Use of IL-18 inhibitors for treating head injuries
United States Patent: 7,655,616
Issued: February 2, 2010
Inventors: Shohami; Esther
(Mevasseret Zion, IL)
Assignee: Ares Trading S.A.
Appl. No.: 10/478,614
Filed: May 23, 2002
PCT Filed: May 23, 2002
PCT No.: PCT/EP02/05666
371(c)(1),(2),(4) Date: April
PCT Pub. No.: WO02/096456
PCT Pub. Date: December 05,
Covidien Pharmaceuticals Outsourcing
The invention relates to the use of
inhibitors of IL-18 in the preparation of a medicament for treatment
and/or prevention of central nervous system injury, in particular of
traumatic head injury.
Description of the
SUMMARY OF THE INVENTION
The present invention relates to the pathophysiological role of IL-18 in
CNS diseases. It is based on the finding that the treatment of mice with
inhibitors of IL-18, either one hour or three days after experimental
closed head injury (CHI), results in an improved recovery and attenuated
extent of brain damage as compared to control animals. The invention
therefore relates to the use of an IL-18 inhibitor for the manufacture of
a medicament for treatment and/or prevention of central nervous system
(CNS) injury, and in particular of traumatic brain injury.
The use of combinations of an IL-18 inhibitor with an interferon and/or
TNF and/or inhibitors of inflammation and/or anfioxidants are also
provided according to the invention. In order to apply gene therapeutic
approaches to deliver the IL-18 inhibitor to diseased tissues or cells,
further aspects of the invention relate to the use of nucleic acid
molecules comprising the coding sequence of an IL-18 inhibitor for the
treatment and/or prevention of the CNS injury. The invention also relates
to the use of cells genetically engineered to express IL-18 inhibitors for
the prevention and/or treatment of CNS injury.
DESCRIPTION OF THE INVENTION
The present invention is based on the finding of a statistically
significant beneficial effect of an IL-18 inhibitor on the recovery from
brain injury in a murine model of closed head injury. In accordance with
the present invention, it has further been found that IL-18 is
up-regulated in the brain and cerebrospinal fluid after traumatic brain
injury, indicating that this pro-inflammatory cytokine plays an important
role in the pathogenesis of brain injury.
Therefore, the invention relates to the use of an IL-18 inhibitor for the
manufacture of a medicament for treatment and/or prevention of central
nervous system (CNS) injury.
The invention further relates to the use of an IL-18 inhibitor for the
manufacture of a medicament for treatment and/or prevention of
complications and late effects of CNS injury.
In preferred embodiments of the invention, the CNS injury is traumatic
brain injury or closed head injury.
In a further preferred embodiment, the CNS injury is spinal cord injury.
In yet a further yet a further preferred embodiment of the invention, the
brain injury is of vascular origin.
Within the context of the present invention, the expression "central
nervous system injury" or "CNS injury" relates to any injury to the brain
or spinal cord, regardless of the age at onset, or the underlying cause.
The underlying cause may e.g. be mechanical, or an infection. CNS injury
and its clinical symptoms and implications have been described in detail
in the "Background of the invention". CNS injury includes e.g. trauma, or
any other damage of the brain or spinal cord, and it may also be called
Brain injury may for example include or result in any one, or more, of the
following: 1. Attention impairment; 2. cognition impairment; 3. language
impairment; 4. memory impairment; 5. conduct disorder; 6. motor disorder;
6. any other neurological dysfunction.
Spinal cord injury may for example result in paraplegia or tetraplegia.
Complications or late effects of CNS injury may also be treated and/or
prevented in accordance with the present invention. Complications and late
effects of brain injuries have been described above in the "Background of
the invention". They include, but are not limited to, coma, meningitis,
post-traumatic epilepsy, post-traumatic dementia, degeneration of nerve
fibers, or post-traumatic syringomyelia, or hemorrhage, for example.
The present invention also relates to the use of IL-18 inhibitors for the
preparation of a medicament for treatment and/or prevention of any injury
to the brain that is vascular in origin, such as hypoxic brain damage with
cerebral infarction, ischemia, cerebrovascular accident, or stroke.
The terms "treating" and "preventing", as used herein, should be
understood as partially or totally preventing, inhibiting, attenuating,
ameliorating or reversing one or more symptoms or cause(s) of CNS injury,
as well as symptoms, diseases or complications accompanying CNS injury.
When "treating" CNS injury, the substances according to the invention are
given after onset of the disease, "prevention" relates to administration
of the substances before signs of disease can be noted in the patient.
Treatment of CNS injury is preferred in accordance with the present
invention. Preferably, in order to treat CNS injury, the IL-18 inhibitor
is administered as soon as possible after CNS injury, e.g. within the
first hour after the injury. However, as shown in the Examples below, one
IL-18 inhibitor was shown to exert its beneficial effect on brain injury
even when administered three days after brain injury occurred. Therefore,
in order to treat CNS injury, it is preferred to administer the IL-18
inhibitor within three days from the injury.
The term "inhibitor of IL-18" within the context of this invention, refers
to any molecule modulating IL-18 production and/or action in such a way
that IL-18 production and/or action is attenuated, reduced, or partially,
substantially or completely prevented or blocked. The term "IL-18
inhibitor" is meant to encompass inhibitors of IL-18 production, as well
as of inhibitors of IL-18 action.
An inhibitor of production can be any molecule negatively affecting the
synthesis, processing or maturation of IL-18. The inhibitors considered
according to the invention can be, for example, suppressors of gene
expression of the interleukin IL-18, antisense mRNAs reducing or
preventing the transcription of the IL-18 mRNA or leading to degradation
of the mRNA, proteins impairing correct folding, or partially or
substantially preventing secretion of IL-18, proteases degrading IL-18,
once it has been synthesized, inhibitors of proteases cleaving pro-IL-18
in order to generate mature IL-18, such as inhibitors of caspase-1, and
An inhibitor of IL-18 action can be an IL-18 antagonist, for example.
Antagonists can either bind to or sequester the IL-18 molecule itself with
sufficient affinity and specificity to partially or substantially
neutralize the IL-18 or IL-18 binding site(s) responsible for IL-18
binding to its ligands (like, e.g. to its receptors). An antagonist may
also inhibit the IL-18 signaling pathway, which is activated within the
cells upon IL-18 binding to its receptor.
Inhibitors of IL-18 action may also be soluble IL-18 receptors or
molecules mimicking the receptors, or agents blocking the IL-18 receptors,
or IL-18 antibodies, such as polyclonal or monoclonal antibodies, or any
other agent or molecule preventing the binding of IL-18 to its targets,
thus diminishing or preventing triggering of the intra- or extracellular
reactions mediated by IL-18.
In a preferred embodiment of the present invention, the inhibitor of IL-18
is selected from inhibitors of caspase-1 (ICE), antibodies directed
against IL-18, antibodies directed against any of the IL-18 receptor
subunits, inhibitors of the IL-18 signaling pathway, antagonists of IL-18
which compete with IL-18, or bind to, and block the IL-18 receptor, and
IL-18 binding proteins, isoforms, muteins, fused proteins, functional
derivatives, active fractions or circularly permutated derivatives, or
The term "IL-18 binding proteins" is used herein synonymously with
"IL18BP". It comprises IL-18 binding proteins as defined in WO 99/09063 or
in Novick et al., 1999, including splice variants and/or isoforms of IL-18
binding proteins, as defined in Kim et al., 2000. In particular, human
isoforms a and c of IL-18BP are useful in accordance with the presence
invention. The proteins useful according to the present invention may be
glycosylated or non-glycosylated, they may be derived from natural
sources, such as urine, or they may preferably be produced recombinantly.
Recombinant expression may be carried out in prokaryotic expression
systems like E. coli, or in eukaryotic, and preferably in mammalian,
expression systems. A cell line particularly well suited for the IL-18
inhibitors of the present invention is the Chinese hamster ovary (CHO)
Recombinant production of the IL-18 inhibitor, when recombinantly
expressed in mammalian cells or cell lines, may preferably be carried out
in serum free cell culture medium.
As used herein the term "muteins" refers to analogs of an IL-18BP, or
analogs of a viral IL-18BP, in which one or more of the amino acid
residues of a natural IL-18BP or viral IL-18BP are replaced by different
amino acid residues, or are deleted, or one or more amino acid residues
are added to the natural sequence of an IL-18BP, or a viral IL-18BP,
without reducing considerably the activity of the resulting products as
compared with the wild type IL-18BP or viral IL-18BP. These muteins are
prepared by known synthesis and/or by site-directed mutagenesis
techniques, or any other known technique suitable therefor.
Any such mutein preferably has a sequence of amino acids sufficiently
duplicative of that of an IL-18BP, or sufficiently duplicative of a viral
IL-18BP, such as to have substantially similar activity to IL-18BP. One
activity of IL-18BP is its capability of binding IL-18. As long as the
mutein has substantial binding activity to IL-18, it can be used in the
purification of IL-18, such as by means of affinity chromatography, and
thus can be considered to have substantially similar activity to IL-18BP.
Thus, it can be determined whether any given mutein has an activity
substantially similar to IL-18BP by means of routine experimentation
comprising subjecting such a mutein, e.g., to a simple sandwich
competition assay to determine whether or not it binds to an appropriately
labeled IL-18, such as radioimmunoassay or ELISA assay. Simple functional
assays for assessing the biological activity of IL-18BP were described in
detail in WO 99/09063, e.g. in examples 2 (binding to IL-18 as assessed by
cross-linking) or 5 (inhibition of IL-18 induced INF-gamma induction in
mononuclear blood cells).
In a preferred embodiment, any such mutein has at least 40% identity or
homology with the sequence of either an IL-18BP or a virally-encoded
IL-18BP homologue. More preferably, it has at least 50%, at least 60%, at
least 70%, at least 80% or, most preferably, at least 90% identity or
Muteins of IL-18BP polypeptides or muteins of viral IL-18BPs, which can be
used in accordance with the present invention, or nucleic acid coding
therefor, include a finite set of substantially corresponding sequences as
substitution peptides or polynucleotides which can be routinely obtained
by one of ordinary skill in the art, without undue experimentation, based
on the teachings and guidance presented herein.
Muteins in accordance with the present invention include proteins encoded
by a nucleic acid, such as DNA or RNA, which hybridizes to DNA or RNA,
which encodes the IL-18 inhibitor, in accordance with the present
invention, under moderately or highly stringent conditions. The term
"stringent conditions" refers to hybridization and subsequent washing
conditions, which those of ordinary skill in the art conventionally refer
to as "stringent". See Ausubel et al., Current Protocols in Molecular
Biology, supra, Interscience, N.Y., .sctn..sctn.6.3 and 6.4 (1987, 1992),
and Sambrook et al. (Sambrook, J. C., Fritsch, E. F., and Maniatis, T.
(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).
Without limitation, examples of stringent conditions include washing
conditions 12-20.degree. C. below the calculated Tm of the hybrid under
study in, e.g., 2.times.SSC and 0.5% SDS for 5 minutes, 2.times.SSC and
0.1% SDS for 15 minutes; 0.1.times.SSC and 0.5% SDS at 37.degree. C. for
30-60 minutes and then, a 0.1.times.SSC and 0.5% SDS at 68.degree. C. for
30-60 minutes. Those of ordinary skill in this art understand that
stringency conditions also depend on the length of the DNA sequences,
oligonucleotide probes (such as 10-40 bases) or mixed oligonucleotide
probes. If mixed probes are used, it is preferable to use tetramethyl
ammonium chloride (TMAC) instead of SSC. See Ausubel, supra.
In a preferred embodiment, any such mutein has at least 40% identity or
homology with the sequence of SEQ ID NO: 1, 2 or 3 of the annexed sequence
listing. More preferably, it has at least 50%, at least 60%, at least 70%,
at least 80% or, most preferably, at least 90% identity or homology
Identity reflects a relationship between two or more polypeptide sequences
or two or more polynucleotide sequences, determined by comparing the
sequences. In general, identity refers to an exact nucleotide to
nucleotide or amino acid to amino acid correspondence of the two
polynucleotides or two polypeptide sequences, respectively, over the
length of the sequences being compared.
For sequences where there is not an exact correspondence, a "% identity"
may be determined. In general, the two sequences to be compared are
aligned to give a maximum correlation between the sequences. This may
include inserting "gaps" in either one or both sequences, to enhance the
degree of alignment. A % identity may be determined over the whole length
of each of the sequences being compared (so-called global alignment), that
is particularly suitable for sequences of the same or very similar length,
or over shorter, defined lengths (so-called local alignment), that is more
suitable for sequences of unequal length.
Methods for comparing the identity and homology of two or more sequences
are well known in the art. Thus for instance, programs available in the
Wisconsin Sequence Analysis Package, version 9.1 (Devereux J et al.,
1984), for example the programs BESTFIT and GAP, may be used to determine
the % identity between two polynucleotides and the % identity and the %
homology between two polypeptide sequences. BESTFIT uses the "local
homology" algorithm of Smith and Waterman (1981) and finds the best single
region of similarity between two sequences. Other programs for determining
identity and/or similarity between sequences are also known in the art,
for instance the BLAST family of programs (Altschul S F et al, 1990,
Altschul S F et al, 1997, accessible through the home page of the NCBI at
www.ncbi.nlm.nih.gov) and FASTA (Pearson W R, 1990; Pearson 1988).
Preferred changes for muteins in accordance with the present invention are
what are known as "conservative" substitutions. Conservative amino acid
substitutions of IL-18BP polypeptides or proteins or viral IL-18BPs, may
include synonymous amino acids within a group which have sufficiently
similar physicochemical properties that substitution between members of
the group will preserve the biological function of the molecule (Grantham,
1974). It is clear that insertions and deletions of amino acids may also
be made in the above-defined sequences without altering their function,
particularly if the insertions or deletions only involve a few amino
acids, e.g., under thirty, and preferably under ten, and do not remove or
displace amino acids which are critical to a functional conformation,
e.g., cysteine residues. Proteins and muteins produced by such deletions
and/or insertions come within the purview of the present invention.
Preferably, the synonymous amino acid groups are those defined in Table 1 (see Original Patent).
More preferably, the synonymous amino acid groups are those defined in
Table 2 (see Original Patent); and most preferably the synonymous amino
acid groups are those defined in Table 3 (see Original Patent).
Examples of production of amino acid substitutions in proteins which can
be used for obtaining muteins of IL-18BP polypeptides or proteins, or
muteins of viral IL-18BPs, for use in the present invention include any
known method steps, such as presented in U.S. Pat. Nos. 4,959,314,
4,588,585 and 4,737,462, to Mark et al; U.S. Pat. No. 5,116,943 to Koths
et al., U.S. Pat. No. 4,965,195 to Namen et al; U.S. Pat. No. 4,879,111 to
Chong et al; and U.S. Pat. No. 5,017,691 to Lee et al; and lysine
substituted proteins presented in U.S. Pat. No. 4,904,584 (Shaw et al).
The term "fused protein" refers to a polypeptide comprising an IL-18BP, or
a viral IL-18BP, or a mutein or fragment thereof, fused with another
protein, which, e.g., has an extended residence time in body fluids. An
IL-18BP or a viral IL-18BP, may thus be fused to another protein,
polypeptide or the like, e.g., an immunoglobulin or a fragment thereof.
"Functional derivatives" as used herein cover derivatives of IL-18BPs or a
viral IL-18BP, and their muteins and fused proteins, which may be prepared
from the functional groups which occur as side chains on the residues or
the N- or C-terminal groups, by means known in the art, and are included
in the invention as long as they remain pharmaceutically acceptable, i.e.
they do not destroy the activity of the protein which is substantially
similar to the activity of IL-18BP, or viral IL-18BPs, and do not confer
toxic properties on compositions containing it.
These derivatives may, for example, include polyethylene glycol
side-chains, which may mask antigenic sites and extend the residence of an
IL-18BP or a viral IL-18BP in body fluids. Other derivatives include
aliphatic esters of the carboxyl groups, amides of the carboxyl groups by
reaction with ammonia or with primary or secondary amines, N-acyl
derivatives of free amino groups of the amino acid residues formed with
acyl moieties (e.g. alkanoyl or carbocyclic aroyl groups) or O-acyl
derivatives of free hydroxyl groups (for example that of seryl or threonyl
residues) formed with acyl moieties.
As "active fractions" of an IL-18BP, or a viral IL-18BP, muteins and fused
proteins, the present invention covers any fragment or precursors of the
polypeptide chain of the protein molecule alone or together with
associated molecules or residues linked thereto, e.g., sugar or phosphate
residues, or aggregates of the protein molecule or the sugar residues by
themselves, provided said fraction has substantially similar activity to
The term "salts" herein refers to both salts of carboxyl groups and to
acid addition salts of amino groups of IL-18 inhibitor molecule, or
analogs thereof. Salts of a carboxyl group may be formed by means known in
the art and include inorganic salts, for example, sodium, calcium,
ammonium, ferric or zinc salts, and the like, and salts with organic bases
as those formed, for example, with amines, such as triethanolamine,
arginine or lysine, piperidine, procaine and the like. Acid addition salts
include, for example, salts with mineral acids, such as, for example,
hydrochloric acid or sulfuric acid, and salts with organic acids, such as,
for example, acetic acid or oxalic acid. Of course, any such salts must
retain the biological activity of OPN relevant to the present invention,
i.e., exert a proliferative effect on oligodendrocytes.
In a further preferred embodiment of the invention, the inhibitor of IL-18
is an IL-18 antibody. Anti-IL-18 antibodies may be polyclonal or
monoclonal, chimeric, humanized, or even fully human. Recombinant
antibodies and fragments thereof are characterized by high affinity
binding to IL-18 in vivo and low toxicity. The antibodies which can be
used in the invention are characterized by their ability to treat patients
for a period sufficient to have good to excellent regression or
alleviation of the pathogenic condition or any symptom or group of
symptoms related to a pathogenic condition, and a low toxicity.
Neutralizing antibodies are readily raised in animals such as rabbits,
goat or mice by immunization with IL-18. Immunized mice are particularly
useful for providing sources of B cells for the manufacture of hybridomas,
which in turn are cultured to produce large quantities of anti-IL-18
Chimeric antibodies are immunoglobulin molecules characterized by two or
more segments or portions derived from different animal species.
Generally, the variable region of the chimeric antibody is derived from a
non-human mammalian antibody, such as murine monoclonal antibody, and the
immunoglobulin constant region is derived from a human immunoglobulin
molecule. Preferably, both regions and the combination have low
immunogenicity as routinely determined (Elliott et al., 1994). Humanized
antibodies are immunoglobulin molecules created by genetic engineering
techniques in which the murine constant regions are replaced with human
counterparts while retaining the murine antigen binding regions. The
resulting mouse-human chimeric antibody preferably have reduced
immunogenicity and improved pharmacokinetics in humans (Knight et al.,
Thus, in a further preferred embodiment, IL-18 antibody is a humanized
IL-18 antibody. Preferred examples of humanized anti-IL-18 antibodies are
described in the European Patent Application EP 0 974 600, for example.
In yet a further preferred embodiment, the IL-18 antibody is fully human.
The technology for producing human antibodies is described in detail e.g.
in WO00/76310, WO99/53049, U.S. Pat. No. 6,162,963 or AU5336100. Fully
human antibodies are preferably recombinant antibodies, produced in
transgenic animals, e.g. xenomice, comprising all or portions of
functional human Ig loci.
In a highly preferred embodiment of the present invention, the inhibitor
of IL-18 is an IL-18BP, or an isoform, a mutein, fused protein, functional
derivative, active fraction or circularly permutated derivative thereof.
These isoforms, muteins, fused proteins or functional derivatives retain
the biological activity of IL-18BP, in particular the binding to IL-18,
and preferably have essentially at least an activity similar to IL-18BP.
Ideally, such proteins have a biological activity which is even increased
in comparison to unmodified IL-18BP. Preferred active fractions have an
activity which is better than the activity of IL-18BP, or which have
further advantages, like a better stability or a lower toxicity or
immunogenicity, or they are easier to produce in large quantities, or
easier to purify.
The sequences of IL-18BP and its splice variants/isoforms can be taken
from WO 99/09063 or from Novick et al., 1999, as well as from Kim et al.,
Functional derivatives of IL-18BP may be conjugated to polymers in order
to improve the properties of the protein, such as the stability,
half-life, bioavailability, tolerance by the human body, or immunogenicity.
To achieve this goal, the functional derivative may comprise at least one
moiety attached to one or more functional groups, which occur as one or
more side chains on the amino acid residues. Such a functional group may
e.g. be Polyethlyenglycol (PEG). PEGylation may be carried out by known
methods, described in WO 92/13095, for example.
Therefore, in a preferred embodiment of the present invention, the
inhibitors of IL-18, and in particular the IL-18BP is PEGylated.
In a further preferred embodiment of the invention, the inhibitor of IL-18
is a fused protein comprising all or part of an IL-18 binding protein,
which is fused to all or part of an immunoglobulin. The person skilled in
the art will understand that the resulting fusion protein retains the
biological activity of IL-18BP, in particular the binding to IL-18. The
fusion may be direct, or via a short linker peptide which can be as short
as 1 to 3 amino acid residues in length or longer, for example, 13 amino
acid residues in length. Said linker may be a tripeptide of the sequence
E-F-M (Glu-Phe-Met), for example, or a 13-amino acid linker sequence
comprising Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met introduced
between the IL-18BP sequence and the immunoglobulin sequence. The
resulting fusion protein has improved properties, such as an extended
residence time in body fluids (half-life), increased specific activity,
increased expression level, or the purification of the fusion protein is
In a preferred embodiment, IL-18BP is fused to the constant region of an
Ig molecule. Preferably, it is fused to heavy chain regions, like the CH2
and CH3 domains of human IgG1, for example. The generation of specific
fusion proteins comprising IL-18BP and a portion of an immunoglobulin are
described in example 11 of WO 99/09063, for example. Other isoforms of Ig
molecules are also suitable for the generation of fusion proteins
according to the present invention, such as isoforms IgG.sub.2 or
IgG.sub.4, or other Ig classes, like IgM or IgA, for example. Fusion
proteins may be monomeric or multimeric, hetero- or homomultimeric.
Interferons are predominantly known for inhibitory effects on viral
replication and cellular proliferation. Interferon-.gamma., for example,
plays an important role in promoting immune and inflammatory responses.
Interferon .beta. (IFN-.beta., an interferon type I), is said to play an
The invention therefore also relates to the use of a combination of an
inhibitor of IL-18 and an interferon in the manufacture of a medicament
for the treatment of CNS injury.
Interferons may also be conjugated to polymers in order to improve the
stability of the proteins. A conjugate between Interferon .beta. and the
polyol Polyethlyenglycol (PEG) has been described in WO99/55377, for
In another preferred embodiment of the invention, the interferon is
interferon-.beta. (IFN-.beta.), and more preferably IFN-.beta.1a.
The inhibitor of IL-18 production and/or action is preferably used
simultaneously, sequentially, or separately with the interferon.
Tumor necrosis factor, has been described in the literature to have both
protective and toxic effects in brain injury (Shohami et al., 1999). In
Example 1 below, TNF injection into mice following severe brain trauma
resulted in a significant decrease of IL-18 levels in the brain, thus
indicating that TNF may have a beneficial effect on the recovery of
traumatic brain injury. Therefore, a preferred embodiment of the invention
relates to the use of an inhibitor of IL-18 in combination with TNF for
the preparation of a medicament for treatment and/or prevention of brain
injury, for simultaneous, sequential or separate use.
The combination of IL-18 inhibitors with TNF alpha is preferred in
accordance with the present invention.
In a further preferred embodiment of the invention, the medicament further
comprises an anti-inflammatory agent, such as an NSAID (nonsteroidal
anti-inflammatory drugs). In a preferred embodiment, a COX-inhibitor, and
most preferably a COX-2 inhibitor, is used in combination with an IL-18
inhibitor. COX-inhibitors are known in the art. Specific COX-2 inhibitors
are disclosed in WO 01/00229, for example. The active components may be
used simultaneously, sequentially, or separately.
Oxidative stress, in particular reactive oxygen species (ROS), have been
described to play a role in the pathophysiology of brain damage (Shohami
et al., 1997).
Therefore, in a preferred embodiment of the present invention, the
medicament--further comprises an antioxidant, for simultaneous,
sequential, or separate use. Many antioxidants are known in the art, such
as vitamins A, C or E, or 5-aminosalicylic acid, or superoxide dismutase.
In a further preferred embodiment of the present invention, the inhibitor
of IL-18 is used in an amount of about 0.001 to 100 mg/kg of body weight,
or about 0.01 to 10 mg/kg of body weight or about 0.1 to 3 mg/kg of body
weight or about 1 to 2 mg/kg of body weight.
In yet a further preferred embodiment, the inhibitor of IL-18 is used in
an amount of about 0.1 to 1000 .mu.g/kg of body weight or 1 to 100 .mu.g/kg
of body weight or about 10 to 50 .mu.g/kg of body weight.
The invention further relates to the use of a nucleic acid molecule
comprising the coding sequence of an IL-18 inhibitor, a mutein, functional
derivative, or active fraction thereof, in the preparation of a medicament
for the prevention and/or treatment of CNS injury.
Preferably, the nucleic acid molecule further comprises a sequence of an
expression vector, e.g. to use gene therapy for administering the IL-18
inhibitor of the invention.
Preferably, the nucleic acid molecule is administered intramuscularly.
In order to treat and/or prevent CNS injury, the gene therapy vector
comprising the sequence of an inhibitor of IL-18 production and/or action
may be injected directly into the diseased tissue, for example, thus
avoiding problems involved in systemic administration of gene therapy
vectors, like dilution of the vectors, reaching and targeting of the
target cells or tissues, and of side effects.
The use of a vector for inducing and/or enhancing the endogenous
production of an inhibitor of IL-18 in a cell normally silent for
expression of an IL-18 inhibitor, or which expresses amounts of the
inhibitor which are not sufficient, are also contemplated according to the
invention for treatment and/or prevention of CNS injury. The vector may
comprise regulatory elements functional in the cells desired to express
the inhibitor or IL-18. Such regulatory sequences or elements may be
promoters or enhancers, for example. The regulatory sequence may then be
introduced into the right locus of the genome by homologous recombination,
thus operably linking the regulatory sequence with the gene, the
expression of which is required to be induced or enhanced. The technology
is usually referred to as "Endogenous Gene Activation" (EGA), and it is
described e.g. in WO 91/09955.
It will be understood by the person skilled in the art that it is also
possible to shut down IL-18 expression directly, without using an
inhibitor of IL-18, with the same technique. To do that, a negative
regulation element, like e.g. a silencing element, may be introduced into
the gene locus of IL-18, thus leading to down-regulation or prevention of
IL-18 expression. The person skilled in the art will understand that such
down-regulation or silencing of IL-18 expression has the same effect as
the use of an IL-18 inhibitor in order to prevent and/or treat disease.
The invention further relates to the use of a cell that has been
genetically modified to produce an inhibitor of IL-18 in the manufacture
of a medicament for the treatment and/or prevention of CNS injury.
The invention further relates to pharmaceutical compositions, particularly
useful for prevention and/or treatment of inflammatory CNS injury, which
comprise a therapeutically effective amount of an inhibitor of IL-18
and/or a therapeutically effective amount of an interferon and/or a
pharmaceutically effective amount of TNF and/or a pharmaceutically
effective amount of an anti-inflammatory agent and/or a pharmaceutically
effective amount of an anti-oxidative agent.
As inhibitor of IL-18, the composition may comprise caspase-1 inhibitors,
antibodies against IL-18, antibodies against any of the IL-18 receptor
subunits, inhibitors of the IL-18 signaling pathway, antagonists of IL-18
which compete with IL-18 and block the IL-18 receptor, and IL-18 binding
proteins, isoforms, muteins, fused proteins, functional derivatives,
active fractions or circularly permutated derivatives thereof having the
IL-18BP and its isoforms, muteins, fused proteins, functional derivatives,
active fractions or circularly permutated derivatives as described above
are the preferred active ingredients of the pharmaceutical compositions.
The interferon comprised in the pharmaceutical composition is preferably
IFN-beta or IFN-alpha.
In yet another preferred embodiment, the pharmaceutical composition
comprises therapeutically effective amounts of TNF alpha. The
pharmaceutical composition according to the invention may further comprise
one or more COX-inhibitors.
The definition of "pharmaceutically acceptable" is meant to encompass any
carrier, which does not interfere with effectiveness of the biological
activity of the active ingredient and that is not toxic to the host to
which it is administered. For example, for parenteral administration, the
active protein(s) may be formulated in a unit dosage form for injection in
vehicles such as saline, dextrose solution, serum albumin and Ringer's
The active ingredients of the pharmaceutical composition according to the
invention can be administered to an individual in a variety of ways. The
routes of administration include intradermal, transdermal (e.g. in slow
release formulations), intramuscular, intraperitoneal, intravenous,
subcutaneous, oral, intracranial, epidural, rectal, topical, and
intranasal routes. Any other therapeutically efficacious route of
administration can be used, for example absorption through epithelial or
endothelial tissues or by gene therapy wherein a DNA molecule encoding the
active agent is administered to the patient (e.g. via a vector), which
causes the active agent to be expressed and secreted in vivo. In addition,
the protein(s) according to the invention can be administered together
with other components of biologically active agents such as
pharmaceutically acceptable surfactants, excipients, carriers, diluents
For parenteral (e.g. intravenous, subcutaneous, intramuscular)
administration, the active protein(s) can be formulated as a solution,
suspension, emulsion or lyophilized powder in association with a
pharmaceutically acceptable parenteral vehicle (e.g. water, saline,
dextrose solution) and additives that maintain isotonicity (e.g. mannitol)
or chemical stability (e.g. preservatives and buffers). The formulation is
sterilized by commonly used techniques.
The bioavailability of the active protein(s) according to the invention
can also be ameliorated by using conjugation procedures which increase the
half-life of the molecule in the human body, for example linking the
molecule to polyethylenglycol, as described in the PCT Patent Application
The therapeutically effective amounts of the active protein(s) will be a
function of many variables, including the type of antagonist, the affinity
of the antagonist for IL-18, any residual cytotoxic activity exhibited by
the antagonists, the route of administration, the clinical condition of
the patient (including the desirability of maintaining a non-toxic level
of endogenous IL-18 activity).
A "therapeutically effective amount" is such that when administered, the
IL-18 inhibitor results in inhibition of the biological activity of IL-18.
The dosage administered, as single or multiple doses, to an individual
will vary depending upon a variety of factors, including IL-18 inhibitor
pharmacokinetic properties, the route of administration, patient
conditions and characteristics (sex, age, body weight, health, size),
extent of symptoms, concurrent treatments, frequency of treatment and the
effect desired. Adjustment and manipulation of established dosage ranges
are well within the ability of those skilled in the art, as well as in
vitro and in vivo methods of determining the inhibition of IL-18 in an
According to the invention, the inhibitor of IL-18 is used in an amount of
about 0.0001 to 100 mg/kg or about 0.01 to 10 mg/kg or body weight, or
about 0.1 to 5 mg/kg of body weight or about 1 to 3 mg/kg of body weight
or about 1 to 2 mg/kg of body weight. Alternatively, the IL-18 inhibitors
may be administered in amounts of about 0.1 to 1000 .mu.g/kg of body
weight or about 1 to 100 .mu.g/kg of body weight or about 10 to 50 .mu.g/kg
of body weight
The route of administration, which is preferred according to the invention
is administration by subcutaneous route. Intramuscular administration is
further preferred according to the invention. In order to administer the
IL-18 inhibitor directly to the place of its action, it is also preferred
to administer it via the intracranial or intrathecal route. The
intracranial route is especially preferred in combination with open head
injury (missile injury of the brain).
In further preferred embodiments, the inhibitor of IL-18 is administered
daily or every other day.
The daily doses are usually given in divided doses or in sustained release
form effective to obtain the desired results. Second or subsequent
administrations can be performed at a dosage which is the same, less than
or greater than the initial or previous dose administered to the
individual. A second or subsequent administration can be administered
during or prior to onset of the disease.
According to the invention, the IL-18 inhibitor can be administered
prophylactically or therapeutically to an individual prior to,
simultaneously or sequentially with other therapeutic regimens or agents
(e.g. multiple drug regimens), in a therapeutically effective amount, in
particular with an interferon and/or a TNF and/or another
anti-inflammatory agent, such as a COX inhibitor and/or an antioxidant.
Depending on the brain injury, the co-administration of a TNF-antagonist
instead of TNF itself can also be conceived (Shohami et al., 1999). Active
agents that are administered simultaneously with other therapeutic agents
can be administered in the same or different compositions.
The invention further relates to a method for the preparation of a
pharmaceutical composition comprising admixing an effective amount of an
IL-18 inhibitor and/or an interferon and/or a TNF antagonist and/or a COX
inhibitor with a pharmaceutically acceptable carrier.
The invention further relates to a method of treatment of CNS injury,
comprising administering a pharmaceutically effective amount of an IL-18
inhibitor to a patient in need thereof.
Claim 1 of 13 Claims
1. A method of treatment of an isolated
closed head injury comprising administering to an individual in need
thereof an effective inhibiting amount of IL-18BP, wherein the IL-18BP is
administered in a single dose at 3 days after the closed head injury.
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