Note: Descriptions are shown in the official language in which they were submitted.
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ANTAGONIST ANTI-CD40 MONOCLONAL ANTIBODY THERAPY
FOR MULTIPLE SCLEROSIS TREATMENT
TECHNICAL FIELD
The invention relates to treatment of B and T cell mediated autoimmune
disease in humans using antibodies to CD40 alone or in combination with an
immunomodulating agent such as interferon-j3-lb.
BACKGROUND OF THE INVENTION
Multiple sclerosis (MS) is one of several diseases believed to result from an
immunoregulatory defect. It results in a chronic, disabling condition of the
central
nervous system, and symptoms can range from mild numbness to paralysis and
loss of
vision. Most patients are diagnosed between the ages of 20 and 40, and
therefore face
a lifetime of symptoms and treatment. The progress of the disease cannot be
predicted, and the treatment in one patient may not be predictive for
treatment of
another patient.
Histologically, the symptoms are associated with destruction of myelin, the
fatty
sheath surrounding nerve fibers. As a result of the damage, the nerve impulses
are
slowed or stopped, producing physical symptoms such as muscle weakness,
tremor,
vision problems, lack of balance, pain, and fatigue, and psychological changes
including mood swings, forgetfulness, and difficulty in concentrating.
Although
existing therapies can alleviate some or all of the symptoms in a given
patient, the
therapy must be carefully monitored and can be contra-indicated if the
symptoms
worsen or the patient experiences a relapse.
At present, 250,000 to 350,000 people in the U.S. are believed to be living
with MS. Because it strikes during a person's most active years, it takes a
huge toll
on the quality of life and productivity of both the pati ent and the family.
Although the
early onset of symptoms is the most common pattern, almost 10% of patients
experience the first symptoms after age 50. In this age group, diagnosis of
often
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complicated by the presence of other conditions, leading to some degree of
misdiagno sis.
The most common treatment for MS at the present time is (3-interferon
(IFN-(3), which exerts its effect through modulation of the immune system.
Specifically, (3-interferon binds to receptors of immune system cells.
However,
(3-interferon only temporarily slows progression of disease in a fraction of
patients.
There is a need in the art for therapeutic compositions such as anti-CD40
antibodies that, alone or in combination with interferon, slow the progression
of disease
in MS patients.
SUMMARY OF THE INVENTION
It is an object of this invention to provide a method far preventing or
treating
an autoimmune disease in a patient, the method comprising administering to a
patient
in need of such treatment a therapeutically effective amount of a human
monoclonal
antibody capable of binding to a human CD40 antigen located on the surface of
a
CD40-bearing cell such as a human B cell or other antigen presenting cell,
wherein
the binding of the antibody to the CD40 antigen prevents the priming of
pathogenic
T cells, alone or in combination with or co-administered with interferon in a
pharmaceutically acceptable excipient.
It is another object of this invention to provide a method for preventing or
treating multiple sclerosis in a patient, the method comprising administering
to a
patient in need of such treatment a therapeutically effective amount of a
human
monoclonal antibody capable of binding to a human CD40 antigen located on the
surface of a human antigen presenting cell, wherein the binding of the
antibody to the
CD40 antigen prevents the priming of pathogenic T cells, alone or in
combination
with or co-administered with interferon. It is a further object of the
invention to
provide a composition for administration to a patient in order to prevent or
treat a B
cell mediated disease, wherein the composition comprises an anti-CD40 antibody
and
a type 1 interferon, in a pharmaceutically acceptable excipient.
In more preferred embodiments of the above objects, the monoclonal antibody
is 15B8, 20C4, 13E4, 12D9, or 9F7. The monoclonal antibody, 15B~, is a human
anti-CD40 monoclonal antibody.
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In other preferred embodiments of the above objects, the interferon is a type
1
interferon. In other preferred embodiments, the type 1 interferon is
interferon-a,
interferon-(3, interferon-~, interferon-cu (interferon-a IIl) or interferon-i
(trophoblast
interferon). In other preferred embodiments of the above objects, the
interferon is
interferon-~3-lb. In other preferred embodiments of the above objects, the
interferon
is interferon-(3-la.
In specific embodiments of the invention, the interferon and antibody
compositions are administered to a tissue of the mammal innervated by the
trigeminal
nerve, the olfactory nerve, or a combination thereof, wherein the interferon
is
absorbed through the tissue and transported to the central nervous system of
the
mammal. In other preferred embodiments of the above objects the interferon and
antibody compositions are administered to a tissue of the mammal by
intravenous
injection. In other preferred embodiments of the above objects the interferon
and
antibody compositions are administered to a tissue of the mammal by
subcutaneous or
intramuscular injection.
The invention provides a method for preventing or alleviating a B cell or
other
antigen presenting cell mediated disease of the central nervous system in a
mammal
wherein said disease is responsive to treatment with interferon, comprising:
administering a composition comprising an interferon to a tissue of the mammal
innervated by the trigeminal nerve, the olfactory nerve, or a combination
thereof,
wherein the interferon is absorbed through the tissue and transported to the
central
nervous system of the mammal; and administering a composition comprising an
anti-
CD40 antibody to said mammal.
In certain embodiments of the method, the tissue comprises a nasal cavity
tissue, a conjunctiva, an oral tissue, or a skin. In other embodiments of the
method,
administering the interferon to the conjunctiva comprises administering the
interferon
between a lower eyelid and an eye.
In further embodiments of the method, administering the interferon to the skin
comprises administering the interferon to a face, a forehead, an upper eyelid,
a lower
eyelid, a dorsum of the nose, a side of the nose, an upper lip, a cheek, a
chin, a scalp,
or a combination thereof.
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In a specific embodiment of the method, administering the interferon to the
oral tissue comprises sublingual administration.
In other embodiments of the method, the interferon is selected from the group
consisting of interferon-alpha (IFN-a), interferon-beta (IFN-[3), and
biologically
active variants thereof, and the anti-CD40 antibody is a monoclonal antibody.
In specific embodiments, the interferon is native human IFN-(3 or a
biologically active variant thereof. In other specific embodiments, the IFN-(3
variant
comprises an amino acid sequence having at least 70% sequence identity to
native
human IFN-(3. In yet a further specific embodiment, the interferon is
administered to
an upper one third of a nasal cavity.
In other embodiments of the method, the interferon is transported to a
cerebellum, a superior colliculus, a periventricular white matter, an optic
nerve, a
midbrain, a pons, an olfactory bulb, an anterior olfactory nucleus, or any
combination
thereof, or to a spinal cord, a brain stem, a cortical structure, a
subcortical structure, or
any combination thereof.
In certain embodiments, the interferon is administered in a dosage range of
about 0.14 nmol/kg of brain weight to about 138 nmol/kg of brain weight. In
specific
embodiments, the interferon is native human IFN-[3 or a biologically active
variant
thereof.
The invention provides a method of preventing or alleviating multiple
sclerosis.
The invention further provides a method of prolonging the effectiveness of
interferon treatment of multiple sclerosis in a human, comprising
administering to the
human a composition comprising an anti-CD40 antibody. In certain embodiments
of
this method, the antibody is selected from the group consisting of 15B8, 20C4,
13E4,
12D9, 9F7 and 5D12.
DETAILED DESCRIPTION OF THE INVENTION
The CD40 antigen is a glycoprotein expressed on the cell surface of B cells
and other antigen-presenting cells, including dendritic cells. During B-cell
differentiation, the molecule is first expressed on pre-B cells and then
disappears from
the cell surface when the B cell becomes a plasma cell. Crosslinking of the
CD40
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molecules with anti-CD40 antibodies mediates a variety of effects on B cells.
The
CD40 antigen is known to be related to the human nerve growth factor (NGF)
receptor and tumor necrosis factor-a (TNF-a) receptor, suggesting that CD40 is
a
receptor for a ligand with important functions in B-cell activation.
CD40 is a key element of immune responses. Engagement of CD40 on
antigen-presenting cells by its ligand, termed CD40L or CD 154, causes
production of
cytokines and up-regulation of co-stimulatory molecules leading to efficient
activation
of T lymphocytes. Engagement of CD40 on B lymphocytes provides a co-
stimulatory
signal to the B cell that drives antibody production. Thus, blocking of CD40
engagement and subsequent T cell activation has the potential to suppress
antibody
and cell mediated immune responses.
CD40L is a member of the TNF family. It is expressed on activated T cells
and B cells, and on endothelial cells, mast cells, eosinophils and basophils.
CD40L is
expressed as both membrane-bound and soluble-secreted forms. Several functions
of
CD40 and CD40L interaction have been identified. In B cells, the interaction
modulates clonal expansion, Ig production, germinal center formation, isotype
switching, induction and maintenance of memory, and affinity maturation. In T
cells,
the interaction modulates activation of T helper cells and cytotoxic T
lymphocytes
(CTLs). In macrophages, the interaction modulates stimulation of co-
stimulatory
functions,. induction of cytokine production, co-stimulation of nitrous oxide
(NO)
generation, induction of metalloproteinase production and rescue from
apoptosis. In
dendritic cells, the interaction modulates maturation, induction of cytokine
production,
antigen-presenting and co-stimulatory function.
The role of CD40-CD40L interaction has been partly elucidated by studying
functional defects of CD40L-deficient humans and mice, and CD40-deficient
mice.
Several effects are seen, including increased susceptibility to intracellular
parasites;
reduced production of IFNy and IL-12; impaired T cell priming to peptide
antigens;
reduced follicular dendritic cell network; absence of germinal center and
impaired Ig
isotype switching to T cell dependent antigens. However, there is normal
antibody
response to T cell independent antigens. Overall, the effects are mediated
through the
impaired immune system. No defect has been identified in other systems. An
anti-
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HuCD40 blocking antibody has been developed, the mouse monoclonal antibody
(SD 12). The antibody inhibits ih vitro MLR and B cell activation and Ig
production.
Several effects of SD12 have been studied in vita°o. First, SD12
causes
inhibition of IgM secretion by human peripheral blood B cells stimulated with
syngeneic human activated T cells. The antibody also causes inhibition of
human
peripheral B cell proliferation stimulated by anti-CD3 activated-Jurkat cells
or CHO-
CD40L cells. Inhibition of cytokine secretions stimulated through CD40 in DCs
or
monocytes was also observed using SD12. Earlier studies provided preliminary
evidence of a role of CD40-CD40L interaction in experimental allergic
encephalomyelitis (EAE) and multiple sclerosis. Gerritse et al.,
(Pt°oc. Natl. Acad.
Sci., 93:2499-2504, 1996) showed that T cells expressing CD40L were present in
MS
brain sections. CD40L+ cells co-localized with CD40-bearing cells (macrophages
or
monocytes) in active lesions (perivascular infiltrates). Anti-CD40L antibody
has been
shown to prevent or reduce EAE in mice. Laman (J. Neuroimmunology X6:30-45,
1998) observed abundant expression of CD40 in perivascular infiltrates of
macrophages in marmoset monkeys with acute EAE.
The present invention relates to a method of treating multiple sclerosis using
anti-CD40 antibodies alone or in combination with a type 1 interferon, for
example,
interferon-~3. Patients with continuing worsening of their disease or
significant side
effects frequently discontinue therapy with interferon-(3. According to the
invention, a
CD401CD40L blockade may have synergistic anti-inflammatory or
immunomodulatory effects with interferon-(3, and may reduce the incidence of
auto-
antibodies (anti-thyroid, anti-hepatic, etc.). Therefore, co-administration of
an anti-
CD40 antibody such as 15B8 with an interferon-/3, such as interferon-/3-lb,
may
reduce the rate of interferon-(3-lb discontinuations.
One target patient population includes all patients currently undergoing
treatment with interferon-(3-lb (for treatment of any form of MS); patients
who have
experienced at least one relapse in the previous six months on interferon-(3-
lb;
patients with at least one enhancing lesion at baseline MRI scan; and patients
with
disease progression in the past six months.
A second target patient population includes all patients newly diagnosed with
MS or with clinically isolated syndromes suggestive of MS. These patients will
be
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treated with anti-CD40 antibody, such as 15B8, co-administered with interferon-
J3-lb.
According to the invention, a CD401CD40L blockade may have synergistic anti-
inflammatory or immunomodulatory effects with interferon-(i. Co-administration
of
anti-CD40 antibody with interferon-(3-lb may prolong time to establishment of
definite MS or result in increased efficacy for treatment of MS compared to
administration of interferon-(3-lb alone.
Another target patient population includes all patients diagnosed with
rheumatoid arthritis or other autoimmune disease. These patients will be
treated with
anti-CD40 antibody, such as 15B8, alone or co-administered with interferon-[3-
lb.
Another target patient population includes all patients diagnosed with central
nervous system (CNS) tumors or gliomas, for example, astrocytomas,
ependymomas,
oligodendrogliomas, and tumors with mixtures of two or more of these cell
types.
These patients will be treated with anti-CD40 antibody, such as 15B8,
co-administered with interferon-(3-lb.
Another target patient population includes all patients diagnosed with Crohn's
disease, Crohn's colitis or chronic ulcerative colitis. These patients will be
treated
with anti-CD40 antibody, such as 15B8, alone or co-administered with
interferon-(3-1 b.
Another target patient population includes all patients diagnosed with
idiopathic pulmonary fibrosis, which may occur as a result of viral infection
or
autoimmune disease. These patients will be treated with anti-CD40 antibody,
such as
15B8, alone or co-administered with interferon-(3-lb.
Other target populations include those undergoing treatment with other
a
interferons. Interferons (IFNs) are a family of molecules encompassing over 20
different proteins and are members of the cytokine family that induce
antiviral,
antiproliferative, antitumor, and/or cytokine effects. IFNs are relatively
small,
species-specific, single chain polypeptides, which are produced in response to
a
variety of inducers, such as mitogens, polypeptides, viruses, and the like. In
humans,
IFNs are produced, for example, as type I interferon (-a, -[3, -cu, or -i) or
type 2
interferon (-y). Synthetic interferons are also known in the art. See, for
example, U.S.
Patent No. 6,114,145, herein incorporated by reference. According to the ' 145
patent,
a synthetic interferon polypeptide has the amino acid sequence of SEQ ID N0:2
and
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in one embodiment is encoded by a polynucleotide of SEQ ID NO:1. Upon
secretion
from mammalian cells, interferon molecules bind to a receptor on the surface
of a
target cell and elicit a chain of events, which can alter the amount and
activity of
protein in the target cell. Such alterations can include, for example, changes
in gene
transcription or enzymatic activity.
Patients undergoing treatment with biologically active variants of interferon
may also be suitable target populations. These variants retain the biological
activity
of the interferon, for example, IFN-a and IFN-(3 variants known in the art and
as
discussed below, and the variants retain the ability to bind their respective
receptor
sites. Such activity may be measured using standard bioassays. Representative
assays detecting the ability of the variant to interact with an interferon
receptor type I
can be found in, for example, U.S. Patent No. 5,766,864, herein incorporated
by
reference. Preferably, the variant has at least the same activity as the
native molecule.
Suitable biologically active variants can be fragments, analogues, and
derivatives of the interferon polypeptides. By "fragment" is intended a
protein
consisting of only a part of the intact interferon polypeptide sequence. The
fragment
can be a C-terminal deletion or N-terminal deletion of the interferon
polypeptide. By
"analogue" is intended either the full length polypeptide or a fragment
thereof,
wherein the analogue comprises a native polypeptide sequence having one or
more
amino acid substitutions, insertions, or deletions. Peptides having one or
more
peptoids (peptide mimics) are also encompassed by the term analogue (see i.e.,
International Publication No. WO 91/04282). By "derivative" is intended any
suitable
modification of the native polypeptide or fragments thereof, or their
respective
analogues, such as glycosylation, phosphorylation, or other addition of
foreign
moieties, so long as the activity is retained.
Preferably, naturally or non-naturally occurring variants of an interferon
have
amino acid sequences that are at least 70%, preferably 80%, more preferably,
85%,
90%, 91%, 92%, 93%, 94% or 95% identical to the amino acid sequence to the
reference molecule, for example, the native human interferon, or to a shorter
portion
of the reference interferon molecule. More preferably, the molecules are 96%,
97%,
98% or 99% identical. Percent sequence identity is determined using the Smith-
Waterman homology search algorithm using an afFne gap search with a gap open
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penalty of 12 and a gap extension penalty ,of 2, BLOSLJM matrix of 62. The
Smith-
Waterman homology search algorithm is taught in Smith and Waterman, Adu Appl.
Math. 2:482-489, 1981. A variant may, for example, differ by as few as 1 to 10
amino
acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino
acid
residue.
With respect to optimal alignment of two amino acid sequences, the
contiguous segment of the variant .amino acid sequence may have additional
amino
acid residues or deleted amino acid residues with respect to the reference
amino acid
sequence. The contiguous segment used for comparison to the reference amino
acid
sequence will include at least 20 contiguous amino acid residues, and may be
30, 40,
50, or more amino acid residues. Corrections for sequence identity associated
with
conservative residue substitutions or gaps can be made (see Smith-Waterman
homology search algorithm).
The art provides substantial guidance regarding the preparation and use of
such variants, as discussed fmrther below. A fragment of an interferon
polypeptide
will generally include at least about 10 contiguous amino acid residues of the
full-
length molecule, preferably about 15-25 contiguous amino acid residues of the
full-
length molecule, and most preferably about 20-50 or more contiguous amino acid
residues of full-length cytokine polypeptide.
For example, conservative amino acid substitutions may be made at one or
more predicted, preferably nonessential amino acid residues. A "nonessential"
amino
acid residue is a residue that can be altered from the wild-type sequence of
an
interferon (i.e., IFN-a or IFN-(3) without altering its biological activity,
whereas an
"essential" amino acid residue is required for biological activity. A
"conservative
amino acid substitution" is one in which the amino acid residue is replaced
with an
amino acid residue having a similar side chain. Families of amino acid
residues
having similar side chains have been defined in the art. These families
include amino
acids with basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-
branched side chains (e.g., threonine, valine, isoleucine), and aromatic side
chains
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(e.g., tyrosine, phenylalanine, tryptophan, histidine). Such substitutions
would not be
made for conserved amino acid residues, or for amino acid residues residing
within a
conserved motif.
Alternatively, variant interferon nucleotide sequences can be made by
introducing mutations randomly along all or part of a cytokine coding
sequence, such
as by saturation mutagenesis, and the resultant mutants can be screened for
cytokine
biological activity to identify mutants that retain activity. Following
mutagenesis, the
encoded protein can be expressed by recombinant means in bacteria, yeast,
insect
cells or mammalian cells.
Alternatively, the interferon can be synthesized chemically, by any of several
techniques that are known to those skilled in the peptide art. See, for
example, Li et
al., Proe. Natl. Acad. Sci. USA 80:2216-2220, 1983, Steward and Young, Solid
Phase
Peptide Synthesis (Pierce Chemical Company, Rockford, Illinois), 1984, and
Baraney
and Merrifield, The Peptides: Analysis, Syv~thesis, Biology, ed. Gross and
Meinhofer,
Vol. 2 (Academic Press, New York, 1980), pp. 3-254, discussing solid-phase
peptide
synthesis techniques; and Bodansky, P~ihciples of Peptide Synthesis (Springer-
Verlag,
Berlin, 1984) and Gross and Meinhofer, eds., The Peptides: Analysis,
Synthesis,
Biology, Vol. 1 (Academic Press, New York, 1980), discussing classical
solution
synthesis. The interferon can also be chemically prepared by the method of
simultaneous multiple peptide synthesis. See, for example, Houghten,
Ps°oe. Natl.
Acad. Sci. USA 82:5131-5135, 1984; and U.S. Patent No. 4,631,211.
The interferon used in the methods of the invention can be from any animal
species including, but not limited to, avian, canine, bovine, porcine, equine,
and
human. Preferably, the interferon is from a mammalian species when the
interferon is
to be used in treatment of a disorder of the CNS, brain or spinal cord, such
as MS, and
more preferably is from a mammal of the same species as the mammal undergoing
treatment for such a disorder.
Interferon-(3
The term "IFN-[3" as used herein refers to mature native human (3-interferon
or
any biologically active variants thereof, which are sometimes referred to in
the art as
IFN-a-like polypeptides, see, e.g., U.S. Patent No. 4,462,940. Human native
IFN-~ or
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variants, which may be naturally occurring (e.g., allelic variants that occur
at the IFN-(3
locus) or recombinantly produced, have amino acid sequences that are similar
to, or
substantially similar to the mature native IFN-(3 sequence. DNA sequences
encoding
native human IFN-(3 are available in the art. See, for example, Goeddel et
al., Nucleic
Acid Res. 8:4057, 1980 and Tanigachi et al., Pf~oc. Japan Acad. Sci. 855:464,
1979.
Fragments of IFN-(3 or truncated forms of IFN-(3 that retain their activity
are also
encompassed. These biologically active fragments or truncated forms of IFN-(3
are
generated by removing amino acid residues from the full-length IFN-[3 amino
acid
sequence using recombinant DNA techiuques well known in the art. IFN-[3
polypeptides may be glycosylated or unglycosylated, as both the glycosylated
and
unglycosylated forms of IFN-(3 show qualitatively similar specific activities
and,
therefore, the glycosyl moieties are not involved in and do not contribute to
the
biological activity of IFN-j3.
The IFN-(3 variants encompassed herein include muteins of the native mature
IFN-(3 sequence, wherein one or more cysteine residues that are not essential
to
biological activity have been deliberately deleted or replaced with other
amino acids
(see, e.g., U.S. Patent No. 4,588,585 and EP218825, herein incorporated by
reference)
to eliminate sites for either intermolecular cross-linking or incorrect
intramolecular
disulfide bond formation. IFN-(i variants of this type include those
containing a
glycine, valine, alanine, leucine, isoleucine, tyrosine, phenylalanine,
histidine,
tryptophan, serine, threonine, or methionine substituted for the cysteine
found at
amino acid 17 of the mature native amino acid sequence. S'erine and threonine
are the
more preferred replacements because of their chemical analogy to cysteine.
Serine
substitutions are most preferred. Thus, IFN-~ variants with one or more
mutations
that improve, for example, their pharmaceutical utility are also encompassed
by the
present invention.
Additional changes can be introduced by mutation into the nucleotide
sequences encoding IFN-(i, thereby leading to changes in the IFN-(3 amino acid
sequence, without altering the biological activity of the interferon. Thus, an
isolated
nucleic acid molecule encoding an IFN-(3 variant having a sequence that
differs from
human IFN-j3 can be created by introducing one or more nucleotide
substitutions,
additions, or deletions into the corresponding nucleotide sequence disclosed
herein,
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such that one or more amino acid substitutions, additions or deletions are
introduced
into the encoded IFN-(3. Mutations can be introduced by standard techniques,
such as
site-directed mutagenesis and PCR-mediated mutagenesis. Such IFN-(3 variants
can
also be employed in the present invention.
Biologically active IFN-(3 variants encompassed by the invention also include
IFN-(3 polypeptides that are covalently linked with, for example, polyethylene
glycol
(PEG) or albumin.
Biologically active variants of IFN-(3 encompassed by the invention should
retain IFN-(i activities, particularly the ability to bind to 1FN-(3 receptors
or retain
immunomodulatory or anti-viral activities. In some embodiments, the IFN-(3
variant
retains at least about 25%, preferably about 50%, and more preferably about
75% or
more of the biological activity of the native IFN-[3 polypeptide. IFN-~i
variants whose
activity is increased in comparison with the activity of the native IFN-[3
polypeptide are
also encompassed. The biological activity of IFN-(3 variants can be measured
by any
method known in the art. Examples of such assays can be found in Fellous et
al., Proc.
Natl. Acad. Sci USA 79:3082-3086, 1982; Czerniecki et al., J. ~rol. 49(2):490-
496,
1984; Mark et al., Proc. Natl Acad. Sci. USA 81:5662-5666, 1984; Branca et
al., Nature
277:221-223, 1981; Williams et al., Nature 282:582-586, 1979; Herberman et
al.,
Nature 277:221-223, 1979; and Anderson et al., J. Biol. them. 257(19):11301-
11304,
1982.
Non-limiting examples of IFN-~3 polypeptides and IFN-(3 variant polypeptides
encompassed by the invention are set forth in Nagata et al., Nature 284:316-
320,
1980; Goeddel et al., Nature 287:411-416, 1980; Yelverton et al., Nucleic
Acids Res.
9:731-741, 1981; Streuli et al., Proc. Natl. Acad. Sci. U.SA. 78:2848-2852,
1981;
EP028033B1, and EP109748B1. See also U.S. Patent Nos. 4,518,584; 4,569,908;
4,588,585; 4,738,844; 4,753,795; 4,769,233; 4,793,995; 4,914,033; 4,959,314;
5,545,723; and 5,814,485. These disclosures are herein incorporated by
reference.
These citations also provide guidance regarding residues and regions of the
IFN-(3
polypeptide that can be altered without the loss of biological activity.
In one embodiment of the present invention, the IFN-(3 used in the methods of
the invention is the mature native human IFN-(3 polypeptide. In another
embodiment,
the IFN-J3 is the mature IFN-(i C17S polypeptide. However, the present
invention
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encompasses other embodiments where the IFN-(i is any biologically active IFN-
(3
polypeptide or variant as described elsewhere herein.
In some embodiments of the present invention, the IFN-[3 is recombinantly
produced. By "recombinantly produced IFN-[3" is intended IFN-(3 that has
comparable biological activity to native 1FN-~i and that has been prepared by
recombinant DNA techniques. IFN-(3 can be produced by culturing a host cell
transformed with an expression vector comprising a nucleotide sequence that
encodes
an IFN-(3 polypeptide. The host cell is one that can transcribe the nucleotide
sequence
and produce the desired protein, and can be prokaryotic (far example, E. eoli)
or
eukaryotic (for example a yeast, insect, or mammalian cell). Examples of
recombinant
production of IFN-(3 are given in Mantei et al., Nature 297:128, 1982; Ohno et
al.,
Nucleic Aeids Res. 10:967, 1982; Smith et al., Mol. Cell. Biol. 3:2156, 1983,
and U.S.
Patent Nos. 4,462,940, 5,702,699, and 5,814,485; herein incorporated by
reference.
Interferon-a
The term "IFN-a" as used herein refers to a biologically active human
a-interferon or any biologically active variants thereof, which are sometimes
referred to
in the art as IFN-a-like polypeptides. Human alpha interferons comprise a
family of
about 30 protein species, encoded by at least 14 different genes and about 16
alleles.
Such IFN-a palypeptides include IFN-aa, IFN-aB, IFN-aC, IFN-aD, IFN-aH, IFN-
aJ,
IFN-aJl, IFN-aJ2 and IFN-alb. Native human IFN-a or variants, which may be
naturally occurring (e.g., allelic variants that occur at the IFN-a locus) or
recombinantly
produced, have amino acid sequences that are similar to, or substaaitially
similar to the
mature native IFN-a sequence. DNA sequences encoding human IFN-a are also
available in the art. See, for example, Gaeddel et al., Natuy~e 290:20-26,
1981 (Genbank
Accession No. V00551 J00209); Nagata et al., Nature 24:3126-320, 1980; Bowden
et
al., Gene 27:87-99, 1984 (Genbank Accession No. NM 000605); and Ohara et al.,
FEBS Letters 211:78-82, 1987; all of which are herein incorporated by
reference.
Fragments of IFN-a or truncated forms of IFN-a that retain their activity are
also
encompassed. These biologically active fragments or truncated forms of IFN-a
are
generated by removing amino acid residues from the full-length IFN-a amino
acid
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sequence using recombinant DNA techniques well known in the art. IFN-a
polypeptides may further be glycosylated or unglycosylated.
Additional changes can be introduced by mutation into the nucleotide
sequences encoding IFN-a, thereby leading to changes in the IFN-a amino acid
sequence, without altering the biological activity of the interferon. Thus, an
isolated
nucleic acid molecule encoding an IFN-a variant having a sequence that differs
from
human IFN-a can be created by introducing one or more nucleotide
substitutions,
additions, or deletions into the corresponding nucleotide sequence disclosed
herein,
such that one or more amino acid substitutions, additions or deletions are
introduced
into the encoded IFN-a. Mutations can be introduced by standard techniques.
Such
variants of IFN-a, include, for example, IFN-a-2a (Roferon-ATM), IFN-a-2b
(Intron
ATM), and IFN-a con-1 (InfergenTM). Another variant useful in the methods of
the
present invention is IFN-a2a, which is disclosed in, for example, EP 43980;
Meada et
al., PNAS 77:7010, 1980; and Levy et al., PNAS 7:6186, 1981; all of which are
herein incorporated by reference. Further variants of IFN-a can be found, for
example, in U.S. Patent No. ~ 5,676,942, herein incorporated by reference.
These
citations also provide guidance regarding residues and regions of the IFN-a
polypeptide that can be altered without the loss of biological activity.
Biologically active IFN-a variants used in the methods of the invention also
include IFN-a polypeptides that have covalently linked with, for example,
polyethylene glycol (PEG) or albumin. See, for example, U.S. Patent No.
5,762,923,
herein incorporated by reference.
Biologically active variants of IFN-a used in the methods of the invention
should retain IFN-a activities, particularly the ability to bind to IFN-a
receptors or
retain immunomodulatory, antiviral, or antiproliferative activities. In some
embodiments, the IFN-a variant retains at least about 25%, preferably about
50%, and
more preferably about 75% or more of the biological activity of the native IFN-
a
polypeptide. IFN-a variants whose activity is increased in comparison with the
activity of the native IFN-a polypeptide are also encompassed. The biological
activity of IFN-a variants can be measured by any method known in the art.
Examples of such assays are describe above.
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In some embodiments of the present invention, the IFN-a is recombinantly
produced. By "recombinantly produced IFN-a" is intended IFN-a that has
comparable biological activity to native IFN-a and that has been prepared by
recombinant DNA techniques. IFN-a can be produced by culturing a host cell
transformed with an expression vector comprising a nucleotide sequence that
encodes
an IFN-a polypeptide. The host cell is one that can transcribe the nucleotide
sequence
and produce the desired protein, and can be prokaryotic (for example, E. coli)
or
eukaryotic (for example a yeast, insect, or mammalian cell). Details of the
cloning of
interferon-cDNA and the direct expression thereof, especially in E. coli, have
in the
meantime been the subject of many publications. Thus, for example, the
preparation
of recombinant interferons is known. See, for example, Nature 295:503-508,
1982;
Nature 284:316-320, 1980; Nature 290:20-26, 1981; Nucleic Acids Res. 8:4057-
4074, 1980, as well as European Patents Nos. 32134, 43980 and 211 148. Further
examples of recombinant production of IFN-a-2 are provided in Nagata et al.,
Nature
284:316, 1980 and European Patent 32,134. All of these references are herein
incorporated by reference.
Pharmaceutical Composition: Interferon
Increases in the amount of interferon in the CNS, brain, and/or spinal cord to
a
therapeutically effective level may be obtained via administration of a
pharmaceutical
composition including a therapeutically effective dose of interferon. By
"therapeutically effective dose" is intended a dose of interferon that
achieves the
desired goal of increasing the amount of interferon in a relevant portion of
the CNS,
brain, and/or spinal cord to a therapeutically effective level enabling a
desired
biological activity of the interferon.
The invention employs a composition for delivery of interferon to the CNS,
brain, and/or spinal cord upon administration to tissue innervated by the
olfactory
and/or trigeminal nerves. The composition can include, for example, any
pharmaceutically acceptable additive, carrier, or adjuvant that is suitable
for
administering an interferon to tissue innervated by the olfactory and/or
trigeminal
nerves. Preferably, the pharmaceutical composition can be employed in
diagnosis,
prevention, or treatment of a disease, disorder, or injury of the CNS, brain,
and/or spinal
CA 02466931 2004-05-19
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cord. Preferably, the composition includes an interferon in combination with a
pharmaceutical carrier, additive, and/or adjuvant that can promote the
transfer of the
interferon within or through tissue innervated by the olfactory and/or
trigeminal nerves.
Alternatively, the interferon may be combined with substances that may assist
in
transporting the interferon to sites of nerve cell damage. The composition can
include
one or several interferons.
The composition typically contains a pharmaceutically acceptable carrier
mixed with the interferon and other components in the pharmaceutical
composition.
By "pharmaceutically acceptable carrier" is 'intended a carrier that is
conventionally
used in the art to facilitate the storage, administration, and/or the healing
effect of the
interferon. A carrier may also reduce any undesirable side effects of the
interferon. A
suitable carrier should be stable, i.e., incapable of reacting with other
ingredients in
the formulation. It should not produce significant local or systemic adverse
effect in
recipients at the dosages and concentrations employed for treatment. Such
carriers
are generally known in the art.
Suitable carriers for this invention include those conventionally used for
large
stable macromolecules such as albumin, gelatin, collagen; polysaccharide,
monosaccharides, polyvinylpyrrolidone, polylactic acid, polyglycolic acid,
polymeric
amino acids, fixed oils, ethyl oleate, liposomes, glucose, sucrose, lactose,
mannose,
dextrose, dextran, cellulose, mannitol, sorbitol, polyethylene glycol (PEG),
and the
like.
Water, saline, aqueous dextrose, and glycols are preferred liquid carriers,
particularly 'for solutions. The carrier can be selected from various oils,
including those
of petroleum, animal, vegetable or synthetic origin, for example, peanut oil,
soybean oil,
mineral oil, sesame oil, and the like. Suitable pharmaceutical excipients
include starch,
cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel,
magnesium stearate, sodium steaxate, glycerol monostearate, sodium chloride,
dried
skim milk, glycerol, propylene glycol, water, ethanol, and the like. The
compositions
can be subjected to conventional pharmaceutical expedients, such as
sterilization, and
can contain conventional pharmaceutical additives, such as preservatives,
stabilizing
interferons, wetting, or emulsifying agents, salts for adjusting osmotic
pressure, buffers,
and the like.
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A composition formulated for intranasal delivery may optionally comprise an
odorant. An odorant agent is combined with the interferon to provide an
odoriferous
sensation, and/or to indicate inhalation of the intranasal preparation to
enhance
delivery of the active interferon to the olfactory neuroepithelium. The
odoriferous
sensation provided by the odorant agent may be pleasant, obnoxious, or
otherwise
malodorous. The odorant receptor neurons are localized to the olfactory
epithelium
that, in humans, occupies only a few square centimeters in the upper part of
the nasal
cavity. The cilia of the olfactory neuronal dendrites that contain the
receptors are
fairly long (about 30-200 um). A 10-30 pin layer of mucus envelops the cilia
that the
odorant agent must penetrate to reach the receptors. See Snyder et al., J.
Biol. Chem.
263:13972-13974, 1998. Use of a lipophilic odorant agent having moderate to
high
affinity for odorant binding protein (OBP) is preferred. OBP has an affinity
for small
lipophilic molecules found in nasal secretions and may act as a carrier to
enhance the
transport of a lipophilic odorant substance, interferon to the olfactory
receptor
neurons. It is also preferred that an odorant agent is capable of associating
with
lipophilic additives such as liposomes and micelles within the preparation to
further
enhance delivery of the interferons by means of OBP to the olfactory
neuroepithelium. OBP may also bind directly to lipophilic agents to enhance
transport of the interferon to olfactory neural receptors.
Suitable odorants having a high affinity for OBP include terpanoids such as
cetralva and citronellol, aldehydes such as amyl cinnamaldehyde and hexyl
cinnamaldehyde, esters such as octyl isovalerate, jasmines such as C1S jasmine
and
jasmal, and musk 89. Other suitable odorant agents include those which may be
capable of stimulating odorant-sensitive enzymes such as adenylate cyclase and
guanylate cyclase, or which may be capable of modifying ion channels within
the
olfactory system to enhance absorption of the interferon.
Other acceptable components in the composition include, but are not limited
to, pharmaceutically acceptable agents that modify isotonicity, including
water, salts,
sugars, polyols, amino acids, and buffers such as phosphate, citrate,
succinate, acetate,
and other organic acids or their salts. Typically, the pharmaceutically
acceptable
carrier also includes one or more stabilizers, reducing agents, anti-oxidants
andlor
anti-oxidant chelating agents. The use of buffers, stabilizers, reducing
agents, anti-
17
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oxidants and chelating agents in the preparation of protein based
compositions,
particularly pharmaceutical compositions, is well known in the art. See Wang
et al., J.
Par~etzt. DrugAssfz. 34(6):452-462, 1980; Wang et al., J. Pa~efzt. Sci. and
Tech. 42:54-
526, 1988 (Supplement); Lachman et al., Df~ug and Cosmetic Industry 102(1):36-
38,
40, 1968 and 146-148; Akers, M.J., J. Parefzt. Sci. aid Tech. 36(5):222-228,
1988; and
Colowick et al. Methods ifz Erzzymology, Vol. XXV, p. 185-188, 1988.
Suitable buffers include acetate, adipate, benzoate, citrate, lactate,
maleate,
phosphate, tartarate, borate, tri(hydroxymethyl aminomethane), succinate,
glycine,
histidine, the salts of various amino acids, or the like, or combinations
thereof. See
Wang (1980) supra at page 455. Suitable salts and isotonicifiers include
sodium
chloride, dextrose, mannitol, sucrose, trehalose, or the like. Where the
carrier is a
liquid, it is preferred that the carrier is hypotonic or isotonic with oral,
conjunctiva) or
dermal fluids and have a pH within the range of 4.5-8.5. Where the carrier is
in
powdered form, it is preferred that the carrier is also within an acceptable
non-toxic
pH range.
Suitable reducing agents, which maintain the reduction of reduced cysteines,
include dithiothreitol (DTT; also known as Cleland's reagent) or
dithioerythritol at
0.01 % to 0.1 % wt/wt; acetylcysteine or cysteine at 0.1 % to 0.5 % and pH 2-
3; and
thioglycerol at 0.1% to 0.5% and pH 3.5-7.0; and glutathione. See Akers (1988)
supra at pages 225 to 226. Suitable antioxidants include sodium bisulfate,
sodium
sulfite, sodium metabisulfite, sodium thiosulfate, sodium formaldehyde
sulfoxylate,
and ascorbic acid. See Akers (1988) supra at pages 225. Suitable chelating
agents,
which chelate trace metals to prevent the trace metal catalyzed oxidation of
reduced
cysteines, include citrate, tartrate, ethylenediaminetetraacetic acid (EDTA)
in its
disodium, tetrasodium, and calcium disodium salts, and diethylenetriamine
pentaacetic acid (DTPA). See, e.g., Wang (1980) sup>~a at pages 457-458 and
460-
461, and Algiers (1988) supra at pages 224-227.
The composition can include one or more preservatives such as phenol, cresol,
p-aminobenzoic acid, BDSA, sorbitrate, chlorhexidine, benzalkonium chloride,
or the
like. Suitable stabilizers include carbohydrates such as trehalose or
glycerol. The
composition can include a stabilizer such as one or more of microcrystalline
cellulose,
magnesium stearate, mannitol, sucrose to stabilize, for example, the physical
form of
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WO 03/045978 PCT/US02/38164
the composition; and one or more of glycine, arginine, hydrolyzed collagen, or
protease inhibitors to stabilize, for example, the chemical structure of the
composition. Suitable suspending additives include carboxymethyl cellulose,
hydroxypropyl methylcellulose, hyaluronic acid, alginate, chondroitin sulfate,
dextran, maltodextrin, dextran sulfate, or the like. The composition can
include an
emulsifier such as polysorbate 20, polysorbate 80, pluronic, triolein, soybean
oil,
lecithins, squalene and squalanes, sorbitan treioleate, or the like. The
composition can
include an antimicrobial such as phenylethyl alcohol, phenol, cresol,
benzalkonium
chloride, phenoxyethanol, chlorhexidine, thimerosol, or the like. Suitable
thickeners
include natural polysaccharides such as mannans, arabinans, alginate,
hyaluronic acid,
dextrose, or the like; and synthetic ones like the PEG hydrogels of low
molecular
weight and aforementioned suspending the interferon.
The composition can include an adjuvant such as cetyl trimethyl ammonium
bromide, BDSA, cholate, deoxycholate, polysorbate 20 and 80, fusidic acid, or
the like,
and in the case of DNA delivery, preferably, a cationic lipid. Suitable sugars
include
glycerol, threose, glucose, galactose, mannitol, and sorbitol. A suitable
protein is human
serum albumin.
Preferred compositions include one or more of a solubility enhancing additive,
preferably a cyclodextrin; a hydrophilic additive, preferably a monosaccharide
or
oligosaccharide; an absorption promoting additive, preferably a cholate, a
deoxycholate, a fusidic acid, or a chitosan; a cationic surfactant, preferably
a cetyl
trimethyl ammonium bromide; a viscosity enhancing additive, preferably to
promote
residence time of the composition at the site of administration, preferably a
carboxymethyl cellulose, a maltodextrin, an alginic acid, a hyaluronic acid,
or a
chondroitin sulfate; or a sustained release matrix, preferably a
polyanhydride, a
polyorthoester, a hydrogel, a particulate slow release depo system, preferably
a
polylactide co-glycolide (PLG), a depo foam, a starch microsphere, or a
cellulose
derived buccal system; a lipid-based carrier, preferably an emulsion, a
liposome, a
niosome, or a micelle. The composition can include a bilayer destabilizing
additive,
preferably a phosphatidyl ethanolamine; a fusogenic additive, preferably a
cholesterol
hemisuccinate.
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Other preferred compositions for sublingual administration including, for
example, a bioadhesive to retain the interferon sublingually; a spray, paint,
or swab
applied to the tongue; retaining a slow dissolving pill or lozenge under the
tongue; or
the like. Other preferred compositions for transdermal administration include
a
bioadhesive to retain the interferon on or in the skin; a spray, paint,
cosmetic, or swab
applied to the skin; or the like.
These lists of carriers and additives is by no means complete and a worker
skilled in the art can choose excipients from the GRAS (generally regarded as
safe)
list of chemicals allowed in the pharmaceutical preparations and those that
are
currently allowed in topical and parenteral formulations.
For the purposes of this invention, the pharmaceutical composition comprising
the interferon can be formulated in a unit dosage and in a form such as a
solution,
suspension, or emulsion. The interferon may be administered to tissue
innervated by
the trigeminal and/or olfactory neurons as a powder, a granule, a solution, a
cream, a
spray (e.g., an aerosol), a gel, an ointment, an infusion, an injection, a
drop, or
sustained-release composition, such as a polymer disk. For buccal
administration, the
compositions can take the form of tablets or lozenges formulated in a
conventional
manner. For administration to the eye or other external tissues, e.g., mouth
and skin, the
compositions can be applied to the infected part of the body of the patient as
a topical
ointment or cream. The compounds can be presented in an ointment, for instance
with a
water-soluble ointment base, or in a cream, for instance with an-oil-in water
cream base.
For conjunctiva) applications, the interferon can be administered in
biodegradable or
non-degradable ocular inserts. The drug may be released by matrix erosion or
passively through a pore as in ethylene-vinylacetate polymer inserts. For
other
mucosal administrations, such as sublingual, powder discs may be placed under
the
tongue.
Other preferred forms of compositions for administration include a suspension
of a particulate, such as an emulsion, a liposome, an insert that releases the
interferon
slowly, and the like. The powder or granular forms of the pharmaceutical
composition may be combined with a solution and with a diluting, dispersing,
or
surface-active interferon. Additional preferred compositions for
administration
include a bioadhesive to retain the interferon at the site of administration;
a spray,
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paint, or swab applied to the mucosa or epithelium; a slow dissolving pill or
lozenge;
or the like. The composition can also be in the form of lyophilized powder,
which can
be converted into a solution, suspension, or emulsion before administration.
The -
pharmaceutical composition including interferon is preferably sterilized by
membrane
filtration and is stored in unit-dose or mufti-dose containers such as sealed
vials or
ampoules.
The method for formulating a pharmaceutical composition is generally known
in the art. A thorough discussion of formulation and selection of
pharmaceutically
acceptable carriers, stabilizers, and isomolytes can be found in Remington's
Pharmaceutical Sciences (18th ed.; Mack Publishing Company, Eaton,
Pennsylvania,
1990), herein incorporated by reference.
The interferon can also be formulated in a sustained-release form to prolong
the presence of the pharmaceutically active interferon in the treated mammal,
generally for longer than one day. Many methods of preparation of a sustained-
release formulation are known in the art and are disclosed in Remington's
Pharmaceutical Sciences (18a' ed.; Mack Publishing Company, Eaton,
Pennsylvania,
1990), herein incorporated by reference.
Generally, the interferon can be entrapped in semipermeable matrices of solid
hydrophobic polymers. The matrices can be shaped into films or microcapsules.
Examples of such matrices include, but are not limited to, polyesters,
copolymers of
L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers 2~:547-
556, 1983), polylactides (U.S. Patent No. 3,773,919 and EP 58,481),
polylactate
polyglycolate (PLGA) such as polylactide-co-glycolide (see, for example, U.S.
Patent
Nos. 4,767,628 and 5,654,008), hydrogels (see, for example, Langer et al., J.
Biomed.
Mater Res. 15:167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982), non-
degradable
ethylene-vinyl acetate (e.g. ethylene vinyl acetate disks and polyethylene-co-
vinyl
acetate)), degradable lactic acid-glycolic acid copolyers such as the Lupron
DepotTM,
poly-D-(-)-3-hydroxybutyric acid (EP 133,988), hyaluronic acid gels (see, for
example, U.S. Patent 4,636,524), alginic acid suspensions, and the like.
Suitable microcapsules can also include hydroxymethylcellulose or gelatin-
microcapsules and polymethyl methacrylate microcapsules prepared by
coacervation
techniques or by interfacial polymerization. See the PCT publication WO
99/24061
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entitled "Method for P~oducihg Sustai~zed release Formulations," wherein a
protein is
encapsulated in PLGA microspheres, herein incorporated by reference. In
addition,
microemulsions or colloidal drug delivery systems such as liposomes and
albumin
microspheres, may also be used. See Remi~rgton's Pharf~zaceutical Sciences
(18th ed.;
Mack Publishing Company Co., Eaton, Pennsylvania, 1990). Other preferred
sustained-release compositions employ a bioadhesive to retain the interferon
at the site
of administration.
., Among the optional substances that may be combined with the interferon in
the pharmaceutical composition are lipophilic substances that can enhance
absorption
of the interferon through the mucosa or epithelium of the nasal cavity, or
along a
neural, lymphatic, or perivascular pathway to damaged nerve cells in the CNS.
The
interferon may be mixed with a lipophilic adjuvant alone or in combination
with a
carrier, or may be combined with one or several types of micelle or liposome
substances. Among the preferred lipophilic substances are cationic liposomes
included of one or more of the following: phosphatidyl choline, lipofectin, a
lipid-
peptoid conjugate, a synthetic phospholipid such as phosphatidyl lysine, or
the like.
These liposomes may include other lipophilic substances such as gangliosides
and
phosphatidylserine (PS). Also preferred are micellar additives such as GM-1
gangliosides and phosphatidylserine (PS), which may be combined with the
interferon
either alone or in combination. GM-1 ganglioside can be included at 1-10 mole
percent in any liposomal compositions or in higher amounts in micellar
structures.
Protein interferons can be either encapsulated in particulate structures or
incorporated
as part of the hydrophobic portion of the structure depending on the
hydrophobicity of
the active interferon.
One preferred liposomal formulation employs Depofoam. Interferon can be
encapsulated in multivesicular liposomes, as disclosed in the WO publication
99112522 entitled "High ahd Low Load Formulations of IGF I i~
Multivesicula~°
Liposomes," herein incorporated by reference. The mean residence time of
interferon
at the site of administration can be prolonged with a Depofoam composition.
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Administering_the Interferon
The total amount of interferon administered per dose should be in a range
sufficient to delivery a biologically relevant amount of the interferon (i.e.,
an amount
sufficient to produce a therapeutical effect). The pharmaceutical composition
having
a unit dose of interferon can be in the form of solution, suspension,
emulsion, or a
sustained-release formulation. The total volume of one dose of the
pharmaceutical
composition can range from about 10 ~.1 to about 100 p,l, for example, for
nasal
aclininistration. It is apparent that the suitable volume can vary with
factors such as
the size of the tissue to which the interferon is administered and the
solubility of the
components in the composition.
It is recognized that the total amount of interferon administered as a unit
dose
to a particular tissue will depend upon the type of pharmaceutical composition
being
administered, that is whether the composition is in the form of, for example,
a
solution, a suspension, an emulsion, or a sustained-release formulation. For
example,
where the pharmaceutical composition comprising a therapeutically effective
amount
of interferon is a sustained-release formulation, interferon is administered
at a higher
concentration. Needle-free subcutaneous administration to an extranasal tissue
innervated by the trigeminal nerve may be accomplished by use of a device
which
employs a supersonic gas jet as a power source to accelerate an agent that is
formulated as a powder or a microparticle into the skin. The characteristics
of such a
delivery method will be determined by the properties of the particle, the
formulation
of the agent and the gas dynamics of the delivery device. Similarly, the
subcutaneous
delivery of an aqueous composition can be accomplished in a needle-free manner
by
employing a gas-spring powered hand held device to produce a high force jet of
fluid
capable of penetrating the skin. Alternatively, a skin-patch formulated to
mediate a
sustained release of a composition can be employed for the transdermal
delivery of a
neuroregulatory agent to a tissue innervated by the trigeminal nerve. Where
the
pharmaceutical composition comprises a therapeutically effective amount of an
agent,
or a combination of agents, in a sustained-release formulation, the agents)
is/are
administered at a higher concentration.
It should be apparent to a person skilled in the art that variations may be
acceptable with respect to the therapeutically effective dose and frequency of
the
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administration of an interferon in this embodiment of the invention. The
amount of
the interferon administered will be inversely correlated with the frequency of
administration. Hence, an increase in the concentration of interferon in a
single
administered dose, or an increase in the mean residence time in the case of a
sustained-release form of interferon, generally will be coupled with a
decrease in the
frequency of administration.
In the practice of the present invention, additional factors should be taken
into
consideration when determining the therapeutically effective dose of
interferon and
frequency of its administration. Such factors include, for example, the size
of the
tissue, the area of the surface of the tissue, the severity of the disease or
disorder, and
the age, height, weight, health, and physical condition of the individual to
be treated.
Generally, a higher dosage is preferred if the tissue is larger or the disease
or disorder
is more severe.
Some minor degree of experimentation may be required to determine the most
effective dose and frequency of dose administration, this being well within
the
capability of one skilled in the art once apprised of the present disclosure.
For the treatment of a disorder of the CNS in a human, including neurologic,
viral, proliferative or immunomodulatory disorders, a therapeutically
effective amount
or dose of interferon is about 0.14 nmol/kg of brain weight to about 138
nmol/kg brain
weight and about 0.14 nmol/kg of brain weight to about 69 nmol/kg of brain
weight. In
some regimens, therapeutically effective doses for administration of
interferon include
about 0.13-140 nmol/kg of brain weight. Fox the treatment of a disorder of the
CNS in
a human, including neurologic, viral, proliferative or immunomodulatory
disorders, the
therapeutically effective amount or dose of IFN-[3 or biologically active
variant thereof
is about 0.14 nmol/kg of brain weight to about 138 nmol/kg of brain weight and
about
0.14 nmol/kg of brain weight to about 69 nmol/kg of brain weight. In some
regimens,
therapeutically effective doses for administration of IFN-[3 include about
0.13-140
nmollkg of brain weight.
It is further recognized that the therapeutically effective amount or dose of
interferon to a human may be lower when the interferon is administered via the
nasal
lymphatics to various tissues of the head and neck for the treatment or
prevention of
disorders or diseases characterized by immune and inflammatory responses
(i.e.,
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diseases resulting in acute or chronic inflammation and/or infiltration by
lymphocytes).
In these embodiments, while the interferon can be administered in the dosage
range
provided above, the interferon may also be administered from about 0.02 to
about 138
pmollkg of brain weight. Alternatively, the interferon may be administered
from about
0.02-140 pmol/kg of brain weight. Similarly, when the interferon is IFN-(3,
the dosage
range may also be from about 0.02 to about 140 pmol/kg of brain weight.
Alternatively,
the interferon may be administered from about 0.02-140 pmol/kg of brain
weight.
These doses depend on factors including the efficiency with which the
interferon is transported to the CNS or lymphatic system. A larger total dose
can be
delivered by multiple administrations of the agent.
_Intermittent Dosing
In another embodiment of the invention, the pharmaceutical composition
comprising the therapeutically effective dose of interferon is administered
intermittently. By "intermittent administration" is intended administration of
a
therapeutically effective dose of interferon, followed by a time period of
discontinuance, which is then followed by another administration of a
therapeutically
effective dose, and so forth. Administration of the therapeutically effective
dose may
be achieved in a continuous manner, as for example with a sustained-release
formulation, or it may be achieved according to a desired daily dosage
regimen, as for
example with one, two, three or more administrations per day. By "time period
of
discontinuance" is intended a discontinuing of the continuous sustained-
released or
daily administration of interferon. The time period of discontinuance may be
longer
or shorter than the period of continuous sustained-release or daily
administration.
During the time period of discontinuance, the interferon level in the relevant
tissue is
substantially below the maximum level obtained during the treatment. The
preferred
length of the discontinuance period depends on the concentration of the
effective dose
and the form of interferon used. The discontinuance period can be at least 2
days,
preferably is at least 4 days, more preferably is at least 1 week and
generally does not
exceed a period of 4 weeks. When a sustained-release formulation is used, the
discontinuance period must be extended to account for the greater residence
time of
interferon at the site of injury. Alternatively, the frequency of
administration of the
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effective dose of the sustained-release formulation can be decreased
accordingly. An
intermittent schedule of administration of interferon can continue until the
desired
therapeutic effect, and ultimately treatment of the disease or disorder, is
achieved.
In yet another embodiment, intermittent administration of the therapeutically
effective dose of interferon is cyclic. By "cyclic" is intended intermittent
administration accompanied by breaks in the administration, with cycles
ranging from
about 1 month to about 2-6 months. For example, the administration schedule
might
be intermittent administration of the effective dose of interferon, wherein a
single
short-term dose is given once per week for 4 weeks, followed by a break in
intermittent administration fox a period of 3 months, followed by intermittent
administration by administration of a single short-term dose given once per
week for
4 weeks, followed by a break in intermittent administration for a period of 2
months,
and so forth. As another example, a single short-term dose may be given once
per
week for 2 weeks, followed by a break in intermittent administration for a
period of 1
month, followed by a single short-term dose given once per week for 2 weeks,
followed by a break in intermittent administration for a period of 1 month,
and so
forth. A cyclic intermittent schedule of administration of interferon to
subject may
continue until the desired therapeutic effect, and ultimately treatment of the
disorder
or disease, is achieved.
Neuronal Transport
One embodiment of the present method includes administration of the
interferon to the subject in a manner such that the interferon is transported
to the
lymphatic system, the lacrimal gland, CNS, brain, and/or spinal cord along a
neural
pathway. A neural pathway includes transport within or along a neuron, through
or by
way of lymphatics running with a neuron, through or by way of a perivascular
space
of a blood vessel running with a neuron or neural pathway, through or by way
of an
adventitia of a blood vessel running with a neuron or neural pathway, or
through an
hemangiolymphatic system. The invention prefers transport of interferon by way
of a
neural pathway, rather than through the circulatory system, so that interferon
that are
unable to, or only poorly, cross the blood-brain barrier from the bloodstream
into the
brain can be delivered to the lymphatic system, CNS, brain, and/or spinal
cord. The
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interferon, once past the blood-brain barrier and in the CNS, can then be
delivered to
various areas of the brain or spinal cord through lymphatic channels, through
a
perivascular space, or transport through or along neurons. In one embodiment,
the
interferon preferably accumulates in areas having the greatest density of
receptor or
binding sites for the interferon.
Use of a neural pathway to transport interferon to the lymphatic system,
lacrimal
gland, brain, spinal cord, or other components of the central nervous system
obviates
the obstacle presented by the blood-brain barrier so that medications that
cannot
normally cross that barrier, can be delivered directly to the brain,
cerebellum, brain
stem, or spinal cord. Although the interferon that is administered may be
absorbed into
the bloodstream as well as the neural pathway, the interferon preferably
provides
minimal effects systemically. In addition, the invention can provide fox
delivery of a
more concentrated level of the interferon to neural cells since the interferon
does not
become diluted in fluids present in the bloodstream. As such, the invention
provides an
improved method for delivering interferon to the lymphatic system, CNS, brain,
and/or
spinal cord.
The Olfactory Neural Pathway
One embodiment of the present method includes delivery of the interferon to
the subject in a manner such that the interferon is transported into the CNS,
brain,
and/or spinal cord along an olfactory neural pathway. Typically, such an
embodiment
includes administering the interferon to tissue innervated by the olfactory
nerve and
inside the nasal cavity. The olfactory neural pathway innervates primarily the
olfactory epithelium in the upper third of the nasal cavity, as described
above.
Application of the interferon to a tissue innervated by the olfactory nerve
can deliver
the interferon to damaged neurons or cells of the CNS, brain, and/or spinal
cord.
Olfactory neurons innervate this tissue and can provide a direct connection to
the
CNS, brain, and/or spinal cord due, it is believed, to their role in
olfaction.
Delivery through the olfactory neural pathway can employ lymphatics that
travel with the olfactory nerve to the various brain areas and from there into
dural
lymphatics associated with portions of the CNS, such as the spinal cord.
Transport
along the olfactory nerve can also deliver interferon to an olfactory bulb. A
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perivascular pathway and/or a hemangiolymphatic pathway, such as lymphatic
channels running within the adventitia of cerebral blood vessels, can provide
an
additional mechanism for transport of therapeutic interferon to the brain and
spinal
cord from tissue innervated by the olfactory nerve.
Interferon can be administered to the olfactory nerve, for example, through
the
olfactory epithelium. Such administration can employ extracellular or
intracellular
(e.g., transneuronal) anterograde and retrograde transport of the interferon
entering
through the olfactory nerves to the brain and its meninges, to the brain stem,
or to the
spinal cord. Once the interferon is dispensed into or onto tissue imiervated
by the
olfactory nerve, the interferon may transport through the tissue and travel
along
olfactory neurons into areas of the CNS including the brain stem, cerebellum,
spinal
cord, olfactory bulb, and cortical and subcortical structures.
Delivery through the olfactory neural pathway can employ movement of
interferon into or across mucosa or epithelium into the olfactory nerve or
into a
lymphatic, a blood vessel perivascular space, a blood vessel adventitia, or a
blood
vessel lymphatic that travels with the olfactory nerve to the brain and from
there into
meningial lymphatics associated with portions of the CNS such as the spinal
cord.
Blood vessel lymphatics include lymphatic channels that are around the blood
vessels
on the outside of the blood vessels. This also is referred to as the
hemangiolymphatic
system. Introduction of interferon into the blood vessel lymphatics does not
necessarily
introduce the interferon into the blood.
The Tri~eminal Neural Pathway
One embodiment of the present method includes delivery of the interferon to
the subject in a manner such that the interferon is transported into the CNS,
brain,
and/or spinal cord along a trigeminal neural pathway. Typically, such an
embodiment
includes administering the interferon to tissue innervated by the trigeminal
nerve
including inside and outside the nasal cavity. The trigeminal neural pathway
innervates various tissues of the head and face, as described above. In
particular, the
trigeminal nerve innervates the nasal, sinusoidal, oral and conjunctiva)
mucosa or
epithelium, and the skin of the face. Application of the interferon to a
tissue
innervated by the trigeminal nerve can deliver the interferon to damaged
neurons or
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cells of the CNS, brain, and/or spinal cord to cells of the lymphatic system.
Trigeminal neurons innervate these tissues and can provide a direct connection
to the
CNS, brain, and/or spinal cord due, it is believed, to their role in the
common
chemical sense including mechanical sensation, thermal sensation and
nociception
(for example detection of hot spices and of noxious chemicals).
Delivery through the trigeminal neural pathway can employ lymphatics that
travel with the trigeminal nerve to the pons and other brain areas and from
there into
dural lymphatics associated with portions of the CNS, such as the spinal cord.
Transport along the trigeminal nerve can also deliver interferons to an
olfactory bulb.
A perivascular pathway and/or a hemangiolymphatic pathway, such as lymphatic
channels running within the adventitia of cerebral blood vessels, can provide
an
additional mechanism for transport of therapeutic interferons to the spinal
cord from
tissue innervated by the trigeminal nerve.
The trigeminal nerve includes large diameter axons, which mediate mechanical
sensation, e.g., touch, and small diameter axons, which mediate pain and
thermal
sensation, both of whose cell bodies are located in the semilunar (or
trigeminal)
ganglion or the mesencephalic trigeminal nucleus in the midbrain. Certain
portions of
the trigeminal nerve extend into the nasal cavity, oral and conjunctiva)
mucosa and/or
epithelium. Other portions of the trigeminal nerve extend into the skin of the
face,
forehead, upper eyelid, lower eyelid, dorsum of the nose, side of the nose,
upper lip,
cheek, chin, scalp and teeth. Individual fibers of the trigeminal nerve
collect into a
large bundle, travel underneath the brain and enter the ventral aspect of the
pons.
Interferon can be administered to the trigeminal nerve, for example, through
the nasal
cavity's, oral, lingual, and/or conjunctiva) mucosa and/or epithelium; or
through the
skin of the face, forehead, upper eyelid, lower eyelid, dorsum of the nose,
side of the
nose, upper lip, cheek, chin, scalp and teeth. Such administration can employ
extracellular or intracellular (e.g., transneuronal) anterograde and
retrograde transport of
the interferon entering through the trigeminal nerves to the brain and its
meninges, to
the brain stem, or to the spinal cord. Once the interferon is dispensed into
or onto tissue
innervated by the trigeminal nerve, the interferon may transport through the
tissue and
travel along trigeminal neurons into areas of the CNS including the brain
stem,
cerebellum, spinal cord, olfactory bulb, and cortical and subcortical
structures.
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Delivery through the trigeminal neural pathway can employ movement of
interferon across skin, mucosa, or epithelium into the trigeminal nerve or
into a
lymphatic, a blood vessel perivascular space, a blood vessel adventitia, or a
blood
vessel lymphatic that travels with the trigeminal nerve to the pons and from
there into
meningial lymphatics associated with portions of the CNS such as the spinal
cord.
Blood vessel lymphatics include lymphatic chamzels that are around the blood
vessels
on the outside of the blood vessels. This also is referred to as the
hemangiolymphatic
system. Introduction of an interferon into the blood vessel lymphatics does
not
necessarily introduce the interferon into the blood.
Neural Pathways and Nasal Administration
In one embodiment, the method of the invention can employ delivery by a
neural pathway, e.g., a trigeminal or olfactory neural pathway, after
administration to
the nasal cavity. Upon administration to the nasal cavity, delivery via the
trigeminal
neural pathway may employ movement of an interferon through the nasal mucosa
and/or epithelium to reach a trigeminal nerve or a perivascular and/or
lymphatic
channel that travels with the nerve. Upon administration to the nasal cavity,
delivery
via the olfactory neural pathway may employ movement of an interferon through
the
nasal mucosa and/or epithelium to reach the olfactory nerve or a perivascular
and/or
lymphatic channel that travels with the nerve.
For example, the interferon can be administered to the nasal cavity in a
manner that employs extracellular or intracellular (e.g., transneuronal)
anterograde
and retrograde transport into and along the trigeminal and/or olfactory nerves
to reach
the brain, the brain stem, or the spinal cord. Once the interferon is
dispensed into or
onto nasal mucosa and/or epithelium innervated by the trigeminal and/or
olfactory
nerve, the interferon may transport through the nasal mucosa and/or epithelium
and
travel along trigeminal and/or olfactory neurons into areas of the CNS
including the
brain stem, cerebellum, spinal cord, olfactory bulb, and cortical and
subcortical
structures. Alternatively, administration to the nasal cavity can result in
delivery of an
interferon into a blood vessel perivascular space or a lymphatic that travels
with the
trigeminal and/or olfactory nerve to the pons, olfactory bulb, and other brain
areas,
and from there into meningeal lymphatics associated with portions of the CNS
such as
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the spinal cord. Transport along the trigeminal and/or olfactory nerve may
also
deliver interferons administered to the nasal cavity to the olfactory bulb,
midbrain,
diencephalon, medulla, and cerebellum. an interferon administered to the nasal
cavity
can enter the ventral dura of the brain and travel in lymphatic channels
within the
dura.
In addition, the method of the invention can be carried out in a way that
employs a perivascular pathway and/or an hemangiolymphatic pathway, such as a
lymphatic channel running within the adventitia of a cerebral blood vessel, to
provide
an additional mechanism for transport of interferon to the spinal cord from
the nasal
mucosa and/or epithelium. an interferon transported by the hemangiolymphatic
pathway does not necessarily enter the circulation. Blood vessel lymphatics
associated with the circle of Willis as well as blood vessels following the
trigeminal
and/or olfactory nerve can also be involved in the transport of the
interferon.
Administration to the nasal cavity employing a neural pathway can deliver an
interferon to the lymphatic system, brain stem, cerebellum, spinal cord, and
cortical
and subcortical structures. The interferon alone may facilitate this movement
into the
CNS, brain, and/or spinal cord. Alternatively, the carrier or other transfer-
promoting
factors may assist in the transport of the interferon into and along the
trigeminal
and/or olfactory neural pathway. Administration to the nasal cavity of a
therapeutic
interferon can bypass the blood-brain barrier through a transport system from
the
nasal mucosa and/or epithelium to the brain and spinal cord.
Neural Pathways and Transdermal Administration
In one embodiment, the method of the invention can employ delivery by a
neural pathway, e.g., a trigeminal neural pathway, after transdermal
administration.
Upon transdermal administration, delivery via the trigeminal neural pathway
may
employ movement of an interferon through the skin to reach a trigeminal nerve
or a
perivascular and/or lymphatic channel that travels with the nerve.
For example, the interferon can be administered transdermally in a manner
that employs extracellular or intracellular (e.g., transneuronal) anterograde
and
retrograde transport into and along the trigeminal nerves to reach the brain,
the brain
stem, or the spinal cord. Once the interferon is dispensed into or onto skin
innervated
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r r l l VJ J.VVG
by the trigeminal nerve, the interferon may transport through the skin and
travel along
trigeminal neurons into areas of the CNS including the brain stem, cerebellum,
spinal
cord, olfactory bulb, and cortical and subcortical structures. Alternatively,
transdermal administration can result in delivery of an interferon into a
blood vessel
perivascular space or a lymphatic that travels with the trigeminal nerve to
the pons,
olfactory bulb, and other brain areas, and from there into meningeal
lymphatics
associated with portions of the CNS such as the spinal cord. Transport along
the
trigeminal nerve may also deliver transdermally administered interferons to
the
olfactory bulb, midbrain, diencephalon, medulla and cerebellum. The ethmoidal
branch of the trigeminal nerve enters the cribriform region. An transdermally
administered interferon can enter the ventral dura of the brain and travel in
lymphatic
channels within the dura.
In addition, the method of the invention can be carried out in a way that
employs a perivascular pathway and/or an hemangiolymphatic pathway, such as a
lymphatic channel running within the adventitia of a cerebral blood vessel, to
provide
an additional mechanism for transport of interferon to the spinal cord from
the skin. an
interferon transported by the hemangiolymphatic pathway does not necessarily
enter the
circulation. Blood vessel lymphatics associated with the circle of Willis as
well as
blood vessels following the trigeminal nerve can also be involved in the
transport of the
interferon.
Transdermal administration employing a neural pathway can deliver an
interferon to the brain stem, cerebellum, spinal cord and cortical and
subcortical
structures. The interferon alone may facilitate this movement into the CNS,
brain,
and/or spinal cord. Alternatively, the carrier or other transfer-promoting
factors may
assist in the transport of the interferon into and along the trigeminal neural
pathway.
Transdermal administration of a therapeutic interferon can bypass the blood-
brain
barrier through a transport system from the skin to the brain and spinal cord.
Neural Pathways and Sublingual Administration
In another embodiment, the method of the invention can employ delivery by a
neural pathway, e.g., a trigeminal neural pathway, after sublingual
administration.
Upon sublingual administration, delivery via the trigeminal neural pathway may
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employ movement of an interferon from under the tongue and across the lingual
epithelium to reach a trigeminal nerve or a perivascular or lymphatic channel
that
travels with the nerve.
For example, the interferon can be administered sublingually in a manner that
employs extracellular or intracellular (e.g., transneuronal) anterograde and
retrograde
transport through the oral mucosa and then into and along the trigeminal
nerves to
reach the brain, the brain stem, or the spinal cord. Once the interferon is
administered
sublingually, the interferon may transport through the oral mucosa by means of
the
peripheral processes of trigeminal neurons into areas of the CNS including the
brain
stem, spinal cord and cortical and subcortical structures. Alternatively,
sublingual
administration can result in delivery of an interferon into lymphatics that
travel with
the trigeminal nerve to the pons and other brain areas and from there into
meningeal
lymphatics associated with portions of the CNS such as the spinal cord.
Transport
along the trigeminal nerve may also deliver sublingually administered
interferons to
the olfactory bulbs, midbrain, diencephalon, medulla and cerebellum. The
ethmoidal
branch of the trigeminal nerve enters the cribriform . region. A sublingually
administered interferon can enter the ventral data of the brain and travel in
lymphatic
chamlels within the data.
In addition, the method of the invention can be carried out in a way that
~0 employs an hemangiolymphatic pathway, such as a lymphatic channel running
within
the adventitia of a cerebral blood vessel, to provide an additional mechanism
for
transport of an interferon to the spinal cord from the oral submucosa. An
interferon
transported by the hemangiolymphatic pathway does not necessarily enter the
circulation. Blood vessel lymphatics associated with the circle of Willis as
well as
blood vessels following the trigeminal nerve can also be involved in the
transport of
the interferon.
Sublingual administration employing a neural pathway can deliver an
interferon to the brain stem, cerebellum, spinal cord and cortical and
subcortical
structures. The interferon alone may facilitate this movement into the CNS,
brain,
and/or spinal cord. Alternatively, the carrier or other transfer-promoting
factors may
assist in the transpout of the interferon into and along the trigeminal neural
pathway.
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Sublingual administration of a therapeutic interferon can bypass the blood-
brain
barrier through a transport system from the oral mucosa to the brain and
spinal cord.
Neural Pathways and Conj~unctival Administration
In another embodiment, the method of the invention can employ delivery by a
neural pathway, e.g. a trigeminal neural pathway, after conjunctiva)
administration.
Upon conjunctiva) administration, delivery via the trigeminal neural pathway
may
employ movement of an interferon from the conjunctiva through the conjunctiva)
epithelium to reach the trigeminal nerves or lymphatic channels that travel
with the
nerve.
For example, the interferon can be administered conjunctivally in a manner
that
employs extracellular or intracellular (e.g., transneuronal) anterograde and
retrograde
transport through the conjunctiva) mucosa and then into and along the
trigeminal nerves
to reach the brain, the brain stem, or the spinal cord. Once the interferon is
administered conjunctivally, the interferon may transport through the
conjunctiva)
mucosa by means of the peripheral processes of trigeminal neurons into areas
of the
CNS including the brain stem, spinal cord and cortical and subcortical
structures.
Alternatively, conjunctiva) administration can result in delivery of an
interferon into
lymphatics that travel with the trigeminal nerve to the pons and other brain
areas and
from there into meningeal lymphatics associated with portions of the CNS such
as the
spinal cord. Transport along the trigeminal nerve may also deliver
conjunctivally
administered interferons to the olfactory bulbs, midbrain, diencephalon,
medulla and
cerebellum. The ethmoidal branch of the trigeminal nerve enters the cribriform
region.
An conjunctivally administered interferon can enter the ventral dura of the
brain and
travel in lymphatic channels within the dura.
In addition, the method of the invention can be carried out in a way that
employs an hemangiolymphatic pathway, such as a lymphatic channel running
within
the adventitia of cerebral blood vessel, to provide an additional mechanism
for
transport of an interferon to the spinal cord from the conjunctiva) submucosa.
An
interferon transported by the hemangiolymphatic pathway does not necessarily
enter
the circulation. Blood vessel lymphatics associated with the circle of Willis
as well as
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blood vessels following the trigeminal nerve can also be involved in the
transport of
the interferon.
Conjunctiva) administration employing a neural pathway can deliver an
inteneron to the brain stem, cerebellum, spinal cord and cortical and
subcortical
structures. The interferon alone may facilitate this movement into the CNS,
brain,
and/or spinal cord. Alternatively, the carrier or other transfer-promoting
factors may
assist in the transport of the interferon into and along the trigeminal neural
pathway.
Conjunctiva) administration of a therapeutic interferon can bypass the blood-
brain
barrier through a transport system from the conjunctiva) mucosa to the brain
and
spinal cord.
Pharmaceutical Composition: Anti-CD40 Antibodies
The anti-CD40 antibodies of the invention are administered at a concentration
that is therapeutically effective to prevent or treat B cell or other antigen
presenting
cell mediated diseases such as multiple sclerosis in a patient also receiving
interferon
therapy as discussed herein. To accomplish this goal, the anti-CD40 antibodies
may
be formulated using a variety of acceptable excipients known in the art.
Typically, the
anti-CD40 antibodies are administered by injection, either intravenously or
parenterally. Methods to accomplish this administration are known to those of
ordinary skill in the art. Compositions comprising anti-CD40 antibodies which
may
be topically or orally administered, or which may be subcutaneously
administered, or
which may be capable of transmission across mucous membranes, are also
suitable.
Befare administration to patients, formulants may be added to the antibodies.
A liquid formulation is preferred. For example, these formulants may include
oils,
polymers, vitamins, carbohydrates, amine acids, salts, buffers, albumin,
surfactants, or
bulking agents. Preferably carbohydrates include sugar or sugar alcohols such
as
mono-, di-, or polysaccharides, or water soluble glucans. The saccharides or
glucans
can include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose,
maltose,
sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble
starch,
hydroxethyl starch and carboxymethylcellulose, or mixtures thereof. Sucrose is
most
preferred. "Sugar alcohol" is defined as a C4 to C8 hydrocarbon having an --OH
group and includes galactitol, inositol, mannitol, xylitol, sorbitol,
glycerol, and
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arabitol. Mannitol is most preferred. These sugars or sugar alcohols mentioned
above
may be used individually or in combination. There is no fixed limit to amount
used as
long as the sugar or sugar alcohol is soluble in the aqueous preparation.
Preferably,
the sugar or sugar alcohol concentration is between 1.0 w/v % and 7.0 w/v %,
more
preferable between 2.0 and 6.0 w/v %. Preferably amino acids include
levorotary (L)
forms of carnitine, arginine, and betaine; however, other amino acids may be
added.
Preferred polymers include polyvinylpyrrolidone (PVP) with an average
molecular
weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average
molecular weight between 3,000 and 5,000. It is also preferred to use a buffer
in the
composition to minimize pH changes in the solution before lyophilization or
after
reconstitution. Most any physiological buffer may be used, but titrate,
phosphate,
succinate, and glutamate buffers or mixtures thereof are preferred. Most
preferred is a
citrate buffer. Preferably, the concentration is from 0.01 to 0.3 molar.
Surfactants that
can be added to the formulation are shown in EP Nos. 270,799 and 268,110.
Additionally, antibodies can be chemically modified by covalent conjugation
to a polymer to increase their circulating half life, for example. Preferred
polymers
are polyoxyethylene polyols and polyethylene glycol (PEG). PEG is soluble in
water
at room temperature and has the general formula: R(O--CHZ --CH2)" O--R where R
can be hydrogen, or a protective group such as an alkyl or alkanol group.
Preferably,
the protective group has between 1 and 8 carbons, more preferably it is
methyl. The
symbol n is a positive integer, preferably between 1 and 1,000, more
preferably
between 2 and 500. The PEG has a preferred average molecular weight between
1000
and 40,000, more preferably between 2000 and 20,000, most preferably between
3,000 and 12,000. Preferably, PEG has at least one hydroxy group, more
preferably it
is a terminal hydroxy group. It is this hydroxy group which is preferably
activated to
react with a free amino group on the inhibitor. However, it will be understood
that the
type and amount of the reactive groups may be varied to achieve a covalently
conjugated PEG/antibody of the present invention. Preferred polymers, and
methods
to attach them to peptides, are shown in U.S. Pat. Nos. 4,766,106; 4,179,337;
4,495,285; and 4,609,546 which are all hereby incorporated by reference in
their
entireties.
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Water soluble polyoxyethylated polyols are also useful in the present
invention.
They include polyoxyethylated sorbitol, polyoxyethylated glucose,
polyoxyethylated
glycerol (POG), etc. POG is preferred, in part because the glycerol backbone
of
polyoxyethylated glycerol is the same backbone occurring naturally in, for
example,
animals and humans in mono-, di-, triglycerides. Therefore, this branching
would not
necessarily be seen as a foreign agent in the body. The POG has a preferred
molecular
weight in the same range as PEG The structure for POG is shown in Knauf et
al., J.
Bio. Chem. 263:15064-15070, 1988, and a discussion of POG/IL-2 conjugates is
found
in U.S. Pat. No. 4,766,106, both of which are hereby incorporated by reference
in their
entireties.
Another drug delivery system for increasing circulatory half life is the
liposome.
Methods of preparing liposome delivery systems are known in the art, see,
e.g:,
Gabizon et al., Cancef° Research 42:4734, 1982; Cafiso, Biochem
Biophys Acta
649:129, 1981; and Szoka, Afzn Rev Bioph~s Ehg 9:467, 1980. Other drug
delivery
systems are known in the art, see, e.g., Poznansky et al., Drug Delivery
Systems (R. L.
Juliano, ed., Oxford, N.Y 1980), pp. 253-31 S; M. L. Poznansky, Pha~~m Revs
36:277,
1984.
After the liquid pharmaceutical composition is prepared, it is preferably
lyophilized to prevent degradation and to preserve sterility. Methods for
lyophilizing
liquid compositions are known to those of ordinary skill in the art. Just
prior to use,
the composition may be reconstituted with a sterile diluent (Ringer's
solution,
distilled water, or sterile saline, for example) which may include additional
ingredients. Upon reconstitution, the composition is preferably administered
to
subjects using those methods that are known to those skilled in the art.
The preferred route of administration is parenterally, but subcutaneous and
intramuscular administration are also suitable. In parenteral administration,
the
compositions of this invention will be formulated in a unit dosage injectable
form
such as a solution, suspension or emulsion, in association with a
pharmaceutically
acceptable parenteral vehicle. Such vehicles are inherently nontoxic and
nontherapeutic. Examples of such vehicles are saline, Ringer's solution,
dextrose
solution, and Hanks' solution. Nonaqueous vehicles such as fixed oils and
ethyloleate
may also be used. A preferred vehicle is 5% dextrose in saline. The vehicle
may
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WO 03/045978 PCT/US02/38164
contain minor mounts of additives such as substances that enhance isotonicity
and
chemical stability, including buffers and preservatives.
The dosage and mode of administration will depend on the individual.
Generally, the compositions are administered so that antibodies are given at a
dose
between 1 ~g/kg and 20 mg/kg, more preferably between 20 ~.g/kg and 10 mg/kg,
most preferably between 1 and 7 mg/kg. Suitably, it is given as an infusion or
as a
bolus dose, to increase circulating levels by 10-20 fold and for 4-6 hours
after the
bolus dose. Continuous infusion may also be used after the bolus dose. If so,
the
antibodies may be infused at a dose between 5 and 20 p.g/lcg/minute, more
preferably
between 7 and 15 ~.g/kghninute. Suitable treatment regimens are disclosed in
WO
00/27428 and WO 00/27433, which are incorporated herein by reference.
Additional suitable formulations and routes of administration are discussed
below and in U.S. Patent No. 5,874,082, incorporated herein by reference.
Typically,
the antibodies are administered by injection, either intravenously or
parenterally. It
may also be possible to obtain compositions which may be topically or orally
administered, or which may be capable of transmission across mucous membranes.
Compositions comprising antibodies may be administered subcutaneously.
Before administration to patients, formulants may be added to the antibodies.
A liquid formulation is preferred. For example, these formulants may include
oils,
polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin,
surfactants, or
bulking agents. Preferably carbohydrates include sugar or sugar alcohols such
as
mono-, di- or polysaccharides, or water soluble glucans. The saccharide or
glucans
can include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose,
maltose,
sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble
starch,
hydroxethyl starch and carboxymethylellulose, or mixtures thereof. Sucrose is
most
preferred. "Sugar alcohol" is defined as a C4 to C8 hydrocarbon having an -OH
group
and includes galacitol, inositol, mannitol, xylitol, sorbitol, glycerol, and
arabitol.
Mannitol is most preferred. These sugars or sugar alcohols mentioned above may
be
used individually or in combination. There is no fixed limit to amount used as
long as
the sugar or sugar alcohol is soluble in the aqueous preparation. Preferably,
the sugar
or sugar alcohol concentration is between 1.0 w/v % and 7.0 w/v %, more
preferable
between 2.0 and 6.0 wlv %. Preferably amino acids include levorotary (L) forms
of
38
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canitine, arginine, and betaine; however, other amino acids may be added.
Preferred
polymers include polyvinylpyrrolidone (PVP) with an average molecular weight
between 2,000 and 3,000, or polyethylene glycol (PEG) with an average
molecular
weight between 3,000 and 5,000. It is also preferred to use a buffer in the
composition to minimize pH changes in the solution before lyophilization or
after
reconstitution. Most any physiological buffer may be used, but citrate,
phosphate,
succinate, and glutamate buffers or mixtures thereof are preferred. Most
preferred is a
citrate buffer. Preferably, the concentration is from 0.01 to 0.3 molar.
Surfactants that
can be added to the formulation are shown in EP Nos. 270,799 and 268,110.
Additionally, antibodies can be chemically modified by covalent conjugation
to a polymer to increase their circulating half life, for example. Preferred
polymers
are disclosed in U.S. Patent Nos. 4,179,337; 4,495,285; and 4,609,546 which
are all
hereby incorporated by reference in their entireties. Preferred polymers are
polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in
water at
room temperature and has the general formula: R(O-CH2-CH2)n0 R where R
can be hydrogen, or a protective group such as an alkyl or aklanol group.
Preferably,
the protective group has between 1 and 8 carbons, more preferably it is
methyl. The
symbol n is a positive integer, preferably between 1 and 1,000, more
preferably
between 2 and 500. The PEG has a preferred average molecular weight between
1,000 and 40,000, more preferably between 2,000 and 20,000, most preferably
between 3,000 and 12,000. Preferably, PEG has at least one hydroxy group, more
preferably it is a terminal hydroxy group. It is this hydroxy group which is
preferably
activated to react with a free amino group on the inhibitor. However, it will
be
understood that the type and amount of the reactive groups may be varied to
achieve a
covalently conjugated PEG/antibody of the present invention.
Water soluble polyoxyethylated polyols are also useful in the present
invention. They include polyoxyethylated sorbitol, polyoxyethylated glucose,
polyoxyethylated glycerol (POG), etc. POG is preferred, in part because the
glycerol
backbone of polyoxyethylated glycerol is the same backbone occurring naturally
in,
for example, animals and humans in mono-, di- and triglycerides. Therefore,
this
branching would not necessarily be seen as a foreign agent in the body. The
POG has
a preferred molecular weight in the same range as PEG. The structure for POG
is
39
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WO 03/045978 PCT/US02/38164
shown in Knauf et al., J. Bio. Chem. 263:15064-15070, X988, and a discussion
of
POG/IL-2 conjugates is found in U.S. Pat. No. 4,766,106, both of which are
hereby
incorporated by reference in their entireties.
Another drug delivery system for increasing circulatory half life is the
liposome. Methods of preparing liposome delivery systems are discussed in
Gabizon
et al., Cancef° Research, 42:4734, 1982; Cafiso, Biochem. Biophys. Acta
649:129,
1981; and Szoka, Any. Rev Biophys. Eng. 9:467, 1980. Other drug delivery
systems
are known in the art and are described in, e.g., Poznansky et al., DRUG
DELIVERY
SYSTEMS (R.L. Juliano, Ed., Oxford, N.Y 1980), pp. 253-315; M.L. Poznansky,
Pharm. Revs. 36:277, 1984.
After the liquid pharmaceutical composition is prepared, it is preferably
lyophilized to prevent degradation and to preserve sterility. Methods for
lyophilizing
liquid compositions are known to those of ordinary skill in -the art. Just
prior to use,
the composition may be reconstituted with a sterile diluent (Ringer's
solution,
distilled water, or sterile saline, for example) which may include additional
ingredients . Upon reconstitution, the composition is preferably administered
to
subjects using those methods that are known to those skilled in the art.
One preferred route of administration is parenterally. In parenteral
administration, the compositions of this invention will be formulated in a
unit dosage
injectable form such as a solution, suspension or emulsion, in association
with a
pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently
nontoxic
and nontherapeutic. Examples of such vehicles are saline, Ringer's solution,
dextrose
solution, and Hanks' solution. Nonaqueous vehicles such as fixed oils and
ethyl
oleate may also be used. A preferred vehicle is 5°6o dextrose in
saline. The vehicle
may contain minor amounts of additives such as substances that enhance
isotonicity
and chemical stability, including buffers and preservatives.
The dosage and mode of administration will depend on the individual.
Generally, the compositions are administered so that antibodies are given at a
dose
between 1 ~,g/kg and 20 mg/kg, more preferably between 20 p.g/leg and 10
mglkg,
most preferably between 0.3 and 7 mg/kg. Preferably, it is given as a bolus
dose, to
increase circulating levels by 10-20 fold and for 4-6 hours after the bolus
dose.
Continuous infusion may also be used after the bolus dose. If so, the
antibodies may
CA 02466931 2004-05-19
WO 03/045978 PCT/US02/38164
be infused at a dose between 5 and 20 ~,g/kg/minute, more preferably between 7
and
15 ~.g/kg/minute. .
The anti-CD40 antibodies suitable for use in the invention include those
disclosed in U.S. Patent Nos. 5,874,082; 6,004,552; 6,056,959; 5,677,165; and
6,051,228, all of which are incorporated herein by reference.
Articles and Methods of Manufacture
The present invention also includes an article of manufacture providing an
interferon and an anti-CD40 antibody for administration to the CNS, brain,
and/or
spinal cord. The article of manufacture can include one or more vials or other
containers that contain a composition suitable for the present method together
with any
carrier, either dried or in liquid form. The interferon and anti-CD40 antibody
will
preferably be supplied in separate vials. However, they may be co-
administered, or
administered within minutes, hours, or days of each other. The article of
manufacture
further includes instructions in the form of a label on the container and/or
in the form of
an insert included in a box in which the container is packaged, for the
carrying out the
method of the invention. The instructions can also be printed on the box in
which the
vial is packaged. The instructions contain information such as sufficient
dosage and
administration information so as to allow the subject or a worker in the field
to
admiuster the interferon and an anti-CD40 antibody. It is anticipated that a
worker in
the field encompasses any doctor, nurse, technician, spouse, or other care-
giver. The
interferon and an anti-CD40 antibody can also be self administered by the
subject.
According to the invention, the interferon and an anti-CD40 antibody can be
used for manufacturing a composition or medicament suitable for intranasal,
conjunctival, transdermal, and/or sublingual administration. For example, a
liquid or
solid composition can be manufactured in several ways, using conventional
techniques. A liquid composition can be manufactured by dissolving an
interferon in
a suitable solvent, such as water, at an appropriate pH, including buffers or
other
excipients, for example to form a solution described herein above.
IFN-(3, like many of the interferons, reportedly serves as an immunomodulator
on a number of target cells (Hall et al., J. Neur~oifnmuhol. 72:11-19, 1997).
For
instance, IFN-[3 appears to exert antiproliferative action on macrophages,
counteract
41
CA 02466931 2004-05-19
WO 03/045978 PCT/US02/38164
"the mitogenic stimulus of certain interferons", augment natural killer cell
activity to
induce an increase in the production of cytotoxic T lymphocytes, and act on
large,
granular lymphocytes to increase killer cell activity. Additionally, IFN-(3
augments the
proliferation of B cells and the secretion of IgM, IgC~ and IgA. It has been
shown to
upregulate class I MHC expression to produce an increase in the presentation
of class I
restricted antigen CD8 cells (Hall et al., J. Nem°oinamunol. 72:11-19,
1997).
Conversely, IFN(3 exerts an inhibitory eiTect on the upregulation of class II
surface
expression. Hence, the immunomodulatory activities of IFN-(3 include, for
example,
influencing systemic immune function, antigen presentation, interferon
production, and
entry of leukocytes into the CNS (Yong et al., Neurology 51:582-689, 1998).
Direct
delivery of the interferon to the lymphatics of the head and neck using the
administration methods of the present invention allows the interferon to
modulate the
immune response, i.e., influence chronic and acute inflammation, wound
healing, and
the autoimmune response; modulate the function by lymphocytes (reduce
lymphocyte
infiltration of the injured tissue); etc.
Given the immunomodulatory role of interferons, the present invention can be
employed to deliver interferons, preferably IFN-(3, and anti-CD40 antibodies
to
various tissues of the head and neck for the treatment and/or prevention of
diseases or
disorders characterized by immune and inflammatory responses. The disorder or
disease of particular interest is multiple sclerosis.
MS presents in the white matter of the CNS and spinal cord as a number of
sclerotic lesions or plaques (Prineas, Demyelinating Diseases, Elsvevier:
Amsterdam
1985; Raine, Multiple Sclerosis, Williams and Wilkins: Baltimore, 1983; Raine
et al.,
J. Nem°oimnaunal. 20:189-201, 1988; and Martin, J. Neural Ti~ansmission
(Supply
49:53-67, 1997). The characteristic MS lesion is inflamed, exhibits axonal
demyelination, axonal degeneration, and is found around small venules. These
characteristics typically evolve early in plaque development and are
hypothesized to
occur as a result of a breakdown in the blood-brain barrier (BBB). As a
consequence
of BBB breakdown, infiltrates consisting of various lymphocytes and
macrophages
enter the brain. The infiltrates cause a decrease in inflammation while
increasing the
presence of glial scar tissue, and elicit incomplete remyelination (Martin, J.
Neural
Tra~snaissio~ (Supply 49:53-67, 1997). Further, it is hypothesized that this
apparent
42
CA 02466931 2004-05-19
WO 03/045978 PCT/US02/38164
immunologic attack targets not only the myelin sheath, but also the
oligodendrocytes
imperative to.CNS myelin production. Interferons are known to effectively
reduce the
symptoms of MS. For example, interferon-(3 (IFN-(3) has received interest as a
treatment fox relapsing-remitting MS. In addition, interest has also developed
in the
use of interferon-i as an effective treatment in autoimmune diseases, such as
MS.
See, for example, U.S. Patent No. 6,060,450, herein incorporated by reference.
The immunomodulating activity of IFN-J3 influences the clinical symptoms of
MS. While the present invention is not bound by the mechanism of IFN-(3
action, the
central nervous system damage that ensues in MS patients is believed to be due
to the
delayed-type hypersensitivity response. This is a cell-mediated response.
First, T
cells are activated by antigens and conveyed to the lymphoid organ
(activation). The
lymphoid organ then activates these T cells while continuing to recruit more T
cells to
its site (recruitment). The activated lymphocytes proliferate and return to
circulation
(expansion). Once returned to circulation, the activated lymphocytes migrate
through
the blood stream, crossing endothelial cells lining the capillaries
(migration). These
migrating lymphocytes and macrophages target, and axe attracted to the area of
inflammation (attraction). Resulting from this attraction, other lymphocytes
continue
to the area of inflammation and tissue is destroyed (tissue destruction).
Subsequently,
the acute response is suppressed (via tissue destruction), and repair of the
area of
inflammation, which is quite limited in MS, may commence (repair) (Kelley, J,
of
Neuroscience Nursing 28:114-120, 1996). Therefore, the migration of activated
lymphocytes from the blood initiates the immune response, thexeby allowing BBB
penetration of activated lymphocytes.
Evidence suggests that the immunomodulatory activity of IFN-(3 inhibits IFN-'y
upregulation by inhibiting the expansion stage of the delayed-type
hypersensitivity
response and thereby influences the clinical symptoms of MS. Particularly, the
reduction of myelin damage appears to occur as a result of two hypothesized
mechanisms of IFN-(3 action: (1) inhibition of 1FN-y-induced macrophage
activation,
and (2) inhibition of monocytotic TNF release (Kelly, J. Neu~osciefzce Nursing
28:114-
120, 1996). Potential sites of IFN-(3 action construed by these hypotheses
involve
systemic immune function, antigen presentation, interferon production, and
entry of
43
CA 02466931 2004-05-19
WO 03/045978 PCT/US02/38164
leukocytes into the CNS (Yong et al., Neurology 51:682-689, 1998). Each of
these sites
has been elaborated in human and animal experiments of MS.
An "effective amount" of an interferon is an amount sufficient to prevent,
treat, reduce and/or ameliorate the symptoms and/or underlying causes of MS.
In
some instances, an "effective amount" is sufficient to eliminate the symptoms
of those
diseases and, perhaps, overcome the disease itself. An important aspect of the
invention is that the dose of interferon may be decreased with the
administration of
the anti-CD40 antibody. In addition, interferon treatment may be continued for
a
longer time if the patient is also receiving anti-CD40 antibody therapy. In
the context
of the present invention, the terms "treat" and "therapy" and the like refer
to alleviate,
slow the progression, prophylaxis, attenuation or cure of existing disease.
Prevent, as
used herein, refers to delaying, slowing, inhibiting, reducing or ameliorating
the onset
of MS. It is preferred that a sufficient quantity of the interferon be applied
in non-
toxic levels in order to provide an effective level of activity within the CNS
to prevent
or treat MS. The methods of the present invention may be used with any
mafnmal.
Exemplary mammals include, but are not limited to rats, cats, dogs, horses,
cows,
sheep, pigs, and more preferably humans.
An effective amount of an interferon to treat MS using the administration
methods of the present invention will be sufficient to reduce or lessen the
clinical
symptoms of MS. For instance, experimental allergic encephalomyelitis (EAE) is
commonly used as an animal model of MS. A therapeutically effect amount of an
interferon delivered by the methods of the present invention will be such as
to improve
the clinical symptoms of EAE in the experimental animal (i.e., rats or mice).
EAE in
rats is scored on a scale of 0-4: 0, clinically normal; 1, flaccid tail
paralysis; 2, hind
limb weakness; 3, hind limb paralysis; 4, front and hind limb afFected. An
effective
amount of interferon delivered by the methods of the present invention will be
effective
if there is at least a 30%, 40%, 50% or greater reduction in the mean
cumulative score
over several days following the onset of disease symptoms in comparison to the
control
group.
Furthermore, effective treatment of MS may be examined in several alternative
ways including extended disability status scale (EDSS) , appearance of
exacerbations,
or MRI. If any of these indicia show that the interferon treatment is losing
its
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effectiveness, or if symptoms worsen, then co-treatment with anti-CD40
antibodies is
warranted. According to the invention, the anti-CD40 antibody treatment will
allow the
continued administration of interferon. Satisfying any of the following
criteria
evidences effective treatment.
The EDSS is a means to grade clinical impairment due to MS (Kurtzke,
Neurology 33:1444, 1983). Eight functional systems are evaluated for the type
and
severity of neurologic impairment. Briefly, prior to treatment, impairment in
the
following systems is evaluated: pyramidal, cerebellar, brainstem, sensory,
bowel and
bladder, visual, cerebral, and other. Follow-ups are conducted at defined
intervals.
The scale ranges from 0 (normal) to 10 (death due to MS). A decrease of one
full step
defines an effective treatment in the context of the present invention
(Kurtzke, Ann.
Neurol. 36:573-579, 1994); a decrease of 0.5 step if EDSS score is >5.5 is
also within
the definition of effective treatment.
Exacerbations are defined as the appearance of a new symptom that is
attributable to MS and accompanied by an ' appropriate new neurologic
abnormality
(IFN-(3 MS Study Group, supra). In addition, the exacerbation must last at
least 24
hours and be preceded by stability or improvement for at least 30 days.
Standard
neurological examinations result in the exacerbations being classified as
either mild,
moderate, or severe according to changes in a Neurological Rating Scale (Sipe
et al.,
Neurology 34:1368, 1984). An annual exacerbation rate and proportion of
exacerbation-free patients are determined. Therapy is deemed to be effective
if there
is a statistically significant difference in the rate or proportion of
exacerbation-free
patients between the treated group and the placebo group for either of these
measurements. In addition, time to first exacerbation and exacerbation
duration and
severity may also be measured. A measure of effectiveness as therapy in this
regard is
a statistically significant difference in the time to first exacerbation or
duration and
severity in the treated group compared to control group.
MRI can be used to measure active lesions using gadolinium-DTPA-enhanced
imaging (McDonald et al., Ann. Neuf~ol. 36:14, 1994) or the location and
extent of
lesions using T1 and T2 -weighted techniques. Briefly, baseline MRIs are
obtained.
The same imaging plane and patient position are used for each subsequent
study.
Areas of lesions are outlined and summed slice by slice for total lesion area.
Three
CA 02466931 2004-05-19
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analyses may be done: evidence of new lesions, rate of appearance of active
lesions,
and percentage change in lesion area (Paty et al., Neurology 43:665, 1993).
Improvement due to therapy is established when there is a statistically
significant
improvement in an individual patient compared to baseline or in a treated
group
versus a placebo group.
The present invention will now be illustrated by reference to the following
examples which set forth particularly advantageous embodiments. However, it
should
be noted that these embodiments are illustrative and are not to be construed
as
restricting the invention in any way.
EXAMPLES
Patient populations are selected on the basis of the following criteria:
symptoms of MS that are incompletely alleviated by administration of
interferon-(31b
or other MS therapeutics, including indicia for discontinuing interferon-(31b
treatment
because of decreased effectiveness. These criteria are evaluated using
standard
methods of measuring MS disease activity and progression. Preferred criteria
for
selecting patients are (a) at least one relapse in the previous six months
despite
treatment with interferon beta-lb or other MS therapeutics; (b) at least one
enhancing
lesion at baseline MRI scan; clinically significant progression in disability
over the
previous six months despite treatment with interferon beta-lb or other MS
therapeutics. The patients are divided into two treatment groups and given a
treatment option as described in Examples 1 and 2.
EXAMPLE 1
TREATMENT WITH ANTI-CD40 ANTIBODY
Patients will be evaluated at baseline for EDSS score, number of enhancing
lesions on MRI scan, and lesion volume on MRI scan. These measures will be
obtained on a repeated basis over the course of 1-2 years. Patients will
receive an
anti-CD40 antibody such as 15B8 in a single cycle at a dose of 0.03 mg/kg to
10
mg/leg via intravenous infusion weekly for four to eight doses. Efficacy of
the anti-
CD40 antibody in reducing the frequency of enhancing lesions and lesion volume
on
MRI and reduction in the proportion of patients experiencing a confirmed
disease
46
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progression will be assessed at yearly intervals. Clinical and MRI course of
patients
receiving the anti-CD40 antibody will be compared against patients receiving a
matching placebo.
EXAMPLE 2
S TREATMENT WITH ANTI-CD40 ANTIBODY AND ~-INTERFERON
Patients will be evaluated at baseline for EDSS score, number of enhancing
lesions on MRI scan, and lesion volume on MRI scan. These measures will be
obtained on a repeated basis over the course of 1-2 years. Some patients will
receive
an anti-CD40 antibody such as 15B8 in a single cycle at a dose of 0.03 mg/kg
to 10
mg/kg via intravenous infusion weekly for four to eight doses. Some patients
will
receive a matching placebo on the same time course as those receiving the anti-
CD40
antibody. All patients will also receive the commercially approved dose of
beta-
interferon (8 MIU subcutaneously every other day for interferon beta-lb
[Betaseron~], 6 MIU every other day intramuscularly for interferon beta-la
[Avonex~] or 12 MIU every other day subcutaneously for interferon beta-la
[Rebif~
if approved for use in the US at the time of this study]. Efficacy of the anti-
CD40
antibody in reducing the frequency of enhancing lesions and lesion volume on
MRI
and reduction in the proportion of patients experiencing a confirmed disease
progression will be assessed at yearly intervals. Clinical and MRI course of
patients
receiving the anti-CD40 antibody plus interferon will be compared against
patients
receiving interferon alone.
EXAMPLE 3
EVALUATION OF PATIENT TREATMENTS
Patients treated as described in Examples 1 and 2 are evaluated to determine
disease progression. In one test, the brain lesions are evaluated by MRI.
The first set of experiments is a proof of concept study to show benefit of
anti-
CD40 antibody either alone in patients who are refractory to other therapies
including
interferon or in combination with continued use of Interferon-(3-lb in
patients at high
risk for discontinuation of therapy (MRI based primary endpoint with clinical
secondary endpoints). A second Phase III pivotal study of 15B8 as monotherapy
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versus add-on therapy following failed treatment with interferon or glatirimer
acetate
(clinical endpoint based primary endpoint with MRI based secondary endpoints)
is
then conducted.
The present invention has been described with reference to specific
embodiments. However, this application is intended to cover those changes and
substitutions which may be made by those skilled in the art without departing
from
the spirit and the scope of the appended claims.
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1
SEQUENCE LISTING
<110> Masuoka, Lorianne K.
Kwan, Min Fun
<120> ANTAGONTST ANTI-CD40 MONOCLONAL ANTIBODY
THERAPY FOR MULTIPLE SCLEROSIS TREATMENT
<130> PP-17035.001/200130.528P1
<140> US
<141> 2001-1l-26
<l60> 2
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 16l
<212> PRT
<213> Homo sapiens
<400> 1
Met Cys Asp Leu Pro Glu Thr His Ser Leu Asp Ser Arg Asn Thr Thr
1 5 10 15
Val Leu Leu His Gln Met Arg Arg Ile Ser Pro Ser Leu Cys Leu Lys
20 25 30
Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Val Lys Gly Ser Lys
35 40 45
Ile Gln Lys Ala His Thr Thr Thr Val Leu His Lys Val Leu Gln Gln
50 55 60
Tle Val Thr Leu Phe Asn Thr Arg Ser Val Gly Trp Asn G1u Thr G1y
65 70 75 80
Leu Glu Lys Leu Phe Thr Glu Phe Tyr Gln His Trp Glu Val Leu Glu
85 90 95
Pro Cys Leu Leu Asn Glu Leu Gly Val Glu Gly Leu Ser Gln Ala Met
100 105 110
Thr Thr Pro Asn Ala Val Lys Ser Tyr Phe Gln Gly Ile Ser Leu Tyr
115 120 125
Leu Glu Lys Lys Glu Glu Ser Leu Cys Thr Trp Glu Val Gly Ala Glu
130 135 140
Ile Met Arg Ser Phe Phe Phe Ser Ser Asn Leu Gln Val Arg Leu Ile
145 150 155 160
Ala
<210> 2
<2l1> 483
<212> DNA
<213> Homo sapiens
CA 02466931 2004-05-19
WO 03/045978 PCT/US02/38164
2
<400> 2
atgtgcgacc tgccggaaac ccactctctg gactctcgta acaccaccgt tctgctgcac 60
cagatgcgtc gtatctctcc gtctctgtgc ctgaaagacc gtcacgactt cggtttcccg 120
caggaagaag ttaaaggttc taaaatccag aaagctcaca ccaccaccgt tctgcacaaa 180
gttctgcagc agatcgttac cctgttcaac acccgttctg ttggttggaa cgaaaccggt 240
ctggaaaaac tgttcaccga attctaccag cactgggaag ttctggaacc gtgcctgctg 300
aacgaactgg gtgttgaagg tctgtctcag gctatgacca ccccgaacgc tgttaaatct 360
tacttccagg gtatctctct gtacctggaa aaaaaagaag aatctctgtg cacctgggaa 420
gttggtgctg aaatcatgcg ttctttcttc ttctcttcta acctgcaggt tcgtctgatc 480
get 483
