts of
recombinantly produced IL-lra (rIL-lra). A~ter a 48 hour
incubation, the supernatants were recovered, and the amounts
of GAG were measured. The~e amounts were normalized for
each cul~ure by dividing the rIL-lra/IL-lB stimulated GAG
release rate by the basal GAG release rate. The results are
depicted in P'ig. 2. As Fig. 2 clearly illus~rates, the
release of GAG from cartilaginous tissue was sharply
-~3-
~ ~ 3 ~
curtailed by an increase in the concentration of rIL-lra
relative to that of IL-lB. For instance~, a ten times molar
excess of rIL-lra over IL-lB (the molecular weights of IL-lB
and rIL-lra are both approximately 17 kD) was sufficient to
return the GAG release rate to the basal level. Similarly,
a 1.5 times molar excess of rIL~-lra over Il-lB was
sufficient to reduce the stimulation of GAG release to 50%
of that observed in the presence of IL-lB alone.
These results were reproduced using car~ilage
derived ~rom several different ~steer~
Lack of Cytotoxiclty of rIL-l~a.
To show that rIL-lra is noncytotoxic, the
inventors took slices from the remaining IL-lB responsive
disks and ~xposed them to varying amount~ of rIL-lra in the
absence of IL-lB. The rate of GAG release was the same as
wher~ neither rIL-lra nor IL-lB was pre~ent.
Next, to show that the effects of IL-lra are
reversible, ~he inventors then removed rIL lra from the
supernatants of culturing slic~s and administered IL-lB
thereto. The slices responded just as they did in thP IL-lB
dose response experiment. Similar results occurred when
cartilage that had been treated with IL-lB and a suf~icient
concentration of rIL-lra to completely block the action o~
IL-lB was subsequently exposed to IL~l~ alone.
ample 2: Demonst~ation o~ the Effects of Human
Inte~leukin 1 Inhlbitor_on Colla~en-Induced
Ar~hrit~s in ~i~e.
Type II collagen-induced arthritis in mice bears
many resemblances to human rheumatoid arthritis and has been
used for several years to study certain aspects of that
disease. J. Stuart t al., The FASEB Journal, Vol. 2, No.
14, pp. 2950-2956, Nove~ber 198S, inco~porated herein by
reference. The potential involve~ent o~ 1 in rheumatoid
arthritis has been noted by S. StimpRon e~ al., The Journal
-24-
21~3~5~
of Immunology, Vol. 140, pp. 2964-2969, No. 9, May 1, 198~,
also incorporated herein by re~erence.
The purpose of this experiment was to demonstrate
that systemic administration of rIL-lra has a mitigating
effect on the pathogenesis of type II collagen-induced
arthritis in mice.
Twenty-four mice DBA/1 mice, purchased from
Jac~son Laboratories, were immunized with 0.1 mg chicX type
II collagen in Freund's complet~. adjuvant. At day fourteen
post immunization, the animals were randomly subdivided into
two groups of twPlve animals each~ The experimental group
was injected intraperitoneally twice daily with
approximately 0.1 mg rIL-lra/kg/injection~ The injections
continued until the animals were sacrificed at day 47 post
immunization (i.e., after 34 days of dosing). Control
animals were injected with an equal volume o~ vehicle tlO mM
sodium phosphate, 150 mM sodium chloride~ on the same
schedule.
Affected limbs were counted and cl.inical scoring
was performed approximately three times weekly during the
in~ e portion of the experiment. Clinical scores from
each animal represent, on a 0-4 point basis, the severity o~
arthritis sustained by each paw as assessed by blinded
obserYers. The clinical scores for each animal from day 2SI
when the first sign~ of clinic lly obserYable arthritis were
notad, through day 47, when the animals w~re sacri~iced, axe
presented in Table 2. The tally of affected limbs and the
total clinical score for each group are also pr~sented in
Tabla 2. These results are graphed as a function of time in
Figs. 3 and 4, respectively. As can clearly be seen, the
incidence and severity o~ the disease were slowed down
considerably by the administration of rIL-lra.
-25-
~3~
Demonstra~ion of the_Effects of Human
Interleukin-l Inhibitor on Streptococcal Cell
Wall (SCW)-Induc~d Reactivatlon of SCW-
Ind~ced Ar~hritis in Rats
Regarding streptococcal cell wall-induced
arthritis, R. L. Wilder in Immunopathoqenetic Mechanisms o~
a~h~i3i~, Chapter 9 entitled "~Experimental Animal Models of
Chronic Arthritis" comments "th~e clinical, histological and
radiological features of the ex]perimental joint disease
closely resemble those observed in adult and juvenile
rheumatoid arthritis".
The experiment described below employs the model
disclosed in Esser, et ~1. Arthritis and Rheumatism, 28:
1401-1411, 1985, specifically incorporated herein by
reference. This model is briefly summarized as follows:
Streptococcal Cell Wall (SCW~ is injected intraarticularly
into the ankle joint of Lewis rats. Saline is injected into
the contralateral joint to provide a control. After a
period of twenty days, in which the initial inflammation
dies away, SCW is again administered, this time by
intravenous injection. Thi-~ dose of SCW is insufficient to
cause joint inflammation by itself and, therefor~, has
little or no ef~ect on the saline injected ankle. In
contrast, however, this dose is capable of reactivating
inflammation and joint destruction in the ankle previously
injected with SCW. To assess the extent of in~lammation
following the second administration o~ S~, the dimensions
of the ankle jaint are ~ea ured daily.
In one o~ many experiments per~ormed with the
above described model, two groups of twelve rats were used.
Each animal was injected in the right ankle with SCW (1.8 ug
rhamnose equivalence) and in the left ankle with an equal
volume o pyrogen-free saline Ankle dimensions were
measured on days 1 through 6.
On day ~0, one group o~ rats was in~ected
-26-
~ ~ 3 ~
intraperitoneally with 1 mq/kg IL-lra in an aqueous vehicle;
the other group was injected intrap~ritoneally with an equal
volume of the vehicle solution only. One hour later, each
animal wa~ injected intravenously with SCW (100 ug rhamnose
equivalence). Ten minutes later, the treatment group was
injected intraperitoneally with 1 mg/kg Il-lra, and th~
control group was injected with vehicle alone. Subcutaneous
injections of Il-lra at 1 mg/kg were given at 2 and 6 hours
post-SCW administration and were repPated every 6 hours
therea~ter for the next 3 days.
Table 3 and Figure 5 show the dimensions of the
saline injected and SCW injected ankles for both the
treatment group and the control group over the course of the
experiment. As expected, the SCW treated ankles in both
groups swelled in responsa to the intravenous in~ection of
SCW. However, the re~ponse differed between treatment
groups. The ankles in the rontrol group swelled by about
30~ of their initial dim~nsions over the first 3 days
whereas the ankles in the treatment group swelled only by
14% over the same period. ~oreover, on days 1 through 5
post-intravenous injection o~ SCW, there was a statistically
s~gnificant (P ~ 0.001 by a two-tailed t-test for
independent means) difference in thQ dimensions of the SCW-
treated and contralateral control ankle~ both groups.
On day 8, the rats were sacrificed and both ankle
were fixed in for~alin. The fixed joint~ w~re decalcified,
stained, and examined. Significant differences in cartilage
erosion, bursiti~, periostitis, and synovitis were found
between the control group and the treat~ent group. 50me of
these differences are s2t forth in Table 4.
~3~elg_~: Demonst~ation of the E~fects o~ Human
In~erleukin-l Inhibitox on Formalin-Immune
Com~lex_Induçed IBD.
The rabbit model of formalin~i~mune complex IBD
-~7-
~39 ~s~3~
has been used to investigate the role of arachidonic acid-
derived inflammatory mediators and to evaluate therapeutic
strategies in IBD. Zipser et al. supra; Brown ~_31- 1987
Gastroenterology 92:54-59; Schwnert et_al. 1988
Prostaglandins 36:565 577, incorporatsd herein by reference.
The experiment described below employs the model
disclosed in Zipser et al. suPril. This model creates
symptoms analogous to active Ulrerative Colitis, and is
briefly summarized as follows: formaldehyde is administered
via a catheter into the colon of rabbits and after a period
o~ time the animals receive an injection of immune complexes
in antigen exces~. Time studies following the induction of
IBD are conducted by sacrificing the animals after 48 hours
and removing the colons. The colons are then histologically
assessed. The effect o~ treatment with IL lra's prior to
and after the ind~ction of IBD on in~lammation, edema and
necrosis was compar~d with non-treated control animals.
nduction of I~D.
Inflammation was induced in the distal colon of
male N~w Zealand rabbits ~2.2-2.5kg) using a modification of
the immune complex method of colitis Kirsnew et al. 1957
Trans. Assoc~ Am. Physicians 70:lO2-llg; Hodgson et al. 1978
Gut 19:225-32, which are incorporated herein by reference.
Four ml of 0.45% (v/v) unbu~fered fo~maldehyde (Electron
Microscopy Sciences, Washington, PA) was administered via a
cath~ter inserted 10 cm lnto the distal colon of
anesthetized rabbits (xylazine and ketamine). Two hours
later, animals r~ceived 0.85 ml o~ immune complexes in
antigen excess through an ear vein. The complexes were
prepared by incubating human serum albumin (500ug/ml) with
rabbit antihuman antisera (ICN Immunobiologicals, Costa
Meas, CA), decanting the supernatant, and redissolving the
pr~cipated immune complexes with an albumin solution
(6mg/ml) as described in Zipser ~ . supra.
Histologic evaluation was per~ormed on a minimum
-2~-
2~3~
o~ two longitudinal sections from each colon. All colon
samples were examined in a blind fashion by a single
pathologist. The mucosa and submucosa were separately
evaluated for infiltration o~ acute inflammatory cells
(neutrophils and eosinophils). A semiquantitive score of
leukocytes (L) per high power f:ield (HPF) was determined ~or
each area examined usinq the following quantitations: 0 = O
or 1; 0.5 - 2-9; 1 - 10-20; 1.5 = 21-30; 2 - 31-40; 2.5 =
41-50; 3 = 51-65; 3.5 = 66-80; 4 = > 81 L/HPF. At a
minimum, eight HPFs of mucosa and submu~osa from each
specimen were s~parately evaluated in each section. The
inflammatory index was calculated by adding the averaged
score for the mucosal and submucosal evaluations. Edema was
semiquantitively assessed on a scale o~ 0 to 4. Necrosis
was expressed as the percent of mucosa involved. After the
administration of formalin, followed by immune complexes,
the distal colon develops acute inflammation. This is
characterized by in~iltration of n~utrophils primarily into
the mucosa and ~ubmucosa, mucus depletion, crypt abscesses,
edema and scatt2red areas of mucosal nscrosis, progressively
increased from 0.3+0.1 (0 hrs) to 4.5+0.7 ~48 hrs)
(p<0.001), from 0.3~0.1 to 3.6+0.3 (p<0.001) and from 0% to
89%(pcO.001) respectively. A subsequent decrease in these
parameters was observed 96 hours after the induction of IBD
(p~0.01 versu~ 48 hours).
T~ea~en~ Yith II- lra ' ~ .
A group of animals were treated intravenously with
IL-lra (5mg/kg; n=8) or the vehicle alone (n=10) at six time
points: 2 hours before and 1, 9, 17, 25, 33 hours a~ter the
administration of the immune complexes. The rabbits were
sacrificed 48 hours after the induction o IBD and the colon
tissue analyzed ~or inflammation.
Treatment o~ rabbits with IL-lra ~ignificantly
r2duced inflammatory index from 3.2~0.4 to 1.4~0.3 (p<0.02~,
-29
edæma from 2.2+0.4 to 0.6~0.3 (p<0.01) and necrosis from
43~10~ to 6.6~3.2% (p~0.03) compared to vehicle-treated IBD
animals, FIG. 6~ This result shows that several of the
indications of IBD may be significantly lassened by
treatment with IL-lra.
Exam~le 5: Bacterial_Cell Wall_Induced IBD in Rats
Unlike many other IBD models, the bacterial cell
wall induced IBD model shows most o~ the indications for
chronic IBD or Crohn's disease. In addition to the
formation of chronic granulomatous response, this model is
subjPct to spontaneous reactivation, anemia and
extraintestinal inflammation.
The Bacterial Cell Wall model essentially as
described by Sartor ~ . supra., was used in this Example
to demonstrate the mitigating affect of IL-lra on IL-l
m~diated IBD. The experiment was per~ormed generally as
follows: the IBD is induced in rats by the intravenous
in;action of a sterile sonicate of peptidoglycan
polysaccharide ~rom group A streptococci. Transient
petechial hemorrhage o~ the colon appears within 2-3 minutes
and resolves by 48-72 hours a~ter injection. A sa~.nplQ group
of animals were tseated with IL-lra following induction of
I8D, and after a period of time the animals were sacrificed,
the colons removed and gross pathology evaluated.
I~duçtion 0~
The ~acterial cell wall material was prepared
according to the procedur~s set forth in Stimpson et al.
1986 I~s~ ~Y~- 51:240-249, incorporated her~in by this
reerence. -Lewis rats are given subsercosal injections with
Streptococcal Cell Walls. The injections result in both
local and sy~temic disorders that include bowel adhesions
and nodules, an increased liver weight and hepatic nodules,
a reduced hematocrit and hemoglobin level, and increased
white blood cell count (WBC), a reduced growth rate, and a
-30-
4 ~ ~
joint swelling characteristic of arthriti~ (see AppendiX
Sartor, et al., Gastroenterology, 89:587 5g5, 198S,
incorporated herein by reference). Three separate protocols
for treatment with Il-lra were per~ormed with this model and
reductions in nodules and adhesions have been observed in
all o~ them; in the last two protocols the reductions in
adhesions were statistically signiicant.
Protocol_A.
Two groups of 12 rats were us~d. On day 1, both
were inj2cted with 15 ug total of Streptococcal cell wall-
derived peptldoglycan polysaccharidP (SCW PG-APS), at seven
sit~s; three areas o the cecum, 2 areas of the Peyer's
patche~, and two areas o~ the ductal ileum. On ~ay ll overt
signs of the disease appeared including joint swelling,
diarrhea, and bloody nose. At thi~ time one group was dosed
subcutaneously with IL-lra (8 mg/kg3 every 12 hours and the
second group was treated identically with placebo (PBS). On
each day the size of the ankle joints were measured. On day
18 the animals were sacrificed and the inte~tines were
scored on a scale o~ 0 to 4 for the presence of granulomas
and adhesions (Table 5). The IL-lra group had fewer nodules
and adhesions. The IL lra group also had smaller livers.
Th~ IL-lra group had a reduced whits blood cell count (WBC~.
p~otocol B.
The protocol was similar to that used in protocol
A ~xcept th~t the amount of PG-APS used was reduced to 12.5
ug and th~ treatment with lL-lra wa~ started at day 8. A5
in protocol A, reductions in secal nodules, intestinal
adhes.ions, liver weights, and WBC were observed (Table 6).
The reduc ion in adhesion was si~nificant at the
p ~ 0.02 level.
Proto~ol C.
The protocol used was again similar to that in
protocol A except that the amount of PG-APS was reduced to
12.5 ug (as in protocol 3) and the traatment yroup was
-31-
~39~
started on IL-lra 8 mg/kg subcutaneous and 2 mg/kg i.v.
i~mediately following the PG-APS injection. Further Il-lra
injections t8 mg/kg) s.c. wsre given at 4, 10 and 18 hours
on day 1, every 8 houx~ on day 2, and then every 12 hours
for the duration of the experiment. FiYe animals in each
group were sacrificed at day 3, and th~ remainder were
sacrificed at day 18 for examination of gut lesions (Table
7). on day 3 there was a signi~icant reduction in a global
parameter representing gut lesions and a reduction in
adhesions that approachad significance (p = O.07). In the
gxoup sacrificed at day 18 the resultc were confused because
no disease appeared in one of the animals in the control
group. However, the reduction in adhesions in the IL-lra
group was still significant at the p ~ 0.02 level and there
was also a signi~icantly greater weight gain in the IL-lra
group.
~XAM~LE S: ~SAI~ Induced IBD in Ra~s.
In an attempt to determine whethPr the anti-
inflammation effects of IL lra would be additive with those
of NSAIDs, rats were treated with indomethacin after the
intravenous injection of PG APS as described in Example 3
above (2 n~g/kg/ at the tims o~ reactivation at 12, 24 and 36
hours post activation, and every 12 hours up to 6 days), Il-
lra (2 mg/kg at 2 and 6 hours, then eve~y 6 hours up to 36
hours and every 12 hours up to 7 days) or a combination of
the two drugs (Figure 7). The group on indomethacin alone
showad a greatar reduction in joint swelling than that on
IL-lra alone. However, the indomethacin group was sick and
two animals died during the course of the experiment. Th~
group receiving both drugs did even better than the group on
indomethacin alone; the joint swelling was less, and the
difference between the two group was statistically
significant on day 4 at p ~ 0.03 and on day 7 and 8 at p~
0.06. No animals were sick in this group and there were
-32-
' "~
2 ~ 8
fewer ulcerations in the mid small intestineS. Ulceration
of ths mid small intestine is a complication in patients on
chronic oral NSAIDs. It appears, therefore, that IL-lra
alleviates some of the IBD-like complications of NSAIDs.
Table 8 shows the e~ects o~ IL-lra on both the
intestinal symptoms--ulc~rs, adhesions, intestinal
t~ickening and myleloperoxidase (MPO) levels--and systemic
symptoms -hematocrit (~CT), hemoglobin (HgB) and WBC
levels--assoclated with the N~AID treatment of PG APS
induced arthritis.
xample 7: Demonstration o~ the ~ects of Human
Interle~kin-1 ReceRtor Antagonist on
Endotoxin Induced S~tic Shock.
Endotoxin induced septic shock studies were
conducted on Blue Chinchilla rabbits. The experimental
protocol did not ~ocu~ on any indications of the induced
septic shock other than group mortality. R~bbits were u~ed
in the study because their sensitivity to pyrogenic and
metabolic effects of endotoxin and oth~r bacterial products
are similar to those of human subjects.
Shock was induced by a single intravenous
injection of endotoxin at time zero. ~he rabbit- were giv~n
periodic intrav~nous inj~ction~ into an ear ~ein at -10
min., at time zaro, and fvr every two hours therPafter ~or a
24 hour period. The result~ of this study can be seen in
TablQ 9.
In Tahle 9, Group A rabbits (n=5) were not given
any endotoxin at time zero, and were ~iven saline injections
free o~ IL-lra at the periodic injection times. Group B
rabbits ~n=lOj were given .5 mg per kg of ~ody weight of
endotoxin at time zero, and the periodic injections were
again free of IL-lra. After 7 days the survival rate o~
ra~bits in Group 3 waq only 20%.
-33-
2~3~
In Groups C-E (n=10) endotoxin was administered at
time zero, and the saline injections contained varying
amounts of IL-lra. The rabbits in group C received a total
of 10 mg per kg of body weight of IL-lra. The rabbits in
group D received a total o~ 30 ~g per kg of body weight o~
Il lra. And finally, the rabbi'ts in group E received a
total o~ 100 m~ per kg o~ body weight of IL-lra. After 7
days, the survival rate of rabb.its in group E was 90~.
This experiment, graphically illustrated in Figure
8, shows that treatment with IL-lra signi~icantly delays and
reduces final mortality rates in rabbits with endotoxin
induced shock.
xam~le 8: ~e~onst~atiQn Q~ t~ g~ct~ of Human
Interleukin L Receptor_~nta~onist on Ischemia
and Re~r~sion Iniur~
In the following example exp~ximental dogs were
subjected to regional myocardial ischemia for two hours and
then reper~used for four hours. The doys were divided into
two groups, one group treated with IL-lra and the other
treatsd with serum albumin i~ the same buffer used ~or the
tes~ group.
Animals were fasted ovsrnight and on the ~ollowing
morning, were anesthetized with 10 ml o~ thiamylal sodium
5~, followed by 2 ml o~ sodium pentobarbital 6%,
intravenously. Additional sodium pentobarbital was
admi~istered during the experiment ~s necessary. Arti~icial
r~spiratio~ wa~ ~aintained with a Harvard respirator. A
le~t thoracotomy was performed through the fifth intercostal
space and polyvinyl catheters placed in the la~t internal
jugular vein for fluid and drug administr2tion, and in the
lsft internal carotid art~ry and femoral arteries for
pressure monitoring and withdrawal of reference blood
samples. A c,atheter wa~ placed in the left atrium for
in~ection of radio active ~icrospheres. The lsft circumflex
-34-
artery was dissected free of surrounding tissue and an
electromagnetic ~low probe was placed on the vessel proximal
to tne ~irst obtuse marginal branch. A~ter an in~ravenous
bolus injection of 50 mg of lidocaine, the circumflex
coronary artery was occluded w:ith the snar~ occluder for 2
hours. Complete occlusion was verified with the
electromagnetic flow probe. The snare was ~hen released
suddenly, allowing reperfusion of the coronary vascular bed
for 4 hours.
Two-dimensional echocardiogra~s and hemodynamic
measurements ~heart rate, blood pressur~ and le~t a~rial
pressure) was d~termined before occlusion, after llO min o~
occlusion, 5 minutes after reperfusion, and 4 hours a~ter
reperfusion. Two-dimensional echocardiography was performed
with the usa of a scanner and a 2.25 MHz transducer. The
transducer was placed on the closed shaved right chest and
was allowed full visualization of the circumferential extent
of the l~ft ventricle in a short~axis pro~ection.
Echocardiographic images were recorded at the midpapillary
muscle position onto a video cassatt~ with use of a Sony
recorder. A two-dimensional echocardiographic analysis was
per. ormed with th~ use o~ A minicomputer-based ~ideo
di~itizing system.
End-diastolic and end systolic frames were
selected for analysis wtth th~ use of the onsPt of the Q
wave in lead $I as a marXer o~ end-diastole and the smallest
le~t ventricular cavity size a~ a marker of end-systole.
Endocardial and epicardial borders for 3 consecutive beats
during normal sinus rhythm was care~ully traced directly
from the video display onto a digiti2ing tablet. Quantitive
analysis was performed with a radial contraction model and a
fixed diastolic center of mas~ at 22.5 d~gree intervals over
the full left ventricular circumference.
The midpoint of the posterior papillary muscle was
chosen as a :Eixed anatomic re~erence and designated as 135
-35-
2 ~ 3 .~
degree~. Wall thickening was computed ~or each of the 22.5
degree sectors with the ~ollowing e~uation: wall thickening
= [(end systolic wall thickness - end diastolic wall
thickness)/ end diastolic wall thickness~ x 100%. The
normal range of wall thickening was determined from a
functional map of the baseline images for three cardiac
cycles and 95% tolerance limits were establi hed in each
animal. These limits were used ~or comparison with
occlusion and reperfusion functi.onal maps and abnormalities
are expressed as the circumfererltial extent of dys~unction
and the degree of dysfunction. The ~xtent of dysfunction
(in degrees~ was measured at th~ intercepts between the
occlusion or reperfusion maps and ths low4r 95% tolerance
limit; the degree of dys~unctio~ (in area units) is the
planimetered area below the lower ~5~ tolerance limit.
Regional myocardial blosd flow was assessed by the
reference withdrawal ~ethod using tracer-tabled microspheres
(15 ~m diamater, New England Nuclear) injected into the left
atrium. The microsphere~ were ultrasonicated and vortex-
agitated before injection. ~icrospheres were injected
before occlusion, a~ter 110 ~in of occlusion, 5 minutes
after reperfusion and 4 hours aftar r perfusion with one of
six available isotopes ll4lCe, 51Cr, 113Sn, 103Ru, 9sNb, 46Sc)~
Simultaneous re~erenc~ arterial samples were withdrawn fro~
the carotid and femoral arteries at ~ constant rate of 7
ml~min with à Harvard withdrawal pump starting 10 sec before
microsphere injection and continuing for 120 sec after
completion o~ th~ injection.
Two adjacent transverse left ventricular slices at
the midpapillary muscle level, corresponding to the
echocardiographic short-axis slices, were selected for blood
flow determination. Each slice was divided into 16 full
thickness 22.5 degree sectors. ~ach sector was then ~urther
divided into epicardial, midmyocardial, and endocardial
samples. The tissua samples were then weighed, placed in
-36-
counting vials, and assayed for radioactivity in a gamma
scintillation count~r. A~ter background and overlap
correction, absolute myocardial blood ~low was calculated
with the following equation: Qm=(Cm x Qr/Cr), where Qm =
myocardial blood ~low (ml/min); Cm = counts/ min in tissue
sample; Qr = withdrawal rate of the re~erence arterial
sample (ml/min); Cr - counts/min in the reference arterial
sampl~. Myocardial blood ~low i5 exprQssed per gram of
tissue ~or each sample.
Just prior to sacri~ice, the le~t circumflex
coronary artery was briefly occluded and monastral blue
pigment (0.5 ml/kg) injected into the left atrium for
d~lineation of the in vivo myocardial area at risk. The
animal th~n received 3000 U o~ heparin and was sacrificed
with an intravenous bol~s of saturated KCI solution and the
heart excised.
~ reatmen~ Gr~u~s. Dog were randomly assigned to
one o~ two groups. In the test group, dogs received a bolus
injection oP ~0 mg Il-lra inhibitor just prior to the onset
of the ischemia and 15 mg IL-lra inhibitor for each hour
until the expariment was terminated. Control animals
~eceived an identical ~u~ntity of endotoxin-~ree, human
albumin dissolved in the same buffer used ~or the test
group.
~ 5~ Y5I~3~5~ A. A~ter death, the
heart oP each dog was 2xcisQd,the l~ft ventricle i~olated
rom surrounding tissue, cooled in a ~reezer for 15 minut~s,
and then sliced into 5 mm transverse sections. The slices
were then weighed and placed in a warm bath of bu~ered
triphenyl tetrazolium chloride ror ten minutes. In th s
t~chnique, viable tissue stains red whil~ nonviable tissue
remains unstained (A~. Heart. J. 101:593). The unstained
zone of infarcted ti~ue i6 outlined on transparent overlay~
and quantitat:ed by planimetry u~ing ~ microcomputer and
corrected for the weight of the heart slice. Infarct size
-37-
is expressed as the percentage of the area of myocardium at
risk (the area at risk of infarction is de~ined as the area
of the myocardium left unstained following the injection o~
monastral blue into th~ left atrium).
NMR Analysis of myca~dial_~d$~. After fixation,
the hearts were cut into 5 to 7 transverse slices
approximately 5 mm thick. Two transmural myocardial tissu~
samples were obtained ~rom the nonischemic zone (positive
msna~tral blue staining) and the central ischemic zone
(n~gative blue staining). The ~picardium for each sample
was dissected away to eliminate possible lipid signal
interf~rence. Each piece was subdivided transmurally
(weighing approximately 500 mg ~ach) with one portion
assessed for % H20 by desiccation te~hnique (wet weight -
dry weight/wet weigh~ whil~ the other wa~ placed into a
clean dry glasc tubeO T1 and Tl relaxation times were
obtained on a IBM PC 20 Minispec spe~tro~et~r (IBM
Instruments, Inc., Danbury, CT) operating at 20 ~Hz and
40C. The location of the sample in the magnet, 90 and
180 radio frequency pulses, and detector phase were
o~timized for each sample before relaxation measurement~
w~re ob~ained. ~1 valu~s w~r~ determir~ed by a ~it o~ 20
inversion data recovery points while T2 values were
d~termined by using a Carr, Purcell, Meiboon-Gill (CPMG~
sequence. In an attempt tQ minimiza effects of di~usion
and mi~cellan~ou~ system instabiliti~, the 180 radio
frequency interpulse spacing was maintained at 180
microseconds. The fraction of echo samples determined were
used as variables to ad~u~t the duration of the CPMG
experiment. Typically, 1 to 150 data points were acquired
as the ec:ho train was delayed to ~ 5% to 25% of its original
amplitllde. T2 values were determined by using a multi
exponantial i~it. Only the dominant component of the
2xponential ~Eit was used ~or 3tatistical analysis. The
r~sults o~ the Tl and T2 analysis were corrected with
--38-
. ..
2~3~
percent water for adjacent tissue samples to veri~y the
accuracy o~ the NMR technique.
~ _loqic and m r~hometr.ic ~valuat1ons. For each
group tested at least 3 animals were ~valuated by light
microscopy. Sections stained with hematoxylin and eosin
from each heart were evaluated ~or n~utrophil accumulation
within the area betwe~n viable and in~arcted tissue.
Stat stic AnalYsis. ~11 data was represented as
-thP mean -~ SEM. Comparisons within groups were made by a
two-way analysis o~ variance; when significant F values are
obtained, paired t tests (corrected ~or multiple comparisons
with the Bonferroni inequality adjustment) will be us~d ~o
determine which maasurem~nt dif~ered significantly ~rom one
another.
Comparison~ between groups were made by unpaired t
test. An exponPntial regression was used to correlate
infarct size data to myocardial blood flow.
The results of the I~-lra treatment regimen on
protecting dog myocardium from occlusion reperfusion injury,
are listed in Table 10 below. As a percentage o~ the left
ventricular ~as~ the in~lux infarct size in the treated
group was reduced to 10.3% as opposed to 18.~ in the
control animals. This represen~s a 40% reduction in the
percent of tha left ventricular ~as~ that was in~arcted.
The percentage of the area at risk in contrast~ was not
markedly chan~ed, 40.5% of t~e left ventricular mass in th~
treated ~roup versus 44.8% in th~ control animals. When the
in~arcted area is calculated as a perc~nt o~ the total area
at ri~k, the numbers similarly-favor th~ IL-lra treated
animals, 24.3~ versus 42% in the control group.
-39~
' '
.
:`
TABLE 10
The effect of IL-lra in reducing the extent at
infarcted tissue in canine coronary occlusion-reperfusion
studies.
ILlra Treated ~ 91 ~I~LY~ LL~L}~L~9
Infarct si~e as a % of
le~t ventricular mass
10.3%-+2.2% 18.2%-~3.3%
Area at risk as a % of
left ventricular mas~
40.5%-+1.7% 44.8%-+1.9%
Infarct size as a
of mass at risk
24.9%-+4.6% 42%-~.3%
Al~hough the present invention has been de~cribed
in connection with preferred embodiments, it is understood
that those skilled in ths art are capable of making
modificatisns and variations without departing from the
scope or spirit of the present invention. Therefore, the
~oregoing d~scription of prefPrred e~bodim~nts is not to be
taken in a limiting sense, and the present invention is best
d~fined by the ~ollowing clai~s and their equivalents.
-4~
-4~ 2 0 3 9 ~ 5 8
The ef~ect of recombinan~ IL~ on ~L-1,B induce~ degradation of Bovine l~ds~l
~, ~n all cases ~IL 1~] -; ng/ml.
(~L-1QJ [IL-LGl Fold Stirnulation
~IL-lb] penod lI/periodl (~/- standard deviation)
0 4.02 ~/- 1.7
n~/rnl 1 2.4 ~/- 0-47
10 ng/rnl 2 1.7 ~- 0.4
2~ ng/ml 5 1.3 +/- 0.4
50 n~/ml 10 1.0 +/~ 0.2
1~0 ng/ml 30 1.1 +/ 0 2
. .
,~ 2039~5~
--42--
., o o .. , .. o .. _ o ., o .. _ ~ o .. o .. ,
~ ~ ~ D -- ~---- ~ ~-- ~ X; ~ ~ ~ ~
~n~_,~ _
V O V ~ .~ D ~ ~OO-~-O O J
O ~ ~. D 9 ~
D ~ ~ ~ O ~ ~ ~ ~ O ~ ~ O n_
.~ o _ .~ ~ ~ o .~ o ~
~so-n~-~0
~-~ ~n~o D~O~DO_
~4~-~ ~O~-OO~
o /~ DD
D ~ ~ ~ ~ ~ ~ D~OV~ ~- ~9
~n~ o~-~ ~ o
~D~n~ ~_-~n
r .~ o ~--o .~ o ~
~ D " n V X
O~ ~ ~ _ 9 ~ V _ _ ~ O ~ V ~ 1~ ~ O ~ ~ ~ ~ ~ ~ O V 1'.
~~ _ ~ ~ ~ D ~ ~ O ~ ~ ~ ~ Dn~o~-~-~o
r - ' - '~ - ''~ ~ ~--~~~-Dn~
~1 ~
g O _~ -o ~ ,
~n--~ D - V ~ D D _ ~ n~ D O O D ~ r
~n~___ D ~ _ O _ O ~ ~ ~ ~ ~ ~ ~ ~ D _
^ ~ _ _ ~ ~ _ ~ O ~ D 1~ D .. ~ ~ _ ~ _
~ N~~----~---- ~ D~O ~0
_ ~ ~ D ~ _ ~ ~ O O O~ .~ ~ O ~ _ ~ ~ ~ 1~ 0 ~ O
~ _ ~ _ _ _ .~ _ ~ ~ _ O _ O ~ O ~ O D ~In~
-D_~ _~_o ~O~o_o~0~
_ ~ ~ O ~ ~ A ~ D ~ ~ a .oo~oo D O D ~ O
_ O ~ D 0 9 _ ~ D O ~ _ ~ O ~ ~ D ~ O D ~ D O ~ ~
.~ ~ _ _ ~ _ _ ~ ~ _ O ~ ~ ~ O O ~ ~ _ ~ ~ ~ --
~ O O _ O ~ O _ ~ _ ~ 0 ~ O C~ -- D _ O D --
_ ~ ~ ~ ~ O O O O O O D ~ O a D ~ D ~ --
n~ D _ ~ ~ D D ~ '. b ~ ~ ~ ~t D
'. .` _ ~ ~ _ D ~ ~n ~ D 1~ ~ ~ D ~ ~ ~ ~ .
O t~ O ~ ~ O ~ D O
s . s .
. ~
: - ;
.
~3~
-~l3 -
Table 3
ANKLE JOINT DIAP~IETER OF RATS INJECTED
WITH SCW AND TREATED WIT11 IL~1rz.0R SALINF
ACCORDING TO PROTOC:OL IN EXAMPLE 3
Joint Diameter (mm) (~_)
SCW Injected Joints Saline In~ected Joints__
. SQ ~aline ~ Sl:) Saline SD
0 5.96 .1 2 6.02 .10 5.95 .17 5.96 .1 6
7.95 .33 7.73 .36 5.94 .15 5.94 .13
2 7.44 .28 7.42 .27 5.98 . l 1 5.95 .17
3 7.~0 .39 7.23 .27 6.00 .1 2 6.01 .07
6 6.78 .27 6.64 .29 6.06 .09 6.06 .13
6.5~ .34 6.63 .18 6.00 .12 5.85 .16
14 6.44 .21 6.36 .17 5.99 .08 5.90 .17
6.46 .18 6.52 .14 5.91 .11 5.87 .~0
21 7.34 .36 7.78 .31 5.73 .18 5.78 .12
2? 8.31 .58 8.70 .43 5.85 .16 ~.96 .22
23 8.55 .B1 9.06 .42 6.02 .19 5.99 .16
24 8.23 .71 8.56 .39 6.03 .13 5.94 .20
8.00 .56 8.16 .43 6.05 .12 6.06 .17
28 7.48 .4~ 7.71 .30 ~.04 .13 5.98 .13
~,~3~
Table ~ -4'1-
EHects ot IL-1rGon Joint Histopathology
Following SCW P~activation of Joint Inflammation
(1 rng/kg 4 times daily on day 20 through 23)
Placebo Group IL~ Group P
.
PalhologyPositives/ 12 ScorePositives/ 12 Score
.
Cartilage Erosion 10 1.0 + .6 3 0.25 + .45 .0023
8Ona Erosion 3 0.25 ~ .45 2 0.17 + .39 NS
Bursitis 11 0.92 ~ .29 3 0.25 ~ .45 .0003
Periostitis 9 0.75 +.4512 0.25 + .45 .013
Synovitis 12 2.21 ~ .84 12 t.08 ~ .47 .OOû52
PMN t2 1.0 12 1.0 NS
.
:.
., . ,. - -; ,
'
~3~
Tabl e
--45--
Effects of IL-lra on Scw ~ nduced enterocolitis
in the rat
Intestinal Cecal Liver W~C
Adhesions ~Q~ ~Qi.ghS_~
IL-lxa 1.7 1.816.9 48.8
P~S 2.2 2.41a.6 57.7
p Yalua 0. 14 0 . 1n o. lg o .13
Por comparison
o~ grs--ps
-- r
2~3~ 4~ ,3
Table ~ -q6-
Effects of IL-lra on SCW-induced enterocolitis
in the rat
Intestinal Cecal Liver W~C
~dh~sions ~Q~lÇ~ Weiqht ~ImL
IL-lra 1.4 1.7 13.3 35.1
P~s 2.2 2.3 14.1 35.~
p value 0.017 0.077 0.23 0.43
rOr co~pariaon
o~ groups
: - .
~, . .
: ::
.
. . .
~39~
TablQ :;a
-~7-
Effects of IL-lra on SCW-induced enterocolitis
in the rat
.
Intestinal Cecal Liver WBC
Adhçsio~s ~Q~ isht (qm~ -
IL-lra 0.8 0.9 0.047 10.7
P~S 1.8 1.0 0,049 10.3
p valua 0.07 0.30 0.24 0.31
for comparlson
of group~
,
~3~4~
--48--
o ~
E ~ ~ T
V~ I I ~t ~ C" qr
oc"l g ~ ~
~,
,e ~
E ~ ~ _ I O
8 ~ i ''
~, ~ o o
:~ ~ o c~
I! ~ ~ o ~ o
C
`E ¦ s r
t~ E E J
~~k~ q _"9_ - 2~39~
~ xperlmental en~o~oxln In~uc~ ~hoc~ in rab~lts; etfects ~ IL-1 ra on
surYival rate
sur~ival (no)
~survival rate
7 days (%)
12h 24h 36h 48h 7d
A (N-S) ~ 5 5 5 5 1~0
8 (N~10) 9 6 3 2 2 ~0
C (N~10) 9 7 4 3 2 20
1: (N=1~) 10 7 6 5 . 40
(N=10) 10 10 10 9 9 90
;
