FIELD OF THE INVENTION
The present invention is in the field of neurological disorders.
It relates to the use of a combination of TNF-alpha and IFN-beta for treatment and/or
prevention of a demyelinating disease, such as multiple sclerosis (MS).
BACKGROUND OF THE INVENTION
Demyelinating diseases are disorders concerning the myelin
sheaths of the nervous system. Myelin sheaths, which cover many nerve fibers, are
composed of lipoprotein layers formed in early life. Myelin is formed by the oligodendroglia
in the CNS and promote transmission of a neural impulse along an axon.
Many congenital metabolic disorders (e.g. phenylketonuria
and other aminoacidurias; Tay-Sachs, Niemann-Pick, and Gaucher's diseases; Hurler's
syndrome; Krabbe's disease and other leukodystrophies) affect the developing myelin
sheath, mainly in the CNS. Unless the biochemical defect can be corrected or compensated
for, permanent, often widespread, neurological deficits result.
Demyelination in later life is a feature of many neurological
disorders; it can result from damage to nerves or myelin due to local injury, ischemia,
toxic agents, or metabolic disorders. Extensive myelin loss is usually followed
by axonal degeneration and often by cell body degeneration, both of which may be
irreversible. However, remyelination occurs in many instances, and repair, regeneration,
and complete recovery of neural function can be rapid. Recovery often occurs after
the segmental demyelination that characterizes many peripheral neuropathies; this
process may account for the exacerbations and remissions of multiple sclerosis (MS).
Central demyelination (i.e. of the spinal cord, brain, or optic nerves) is the predominant
finding in the primary demyelinating diseases, whose etiology is unknown. The most
well known demyelinating disease is MS (see below).
Further demyelinating diseases comprise:
- Acute disseminated encephalomyelitis, which is characterized by perivascular
CNS demyelination, and which can occur spontaneously but usually follows a viral
infection or viral vaccination;
- Acute inflammatory peripheral neuropathies that follow a viral vaccination or
the Guillain-Barré syndrome, they affect only peripheral structures;
- Adrenoleukodystrophy and adrenomyeloneuropathy, which are rare X-linked recessive
metabolic disorders characterized by adrenal gland dysfunction and widespread demyelination
of the nervous system;
- Leber's hereditary optic atrophy and related mitochondrial disorders, which
are characterized primarily by bilateral loss of central vision, and which can resemble
the optic neuritis in MS; and
- HTLV-associated myelopathy, a slowly progressive spinal cord disease associated
with infection by the human T-cell lymphotrophic virus, that is characterized by
spastic weakness of both legs.
Multiple sclerosis (MS) is a slowly progressive CNS disease
characterized by disseminated patches of demyelination in the brain and spinal cord,
resulting in multiple and varied neurological symptoms and signs, usually with remissions
and exacerbation (see The Merck Manual Home Edition, www.merck.com).
The cause is unknown but an immunological abnormality is
suspected, with few clues presently indicating a specific mechanism. Postulated
causes include infection by a slow or latent virus, and myelinolysis by enzymes.
IgG is usually elevated in the CSF, and elevated titers have been associated with
a variety of viruses, including measles. The significance of these findings and
of reported associations with HLA allotypes and altered number of T cells is unclear,
and the evidence somewhat conflicting. An increased family incidence suggests genetic
susceptibility; women are somewhat more often affected than men. Environmental factors
seem to be present. Although age at onset generally is from 20 to 40 years, MS has
been linked to the geographic area where a patient's first 15 years are spent. Relocation
after age 15 does not alter the risk.
Plaques or islands of demyelination with destruction of
oligodendroglia and perivascular inflammation are disseminated through the CNS,
primarily in the white matter, with a predilection for the lateral ad posterior
columns (especially in the cervical and dorsal regions), the optic nerves, and periventricular
areas. Tracts in the midbrain, pons, and cerebellum also are affected, and gray
matter in both cerebrum and cord may be affected.
Cell bodies and axons are usually preserved, especially
in early lesions. Later, axons may be destroyed, especially in the long tracts,
and a fibrous gliosis gives the tracts their "sclerotic" appearance. Both early
and late lesions may be found simultaneously. Chemical changes in lipid and protein
constituents of myelin have been demonstrated in and around the plaques.
The disease is characterized by various symptoms and signs
of CNS dysfunction, with remissions and recurring exacerbations. The most common
presenting symptoms are paresthesias in one or more extremities, in the trunk, or
on one side of the face; weakness or clumsiness of a leg or hand; or visual disturbances,
e.g., partial blindness and pain in one eye (retrobulbar optic neuritis), dimness
of vision, or scotomas. Other common early symptoms are ocular palsy resulting in
double vision (diplopia), transient weakness of one or more extremities, slight
stiffness or unusual fatigability of a limb, minor gait disturbances, difficulty
with bladder control, vertigo, and mild emotional disturbances; all indicate scattered
CNS involvement and often occur months or years before the disease is recognized.
The course is highly varied, unpredictable, and, in most
patients, remittent. Life span is probably not shortened except in the most severe
cases. At first, months or years of remission may separate episodes, especially
when the disease begins with retrobulbar optic neuritis. Remissions can last >
10 years. However, some patients have frequent attacks and are rapidly incapacitated;
for a few, particularly for male patients with onset in middle age, the course can
be rapidly progressive. Exposure to excess heat from fever or the environment sometimes
Diagnosis is indirect, by deduction from clinical and laboratory
features. MRI, the most sensitive diagnostic imaging technique, may show plaques.
Gadolinium-contrast enhancement can distinguish areas of active inflammation from
older brain plaques. MS lesions may also be visible on contrast-enhanced CT scans,
in which sensitivity may be increased by giving twice the iodine dose and delaying
scanning (double-dose delayed CT scan).
CSF is abnormal in the majority of patients. IgG may be
> 13%, and lymphocytes and protein content may be slightly increased. Oligoclonal
bands, which indicate IgG synthesis within the blood-brain barrier, may be detected
by agarose electrophoresis of CSF in up to 90% of patients with MS, but absence
of these bands does not rule out MS. IgG levels correlate with disease severity.
Myelin basic protein may be elevated during active demyelination.
Spontaneous remissions and fluctuating symptoms make treatments
difficult to evaluate. Corticosteroids are the main form of therapy. They may shorten
the symptomatic period during attacks, although they may not affect eventual long-term
disability. Patients presenting with acute severe optic neuritis may delay the onset
of MS by using high-dose IV corticosteroids.
Immunosuppressive drugs (methotrexate, azathioprine, cyclophosphamide,
cladribine) are generally used for more severe progressive forms. Immunomodulatory
therapy with interferon-&bgr; reduc es the frequency of relapses in MS. Other
promising treatments still under investigation include other interferons, oral myelin,
and glatiramer to help keep the body from attacking its own myelin. Glatiramer is
a synthetic co-polymer with similarities to myelin basic protein and is administered
by daily subcutaneous injection. Its main action is thought to be suppression of
the immune response against myelin to promote immune tolerance (Clegg and Bryant,
Interferons are cytokines, i.e. soluble proteins that transmit
messages between cells and play an essential role in the immune system by helping
to destroy microorganisms that cause infection and repairing any resulting damage.
Interferons are naturally secreted by infected cells and were first identified in
1957. Their name is derived from the fact that they "interfere" with viral replication
Interferons exhibit both antiviral and antiproliferative
activity. On the basis of biochemical and immunological properties, the naturally-occurring
human interferons are grouped into three major classes: interferon-alpha (leukocyte),
interferon-beta (fibroblast) and interferon-gamma (immune). Alpha-interferon is
currently approved in the United States and other countries for the treatment of
hairy cell leukemia, venereal warts, Kaposi's Sarcoma (a cancer commonly afflicting
patients suffering from Acquired Immune Deficiency Syndrome (AIDS)), and chronic
non-A, non-B hepatitis.
Further, interferons (IFNs) are glycoproteins produced
by the body in response to a viral infection. They inhibit the multiplication of
viruses in protected cells. Consisting of a lower molecular weight protein, IFNs
are remarkably non specific in their action, i.e. IFN induced by one virus is effective
against a broad range of other viruses. They are however species-specific, i.e.
IFN produced by one species will only stimulate antiviral activity in cells of the
same or a closely related species. IFNs were the first group of cytokines to be
exploited for their potential anti-tumor and antiviral activities.
The three major IFNs are referred to as IFN-&agr;, IFN-&bgr;
and IFN-&ggr;. Such main kinds of IFNs were initially classified according to
their cells of origin (leukocyte, fibroblast or T cell). However, it became clear
that several types may be produced by one cell. Hence leukocyte IFN is now called
IFN-&agr;, fibroblast IFN is IFN-&bgr; and T cell IFN is IFN-&ggr;. There
is also a fourth type of IFN, lymphoblastoid IFN, produced in the "Namalwa" cell
line (derived from Burkitt's lymphoma), which seems to produce a mixture of both
leukocyte and fibroblast IFN.
The interferon unit or International unit for interferon
(U or IU, for international unit) has been reported as a measure of IFN activity
defined as the amount necessary to protect 50% of the cells against viral damage.
The assay that may be used to measure bioactivity is the cytopathic effect inhibition
assay as described (Rubinstein, et al. 1981; Familletti,P. C., et al., 1981). In
this antiviral assays for interferon about 1 unit/ml of interferon is the quantity
necessary to produce a cytopathic effect of 50%. The units are determined with respect
to the international reference standard for Hu-IFN-beta provided by the National
Institutes of Health (Pestka, S. 1986).
Every class of IFN contains several distinct types. IFN-&bgr;
and IFN-&ggr; are each the product of a single gene.
The proteins classified as IFNs-&agr;, are the most diverse
group, containing about 15 types. There is a cluster of IFN-&agr; genes on chromosome
9, containing at least 23 members, of which 15 are active and transcribed. Mature
IFNs-&agr; are not glycosylated.
IFNs-&agr; and IFN-&bgr; are all the same length (165
or 166 amino acids) with similar biological activities. IFNs-&ggr; are 146 amino
acids in length, and resemble the &agr; and &bgr; classes less closely. Only
IFNs-&ggr; can activate macrophages or induce the maturation of killer T cells.
In effect, these new types of therapeutic agents can be called biologic response
modifiers (BRMs), because they have an effect on the response of the organism to
the tumor, affecting recognition via immunomodulation.
In particular, human fibroblast interferon (IFN-&bgr;)
has antiviral activity and can also stimulate natural killer cells against neoplastic
cells. It is a polypeptide of about 20,000 Da induced by viruses and double-stranded
RNAs. From the nucleotide sequence of the gene for fibroblast interferon, cloned
by recombinant DNA technology, (Derynk et al. 1980) deduced the complete amino acid
sequence of the protein. It is 166 amino acid long.
Shepard et al. (1981) described a mutation at base 842
(Cys → Tyr at position 141) that abolished its anti-viral activity, and a
variant clone with a deletion of nucleotides 1119-1121.
Mark et al. (1984) inserted an artificial mutation by replacing
base 469 (T) with (A) causing an amino acid switch from Cys → Ser at position
17. The resulting IFN-&bgr; was reported to be as active as the 'native' IFN-&bgr;
and stable during long-term storage (-70°C).
Rebif® (recombinant human interferon-&bgr;) is a
recent development in interferon therapy for multiple sclerosis (MS) and represents
a significant advance in treatment. Rebif® is interferon(IFN)-beta 1a, produced
from mammalian cell lines. It was established that interferon beta-1a given subcutaneously
three times per week is efficacious in the treatment of Relapsing-Remitting Multiple
Sclerosis (RR-MS). Interferon beta-1a can have a positive effect on the long-term
course of MS by reducing number and severity of relapses and reducing the burden
of the disease and disease activity as measured by MRI (The Lancet, 1998).
Tumor Necrosis Factor, or TNF, previously called Cachectin,
is a pleiotropic cytokine released by activated T cells and macrophages. TNF is
a member of the interferon, interleukin and colony stimulating factor cytokine network,
which has a key role in signaling with regard to the pathogenesis of many infectious
and inflammatory diseases by inducing a number of proinflammatory changes, including
production of other cytokine and adhesion molecule (Fiers, 1991).
The term TNF collectively can mean, both Tumor Necrosis
Factor-alpha or Tumor Necrosis Factor-beta from animals or humans, together with
naturally occurring alleles thereof (Pennica et al., 1984, Wallach et al., 1986,
Beutler, B. and Cerami, A. (1987)). TNF-beta, also called lymphotoxin, has a similar
activity but is produced by different cell types (lymphocytes and Natural Killer
cells) in response to antigenic or mitogenic stimuli (Gray et al., 1984).
Thus, Tumor Necrosis Factor (TNF-&agr;) and Lymphotoxin
(TNF-&bgr;) are cytokines which have many effects on cells. Some of their effects
are likely to be beneficial to the organism: they may destroy, for example, tumor
cells or virus infected cells and augment antibacterial activities of granulocytes.
In this way, TNF contributes to the defense of the organism against infectious agents
and to recovery from injury. But both TNF-&agr; and TNF-&bgr; have also been
described to have deleterious effects. There is evidence that overproduction of
TNF-&agr; can play a major pathogenic role in several diseases. In some diseases,
TNF may cause excessive loss of weight (cachexia) by suppressing activities of adipocytes
and by causing anorexia and TNF-&agr; was thus called cachectin. It was also described
as a mediator of the damage to tissues in rheumatic diseases and as a major mediator
of the damage observed in graft-versus-host reactions.
TNF is expressed as a mature 17 kDa protein that is active
as a trimer. This complex exerts its biological activity by aggregating their cell
surface receptors, which mediate specific effects in different organs and tissues.
TNF exerts its activity, which is required for the normal
development and function of immune system, by binding a family of membrane bound
receptor molecules including p55 TNF receptor I, defined in the literature also
TNF-RI, and p75 TNF receptor, defined in the literature also TNF-RII (Bazzoni and
Beutler, 1996). The dominance of TNF-RI in transducing TNF signal is suggested by
the ability of agonistic antibodies specific for this receptor to mimic the majority
of TNF induced responses (Shalaby et al., 1990). By binding to its membrane-bound
receptors, TNF triggers the signaling pathway through cytoplasmic mediators like
TRADD and TRAP-1 (for TNF-RI) or TRAF-1 and TRAF-2 (for TNF-Rll), leading to different
cell response, like T cell proliferation, tumor-cell lysis in vitro, dermal necrosis,
insuline resistance, apoptosis. The extracellular portions of both TNF receptors
can be shed and these soluble receptors retain the ability to bind TNF, inactivating
TNF activity by formation of high affinity complexes, thereby reducing the binding
of TNF to target cell membrane receptors (Nophar et al., 1990).
Based on the finding that TNF-alpha immunoreactivity has
been found in high levels in MS lesions, TNF has been described to play a role in
the pathogenesis of multiple sclerosis (Darlington, 1999). Therefore, it was generally
thought that TNF should be blocked or reduced in order to treat MS, and TNF blocking
agents have been suggested for treatment of multiple sclerosis (Selmaj et al., 1995).
However, experiments using mice lacking TNF, so-called TNF -/- mice, showed that
these mice developed severe neurological impairment with extensive inflammation
and demyelination upon induction of a MS like disease with the protein MOG (Liu
et al., 1998).
Truncated forms of the TNF-RI (p55) and TNF-RII (p75) receptors
mentioned above are described e.g. in
. These soluble receptors are called TBPI and TBPII, respectively (Engelmann
et al., 1990). The natural and recombinant soluble TNF receptor molecules, and methods
of their production have been described e.g. in the European Patents
EP 308 378
EP 398 327
EP 433 900
EP 398 327
describes that TBPs are not only inhibitors of TNF activity, but also
maintain the beneficial effect of TNF. It has also been described that the soluble
TNF-receptors stabilize the bioactivity of TNF and thus augment some of its effects
(Aderka et al., 1992).
A TNF-like activity was also shown for antibodies directed
against the soluble forms of the TNF-receptors (Engelmann et al., 1990).
In addition to this,
EP 880 970
discloses the use of TBPs for treatment of multiple sclerosis.
SUMMARY OF THE INVENTION
The present invention is based on the finding that the
administration of Tumor Necrosis Factor (TNF) in combination with an interferon
(IFN) has a beneficial effect on remyelination and significantly reduces clinical
signs of the disease in an in vivo model of multiple sclerosis. It has been surprisingly
found that TNF potentiates the therapeutic effect of IFN in multiple sclerosis.
It has further been shown that interferon exerts its beneficial effect also at sub-therapeutic
dosage, when administered in combination with TNF.
Therefore, it is a first object of the present invention
to use Tumor Necrosis Factor (TNF) alpha, in combination with an interferon (IFN)
beta, or an isoform, mutein, fused protein, functional derivative, active fraction
or salt thereof, for the manufacture of a medicament for treatment and/or prevention
of a demyelinating disease, for simultaneous, sequential or separate use.
It is a second object of the present invention to provide
for a pharmaceutical composition containing TNF alpha in combination with an effective
amount of an IFN beta, in the presence of one or more pharmaceutically acceptable
BRIEF DESCRIPTION OF THE DRAWINGS
DETAILED DESCRIPTION OF THE INVENTION
- shows the clinical scores measured daily during an experimental period of 35
days in the murine EAE model after subcutaneous (s.c.) daily administration of either
20000 U/mouse of murine IFN-beta (open squares) or vehicle (filled diamonds).
- Fig. 2
- shows the disease development in the murine EAE model after administration of
IFN-beta (subcutaneous daily administration) or TNF-alpha (intravenous administration,
every other day) alone or in combination. Fig. 2 A shows the mortality rate in this
experiment. Fig. 2 B shows the clinical scores during an experimental period of
35 days after subcutaneous (s.c.) and intravenous (i.v.) administration of vehicle
(filled diamonds), s.c. administration of 5,000 U/mouse of murine IFN-beta (open
diamonds), intravenous (i.v.) administration of 0.1 µg/mouse of murine TNF-alpha
(filled triangles) or administration of both 0.1 µg/mouse mTNF-alpha i.v. and
5,000 U/mouse mlFN-beta s.c. (open triangles). A clinical score of 5 was assigned
to dead animals from the day of death until the end of the experiment (Fig. 2 B).
Fig. 2 C shows the results of the histological analysis of the spinal cord, measuring
the extent of inflammation (hatched horizontally) expressed as number of perivascular
inflammatory infiltrates (PII) or demyelination (hatched vertically), expressed
as percent demyelination (% Dem). N.E. = not evaluated.
- shows the data of Fig. 2, evaluated in a different way. Those animals dying
during the experimental period were not scored, but dropped out completely.
- shows the disease development in the murine EAE model after administration of
IFN-beta (subcutaneous daily administration) or TNF-alpha (intraperitoneal administration,
every other day) alone or in combination. Fig. 4 A shows the mortality rate in this
experiment. Fig. 4 B shows the clinical scores during an experimental period of
35 days after subcutaneous (s.c.) and intraperitoneal (i.p.) administration of vehicle
(filled diamonds), s.c. administration of 5,000 U/mouse of murine IFN-beta (open
diamonds), i.p. administration of 0.1 µg/mouse of murine TNF-alpha (filled
squares) or administration of both 0.1 µg/mouse mTNF-alpha ip.. and 5,000 U/mouse
mlFN-beta s.c. (open squares). A clinical score of 5 was assigned to dead animals
from the day of death until the end of the experiment (Fig. 4 B). Fig. 4 C shows
the results of the histological analysis of the spinal cord, measuring the extent
of inflammation (hatched horizontally) expressed as number of perivascular inflammatory
infiltrates (PII) or demyelination (hatched vertically), expressed as percent demyelination
(% Dem). N.E. = not evaluated.
- shows the data of Fig. 4, evaluated in a different way. Those animals dying
during the experimental period were not scored, but dropped out completely.
In accordance with the present invention, it has been surprisingly
found that TNF and interferon, when administered in combination, have a pronounced
beneficial effect on the clinical severity of multiple sclerosis. It was shown that
TNF enhances the therapeutic activity of IFN in an in vivo model of multiple sclerosis.
Therefore, the invention relates to the use of Tumor Necrosis
Factor (TNF) alpha, in combination with an interferon (IFN) beta, for the manufacture
of a medicament for treatment and/or prevention of a demyelinating disease. In accordance
with the present invention, the TNF alpha, and the interferon may be used simultaneously,
sequentially or separately.
The term "prevention" within the context of this invention
refers not only to a complete prevention of the disease or one or more symptoms
of the disease, but also to any partial or substantial prevention, attenuation,
reduction, decrease or diminishing of the effect before or at early onset of disease.
The term "treatment" within the context of this invention
refers to any beneficial effect on progression of disease, including attenuation,
reduction, decrease or diminishing of the pathological development after onset of
A "demyelinating disease", as used in the context of the
present invention, is a disease involving abnormalities in myelin sheaths of the
nervous system, in particular destruction of myelin, as described in detail in the
"Background of the Invention" above.
An "interferon" or "IFN", as used herein, is intended to
include any molecule defined as such in the literature, comprising for example any
types of IFNs mentioned in the above section "Background of the Invention". IFN-&bgr;
is included in the above definition. IFN-&bgr; is the IFN according to the present
invention as claimed. The use of interferons of human origin is also preferred in
accordance with the present invention. The term interferon, as used herein, is intended
to encompass salts, functional derivatives, variants, analogs and active fragments
The term "interferon-beta (IFN-&bgr;)", as used herein,
is intended to include human fibroblast interferon, as obtained by isolation from
biological fluids or as obtained by DNA recombinant techniques from prokaryotic
or eukaryotic host cells, as well as its salts, functional derivatives, variants,
analogs and active fragments.
A "Tumor Necrosis Factor" or "TNF", as used herein, shall
mean Tumor Necrosis Factor-alpha from animals or humans, together with naturally
occurring alleles thereof (Pennica et al., 1984), as well as its salts, functional
derivatives, variants, analogs and active fragments. The use of human TNF is preferred
in accordance with the present invention.
In a preferred embodiment, Tumor Necrosis Factor (TNF)
alpha is an isoform, mutein, fused protein, functional derivative, active fraction
or salt thereof.
In the following, TNF and IFN, may also be referred to
as "substance(s) of the invention".
As used herein the term "muteins" refers to analogs of
a substance according to the invention, in which one or more of the amino acid residues
of a natural substance of the invention are replaced by different amino acid residues,
or are deleted, or one or more amino acid residues are added to the natural sequence
of substance of the invention, without changing considerably the activity of the
resulting products as compared to the wild type substance of the invention. 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 a substance of the invention, such as to have
substantially similar or even better activity to a substance of the invention. The
biological function of interferon and TNF are well known to the person skilled in
the art, and biological standards are established and available e.g. from the National
Institute for Biological Standards and Control (http://immunology.org/links/NIBSC).
Bioassays for the determination of IFN or TNF activity
have been described. An IFN assay may for example be carried out as described by
Rubinstein et al., 1981. The cytotoxic activity of TNF can be measured according
to Flick and Gifford, 1984, for instance. The effect of a TBP may e.g. be tested
as described in
EP 308 378
EP 398 327
. Thus, it can be determined whether any given mutein has substantially
a similar, or even a better, activity than TNF or IFN by means of routine experimentation.
Muteins of a substance of the invention, 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
Preferred changes for muteins in accordance with the present
invention are what are known as "conservative" substitutions. Conservative amino
acid substitutions of polypeptides or proteins of the invention, 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. 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 I. More preferably, the synonymous amino acid groups are those
defined in Table II; and most preferably the synonymous amino acid groups are those
defined in Table III.
Preferred Groups of Synonymous Amino Acids
Ser, Thr, Gly, Asn
Arg, Gln, Lys, Glu, His
Ile, Phe, Tyr, Met, Val, Leu
Gly, Ala, Thr, Pro
Pro, Ser, Ala, Gly, His, Gln, Thr
Gly, Thr, Pro, Ala
Met, Tyr, Phe, Ile, Leu, Val
Ala, Thr, Pro, Ser, Gly
Met, Tyr, Phe, Val, Leu, Ile
Trp, Met, Tyr, Ile, Val, Leu, Phe
Trp, Met, Phe, lie, Val, Leu, Tyr
Ser, Thr, Cys
Glu, Lys, Gln, Thr, Arg, His
Glu, Lys, Asn, His, Thr, Arg, Gln
Gln, Asp, Ser, Asn
Glu, Gln, His, Arg, Lys
Glu, Asn, Asp
Asp, Lys, Asn, Gln, His, Arg, Glu
Phe, Ile, Val, Leu, Met
More Preferred Groups of Synonymous Amino Acids
His, Lys, Arg
Leu, Ile, Phe, Met
Val, Met, Ile
Ile, Met, Phe, Val, Leu
Met, Tyr, Ile, Leu, Phe
His, Gln, Arg
Glu, Gln, His
Met, Phe, Ile, Val, Leu
Most Preferred Groups of Synonymous Amino Acids
Leu, Ile, Met
Ile, Met, Leu
Met, Ile, Leu
Examples of production of amino acid substitutions in proteins
which can be used for obtaining muteins a substance of the invention, for use in
the present invention include any known method steps, such as presented in
US patents 4,959,314
4,737,462, to Mark et al
5,116,943 to Koths et al.
4,965,195 to Namen et al
4,879,111 to Chong et al
5,017,691 to Lee et al
; and lysine substituted proteins presented in
US patent No. 4,904,584 (Shaw et al
). Specific muteins of TNF-alpha have been described in
, for instance. Specific muteins of IFN-beta have been described, for example
by Mark et al., 1984.
The term "fused protein" refers to a polypeptide comprising
a substance of the invention, or a mutein thereof, fused to another protein, which
e.g., has an extended residence time in body fluids. A substance of the invention
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 a substance of the invention, 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
a substance of the invention, and do not confer toxic properties on compositions
containing it. These derivatives are polyethylene glycol side-chains, which may
mask antigenic sites and extend the residence of a substance of the invention in
body fluids; or 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 a substance of the invention,
or 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 no significantly reduced activity as compared to the corresponding
substance of the invention.
The term "salts" herein refers to both salts of carboxyl
groups and to acid addition salts of amino groups of the proteins described above
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 the proteins (IFN and TNF, respectively) relevant
to the present invention, i.e., the ability to bind to the corresponding receptor
and initiate receptor signaling.
Demyelinating diseases according to the invention may be
e.g. multiple sclerosis, acute disseminated encephalomyelitis, acute inflammatory
peripheral neuropathies adrenoleukodystrophy and adrenomyeloneuropathy, Leber's
hereditary optic atrophy, or HTLV-associated myelopathy, as described in the introduction.
They may preferably be neuropathies with abnormal myelination. They may concern
the peripheral or the central nervous system.
The most common demyelinating disease is multiple sclerosis.
Therefore, the invention, the combination of a TNF and an interferon is used for
treatment and/or prevention of multiple sclerosis (MS). In accordance with the present
invention, MS may have a chronic progressive disease development. It may also be
relapsing-remitting multiple sclerosis.
In accordance with the present invention, the use of recombinant
human IFN-beta and recombinant human TNF-alpha are especially preferred.
A special kind of interferon variant has been described
recently. The so-called "consensus interferons" are non-naturally occurring variants
of IFN (
). Consensus interferons were shown to be effective in the treatment of
Therefore, in a preferred embodiment of the invention,
TNF is used in combination with a consensus interferon.
As used herein, human interferon consensus (IFN-con) shall
mean a non-naturally-occurring polypeptide, which predominantly includes those amino
acid residues that are common to a subset of IFN-alpha's representative of the majority
of the naturally-occurring human leukocyte interferon subtype sequences and which
includes, at one or more of those positions where there is no amino acid common
to all subtypes, an amino acid which predominantly occurs at that position and in
no event includes any amino acid residue which is not existant in that position
in at least one naturally-occurring subtype. IFN-con encompasses but is not limited
to the amino acid sequences designated IFN-con1, IFN-con2 and IFN-con3 which are
. DNA sequences encoding IFN-con may be produced as described in the above-mentioned
patents, or by other standard methods.
In a further preferred embodiment, the fused protein comprises
an lg fusion. 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 sequence of the substances of the invention 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 facilitated.
In a preferred embodiment, IFN and/or TNF is fused to the
constant region of an lg molecule. Preferably, it is fused to heavy chain regions,
like the CH2 and CH3 domains of human IgG1, for example. Other isoforms of Ig molecules
are also suitable for the generation of fusion proteins according to the present
invention, such as isoforms IgG2 or IgG4, or other lg classes,
like IgM or IgA, for example. Fusion proteins may be monomeric or multimeric, hetero-
The present invention relates to the combined treatment
of a TNF and an IFN. The therapeutic entities could also be liked to each other
in order to be able to administer one single molecule, be it monomeric or multimeric,
instead of two or three separate molecules. A multimeric fusion protein could comprise
a TNF fused to an lg moiety, as well as an IFN fused to an lg moiety. If expressed
together, the resulting fusion protein, which may be linked by disulfide bridges,
for instance, will comprise both TNF and IFN. The compounds of the present invention
may further be linked by any other cross-linking agent or moiety, such as a polyethylene
molecule, for instance.
In a further preferred embodiment, the functional derivative
comprises at least one moiety attached to one or more functional groups, which occur
as one or more side chains on the amino acid residues. The moiety is a polyethylene
(PEG) moiety. PEGylation may be carried out by known methods, such as the ones described
, for example.
Standard dosages of human IFN-beta presently used in the
treatment of relapsing-remitting MS are ranging from 80 000 IU/kg and 200 000 IU/kg
per day or 6 MIU (million international units) and 12 MIU per person per day or
22 to 44 mg per person. In accordance with the present invention, it has been surprisingly
shown that TNF enhances the therapeutic effect of IFN in an established model of
multiple sclerosis. Therefore, in accordance with the present invention, IFN may
be administered at a dosage of about 1 to 50 mg, preferably of about 10 to 30 mg,
more preferably of about 10 to 20 mg per person per day, together with TNF. The
preferred route of administration is subcutaneous administration, administered three
times a week. A further preferred route of administration is the intramuscular administration,
which may be applied once a week.
TNF-alpha therapy has been used in cancer treatment so
far. From studies in cancer patients, it is known that toxicity of r-TNF-alpha treatment
is variable and not always dose-dependent. Hepatic and cardiovascular toxicity have
been generally found to increase with increasing dose, but constitutional symptoms
like fever, chills, or rigors seem not to be dose-related. The maximum tolerated
dose of r-TNF-alpha, administered intravenously over 30 min, is reported to be between
100 and 300 µg/m2 (Feinberg et a., 1988 ; Gamm et al., 1991 ; Schiller
et al., 1991).
Therefore, in a further preferred embodiment, TNF-alpha
is administered in a sub-toxic concentration. More preferably, the sub-toxic concentration
is less than 100 µg/m2, preferably less than 50 µg/m2,
more preferably less than 10 µg/m2, and most preferably less than
The administration of such active ingredients may be by
intravenous, intramuscular or subcutaneous route. The preferred route of administration
for IFN and/or TNF is the subcutaneous route. For TNF, a further preferred route
is the intravenous administration.
Corticosteroids are therapeutically efficacious in the
treatment of demyelinating diseases. Therefore, the medicament of the invention
may further comprise a corticosteroid, for simultaneous, sequential, or separate
use. As corticosteroid treatment, oral prednisone 60 to 100 mg/day tapered over
2 to 3 weeks or IV methylprednisolone 500 to 1000 mg/day for 3 to 5 days may be
administered, for instance.
Glatiramer is a synthetic co-polymer with similarities
to myelin basic protein and is administered by daily subcutaneous injection. It
has also been proved to have a therapeutic effect in multiple scleorsis. In a preferred
embodiment of the invention, the medicament further comprises glatiramer, for sequential,
separate or simultaneous use.
The invention further relates to a pharmaceutical composition
comprising an agent having, stimulating or maintaining TNF activity, in combination
an IFN, in the presence of one or more pharmaceutically acceptable excipients. Preferably,
the pharmaceutical composition of the invention may comprise a TNF, in combination
with an Interferon.
The pharmaceutical composition of the invention may further
comprise a corticosteroid and/or glatiramer.
The term "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 solution.
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, epidural, 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 and vehicles.
The subcutaneous route is preferred in accordance with
the present invention.
Another possibility of carrying out the present invention
is to activate endogenously the genes for the compounds of the invention, i.e. TNF
and/or IFN. In this case, a vector for inducing and/or enhancing the endogenous
production of TNF and/or IFN in a cell normally silent for expression of TNF and/or
IFN, or which expresses amounts of TNF and/or IFN which are not sufficient, are
is used for treatment of a demyelinating disease. The vector may comprise regulatory
sequences functional in the cells desired to express TNF and/or IFN. Such regulatory
sequences 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" (E.G.A), and it is described e.g. in
The invention further relates to the use of a cell that
has been genetically modified to produce IFN and/or TNF in the manufacture of a
medicament for the treatment and/or prevention of neurological diseases.
For parenteral (e.g. intravenous, subcutaneous, intramuscular)
administration, the active protein(s) can be formulated as a solution, suspension,
emulsion or lyophilised 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 WO 92/13095
The dosage administered, as single or multiple doses, to
an individual will vary depending upon a variety of factors, including 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.
The substances of the invention may be administered daily
or every other day, of less frequent. Preferably, one or more of the substances
of the invention are administered one, twice or three times per week.
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 substances of the invention
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. Active agents that are administered
simultaneously with other therapeutic agents can be administered in the same or
The invention further relates to a method of treatment
and/or prevention of a demyelinating disease comprising administering to a host
in need thereof a therapeutically effective amount of an agent having, stimulating
or maintaining TNF activity, and a therapeutically effective amount of an interferon.
Reference to known method steps, conventional methods steps,
known methods or conventional methods is not any way an admission that any aspect,
description or embodiment of the present invention is disclosed, taught or suggested
in the relevant art.
The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others can, by applying
knowledge within the skill of the art (including the contents of the references
cited herein), readily modify and/or adapt for various application such specific
embodiments, without undue experimentation, without departing from the general concept
of the present invention.
Having now described the invention, it will be more readily
understood by reference to the following examples that are provided by way of illustration.
Effect of TNF-alpha alone, or in combination with IFN-beta, in an in vivo model
of Multiple Sclerosis
The effect of TNF-alpha, either alone or in combination
with IFN-beta, on disease development was assayed using an established animal model
of multiple sclerosis (MS). The experimental autoimmune encephalomyelitis (EAE)
model is a murine chronic demyelinating model.
EAE induction protocol
Experimental autoimmune encephalomyelitis (EAE) was induced
in groups of mice as follows: groups of C57black6/J female mice were immunized subcutaneously
into the right flank at day 0 with 200 µl emulsion, containing 200 µg
of a synthetic peptide corresponding to Myelin Oligodendrocyte Glycoprotein (MOG
35-55 (Neosystem, Strasbourg, France) in Complete Freund's Adjuvant containing 5
mg/ml of H37RA Mycobaterium tuberculosis.
Immediately, and at day 2, the animals received an intraperitoneal
injection of 500 ng pertussis toxin dissolved in 400 µl of pertussis buffer
(0.5 M NaCl, 0.015 M Tris pH 7.5, 0.017% Triton X-100). At day 7, the animals received
a boost of identical amount (200 µl) of emulsion, containing 200 µg MOG35-55
peptide in Complete Freund's Adjuvant, into the left flank.
Treatment with all the drugs was started individually in
each animal when reaching a clinical score ≥ 1. These animals were treated
daily with 200 µl of either:
- 1) phosphate buffered saline (PBS), administered s.c. (subcutaneous) and i.v.
- 2) murine mIFN-beta at 20,000 U/mouse, administered s.c.;
- 3) murine mIFN-beta at 5,000 U/mouse, administered s.c.;
- 4) r-mTNFa at 0.1 µg/mouse, administered i.v. or i.p.
- 5) combined r-mTNF-alpha (0.1 µg/mouse, i.v. or i.p., respectively) and
mIFNbeta (5,000 U/mouse, s.c.).
Two routes of administration were chosen for TNF-alpha:
The i.v. route in was chosen on the basis of results published by
Liu et al. (Nature Medicine 1998 4: 78-83
). The i.p. route was chosen to overcome the toxicity problems met during
studies with higher doses of TNF-alpha.
PBS was used as vehicle and all substances were injected
subcutaneously in the neck, whereby the group treated with two substances was injected
twice into two different sites. Animals were scored daily for neurological signs
according to the scale indicated below. Weight loss and clinical score of individual
animals were monitored daily up to day 35 after disease onset, using international
standard of scoring by following criteria:
0 = no signs of disease
1 = tail weakness or paralysis
2 = tail paralysis + hindlimb(s) weakness or partial paralysis
3 = tail paralysis + complete hindlimb paralysis
3.5 = tail paralysis + hindlimb paralysis + incontinence
4 = tail paralysis + hindlimbs paralysis + weakness or paralysis of forelimbs
5 = moribund
Differences among experimental groups in the time-course
of clinical score were analyzed by Kruskal-Wallis test followed, in case of significance,
by the the pairwise Wilcoxon test, at each measurement time.
At the end of the treatment period, each animal was anesthetised
with an i.p. injection of sodium pentobarbital (about 50 mg/kg) and, after blood
sampling, transcardially perfused-fixed with 4% paraformaldehyde via the left ventricle.
Fixed spinal cords were carefully dissected out. Spinal cord slices (10 to 12 slices
per animal) were embedded in paraffin blocks, sectioned and stained with hematoxylin
and eosin for evaluation of inflammatory signs and with Kluver-PAS staining (Luxol
Fast Blue plus Periodic Acid Schiff stainings) for detection of demyelination. In
the spinal cord, the total area of all slices was measured for each animal as points
of intersection of a 10x10 grid at a magnification of 0.4x0.4 mm per grid. The perivascular
inflammatory infiltrates (PII) were counted in each slice in order to obtain a total
value for each animal and evaluated as number of infiltrates per mm2.
Demyelination are as were measured for each animal as points of intersection of
10x10 grid at a magnification of 0.1x0.1 mm per grid and were expressed as a percentage
of total demyelination (Dem) area over the total area of the slices. Differences
among experimental groups were assessed by one-way ANOVA followed by Newman-Keuls
In experiments aimed at finding the final study protocol,
the first animals became sick when treated with r-mTNF-alpha at doses of 1 and 10
µg/mouse i.v. every second day. Since after one or two administrations, all
treated animals died, it was decided to reduce the doses to 0.1 µg/mouse and
to administer the compound by i.p and i.v. routes in two experimental groups, respectively.
The following study protocol was applied:
Treatment period (Days)
Every second day
Every second day
Every second day
Fig. 1 depicts the result of a positive control experiment,
wherein 20,000 IU interferon-beta/mouse were administered subcutaneously every day.
As compared to the administration of vehicle only, and as expected, IFN-beta showed
a beneficial effect on the development of the disease from day 12 on, as shown by
a marked reduction in clinical score (open squares).
The effects of an intravenous administration of a four
times lower amount of IFN-beta, i.e. of 5,000 IU/mouse, are shown in Figs. 2 and
3. Two ways of evaluating the results were used: either a clinical score of 5 was
assigned to dead animals from the day of death until the end of experiment, the
outcome of this way of data evaluation are depicted in Fig. 2. In the second way
of data evaluation the animals were scored until their death and then dropped out
completely. The results are shown in Fig. 3.
Figs. 2/3 A show the mortality rate in this series of experiments.
As expected in this animal model, the mortality rate was variable. However, a strikingly
low mortality rate was achieved in those animals receiving administration of both
TNF-alpha and IFN-beta.
Figs. 2/3 B shows the development of clinical score over
the experimental period of 35 days. The administration of 5,000 IU/mouse s.c. every
day did not result in any amelioration of the clinical score (open diamonds).An
administration of 0.1 µg/mouse of TNF-alpha intravenously (Fig. 2 B, filled
triangles) resulted in a slight improvement of clinical score as compared to vehicle,
in particular at later stages of the disease (Fig. 3 B). However, this improvement
was not statistically significant, especially given the high mortality rate of mice
which received TNF-alpha alone.
Combined administration of TNF-alpha (i.v.) and IFN-beta
(s.c.) resulted in a significant improvement (at interval days 6-14 and 19-35, Fig.
2 B, and from day 6 through day 35, Fig. 3 B) of clinical scores during the test
period of 35 days (open triangles).
The histological analysis is depicted in Figs. 2/3 C. The
histology of the spinal cord showed that treatment with IFN-beta alone, and the
combination of TNF-alpha and IFN-beta in combination, resulted in a reduced extent
of inflammation (p<0.01 versus the vehicle-treated group, only for the
combined treatment) and demyelination.
Figs. 4/5 depict the experiment in which 5,000 U/mouse
of IFN-beta s.c. and 0.1 µg/mouse of TNF-alpha i.p. were administered either
alone, or in combination. Fig. 4 illustrates the data analysis counting dead animals
as clinical score 5, Fig. 5 shows the analysis of the same experiment, except that
dead animals were not scored at all anymore.
Administration of IFN-beta in combination with TNF-alpha
(open squares) resulted in a slight improvement of the clinical score (with significant
improvements in the interval days 8-9, Fig. 4 B, and, days 4-15, 16 and 21-35, Fig.
5 B), i.e. the disease development, as compared to the administration of vehicle
only. The beneficial effect of the combined treatment could also be observed in
the histological analysis of the spinal cords (Figs. 4/5 C), showing in particular
a significantly reduced extent of inflammation (p<0.01). Taken together, TNF-alpha
seems to have a lower effect when administered intraperitoneally as compared to
the intravenous route.
Administration of sub-therapeutic amounts of IFN-beta,
in combination with sub-toxic amounts of TNF-alpha, produced a remarkable and long-lasting
improvement of the disease, as expressed by reduced clinical scores as well as reduced
inflammation and demyelination in the spinal cords of the animals. Therefore, the
results presented above show a clear beneficial effect of treatment with a combination
of TNF-alpha and IFN-beta, reducing clinical signs of chronic EAE in mice after
immunization with MOG. Therefore, TNF enhances the therapeutic effect of interferon
in multiple sclerosis. Thus, a combined treatment with TNF and IFN is suggested
for treatment of demyelinating diseases such as multiple sclerosis.
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