interferon-tau compositions and methods
United States Patent: 7,214,367
Issued: May 8, 2007
Inventors: Soos; Jeanne M.
(Waltham, MA), Schiffenbauer; Joel (Gainesville, FL), Johnson; Howard
Marcellus (Gainesville, FL)
Assignee: University of
Appl. No.: 10/694,247
Filed: October 27, 2003
Web Seminars -- Pharm/Biotech/etc.
The present invention includes
interferon-tau (IFN.tau.) pharmaceutical compositions useful for oral
administration to treat cancers, autoimmune disorders (particularly
multiple sclerosis), cell proliferative disorders and viral disease.
SUMMARY OF THE
In one aspect, the present invention
includes an improvement in a method of treating a disease condition in a
mammal (e.g., mouse, dog or human) responsive to treatment by interferon-tau
(IFN.tau.). The improvement comprises orally administering a
therapeutically-effective amount of IFN.tau.. The orally-administered
IFN.tau. is preferably ingested by the mammal. In a general embodiment,
the IFN.tau. is orally-administered at a dosage of between about
1.times.10.sup.5 and about 1.times.10.sup.8 units per day, preferably at a
dosage of between about 1.times.10.sup.6 and about 1.times.10.sup.7 units
per day. The IFN.tau. may be, for example, ovine IFN.tau. (OvIFN.tau.),
e.g., a polypeptide having the sequence represented as SEQ ID NO:2, or a
human IFN.tau. (HuIFN.tau.), e.g., a polypeptide having the sequence
represented as SEQ ID NO:4 or SEQ ID NO:6.
In one embodiment, the disease condition is an immune system disorder,
such as an autoimmune disorder (e.g., multiple sclerosis (MS), type I
(insulin dependent) diabetes mellitus, lupus erythematosus, amyotrophic
lateral sclerosis, Crohn's disease, rheumatoid arthritis, stomatitis,
asthma, allergies or psoriasis). MS is particularly amenable to treatment
using the methods of the present invention.
In another embodiment, the disease condition is a cell proliferation
disorder, such as a cancer (e.g., hairy cell leukemia, Kaposi's Sarcoma,
chronic myelogenous leukemia, multiple myeloma, superficial bladder
cancer, skin cancer (basal cell carcinoma and malignant melanoma), renal
cell carcinoma, ovarian cancer, low grade lymphocytic and cutaneous T cell
lymphoma, and glioma).
In yet another embodiment, the disease condition is a viral disease (e.g.,
hepatitis A, hepatitis B, hepatitis C, non-A, non-B, non-C hepatitis,
Epstein-Barr viral infection, HIV infection, herpes virus (EB, CML, herpes
simplex), papilloma, poxvirus, picorna virus, adeno virus, rhino virus,
HTLV I, HTLV II, and human rotavirus).
In another aspect, the invention includes a method of treating an
autoimmune disorder in a subject (e.g., a human subject), by orally
administering a therapeutically-effective amount of interferon-tau (IFN.tau.)
to the subject. The orally-administered IFN.tau. is preferably ingested by
the subject. Examples of autoimmune conditions amenable to treatment,
dosages, and sources of IFN.tau. are as presented above.
The invention also includes a method of decreasing the severity or
frequency of a relapse of multiple sclerosis (MS) in a human suffering
from MS, by orally administering a therapeutically-effective amount of
interferon-tau (IFN.tau.) to the human. Examples of dosages and sources of
IFN.tau. are as presented above.
In another aspect, the invention includes a method of treating a cell
proliferation disorder in a subject (e.g., a human subject), by orally
administering a therapeutically-effective amount of interferon-tau (IFN.tau.)
to the subject. The orally-administered IFN.tau. is preferably ingested by
the subject. Examples of cell proliferation disorders amenable to
treatment, dosages, and sources of IFN.tau. are as presented above.
In still another aspect, the invention includes a method of treating a
viral disease in a subject (e.g., a human subject), by orally
administering a therapeutically-effective amount of interferon-tau (IFN.tau.)
to the subject. The orally-administered IFN.tau. is preferably ingested by
the subject. Examples of viral diseases amenable to treatment, dosages,
and sources of IFN.tau. are as presented above.
A further aspect of the invention includes a method of enhancing fertility
in a female mammal (e.g., a human female), by orally administering a
therapeutically-effective amount of interferon-tau (IFN.tau.) to the
mammal. Examples of dosages and sources of IFN.tau. are as presented
OF THE INVENTION
The first IFN.tau. to be identified was ovine IFN.tau. (OvIFN.tau.).
Several isoforms of the 18 19 kDa protein were identified in conceptus
(the embryo and surrounding membranes) homogenates (Martal., et al.,
1979). Subsequently, a low molecular weight protein released into
conceptus culture medium was purified and shown to be both heat labile and
susceptible to proteases (Godkin, et al., 1982). OvIFN.tau. was originally
called ovine trophoblast protein-one (oTP-1) because it was the primary
secretory protein initially produced by trophectoderm of the sheep
conceptus during the critical period of maternal recognition in sheep. One
isolate of mature OvIFN.tau. is 172 amino acids in length (SEQ ID NO:2).
IFN.tau.s with similar characteristics and activities have been isolated
from other ruminant species including cows and goats (Bartol, et al.,
1985; Gnatek, et al., 1989; Helmer, et al., 1987; and Imakawa, et al.,
1989). Bovine IFN.tau. (BoIFN.tau.) and OvIFN.tau. have (i) have similar
functions in maternal recognition of pregnancy, and (ii) share a high
degree of amino acid and nucleotide sequence homology between mature
proteins. The nucleic acid sequence homology between OvIFN.tau. and
BoIFN.tau. is 76.3% for the 5' non-coding region, 89.7% for the coding
region, and 91.9% for the 3' non-coding region. The amino acid sequence
homology is 80.4%.
Antisera to all the IFN.tau.s cross-react. This is not unexpected since
the species specific forms of IFN.tau. are more closely homologous to each
other than to the IFNs.alpha. from the identical species (Roberts, et al.,
1992). Relative to other interferons, OvIFN.tau. shares about 45 to 68%
amino acid homology with Interferon-.alpha. and the greatest sequence
similarity with the interferon-.omega.s (IFN.omega.s) of about 68%.
While IFN.tau. displays many of the activities classically associated with
type I IFNs (see Table 1, above -- see Original Patent), considerable
differences exist between it and the other type I IFNs. The most prominent
difference is its role in pregnancy, detailed above. Also different is
viral induction. All type I IFNs, except IFN.tau., are induced readily by
virus and dsRNA (Roberts, et al., 1992). Induced IFN.alpha. and IFN.beta.
expression is transient, lasting approximately a few hours. In contrast,
IFN.tau. synthesis, once induced, is maintained over a period of days (Godkin,
et al., 1982). On a per-cell basis, 300-fold more IFN.tau. is produced
than other type I IFNs (Cross and Roberts, 1991).
Other differences may exist in the regulatory regions of the IFN.tau.
gene. For example, transfection of the human trophoblast cell line JAR
with the gene for bovine IFN.tau. resulted in antiviral activity while
transfection with the bovine IFN.beta. gene did not. This implies unique
transacting factors involved in IFN.tau. gene expression. Consistent with
this is the observation that while the proximal promoter region (from 126
to the transcriptional start site) of IFN.tau. is highly homologous to
that of IFN.alpha. and IFN.beta.; the region from -126 to -450 is not
homologous and enhances only IFN.tau. expression (Cross and Roberts,
1991). Thus, different regulatory factors appear to be involved in IFN.tau.
expression as compared with the other type I IFNs.
IFN.tau. expression may also differ between species. For example, although
IFN.tau. expression is restricted to a particular stage (primarily days 13
21) of conceptus development in ruminants (Godkin, et al., 1982),
preliminary studies suggest that the human form of IFN.tau. is
constitutively expressed throughout pregnancy (Whaley, et al., 1994).
B. Production of IFN.tau.
IFN.tau. polypeptides suitable for use in the methods of the present
invention may produced in any of a number of ways. For example, they may
be purified from animal tissues in which they are expressed, synthesized
using a peptide synthesizer or produced recombinantly.
Recombinant IFN.tau. protein may be produced from any selected IFN.tau.
polynucleotide fragment using a suitable expression system, such as
bacterial or yeast cells. The isolation of IFN.tau. nucleotide and
polypeptide sequences is described in Bazer, et al. (1994). For example,
Bazer, et al., describe the identification and isolation of a human
IFN.tau. gene. A synthetic nucleotide sequence encoding a mature human
interferon-.tau. (HuIFN.tau.) protein is presented herein as SEQ ID NO:3.
SEQ ID NO:4 is the corresponding amino acid sequence for a mature
HuIFN.tau.1 protein. SEQ ID NO:5 is the nucleotide sequence, excluding
leader sequence, of genomic DNA clone HuIFN.tau.3, a natural HuIFN.tau.
gene, and SEQ ID NO:6 is the predicted amino acid sequence of a mature
human IFN.tau. protein encoded by the sequence represented as SEQ ID NO:5.
To make an IFN.tau. expression vector, an IFN.tau. coding sequence (e.g,
SEQ ID NO:1) is placed in an expression vector, e.g., a bacterial
expression vector, and expressed according to standard methods. Examples
of suitable vectors include lambda gt11 (Promega, Madison Wis.); pGEX
(Smith, et al., 1985); pGEMEX (Promega); and pBS (Stratagene, La Jolla
Calif.) vectors. Other bacterial expression vectors containing suitable
promoters, such as the T7 RNA polymerase promoter or the tac promoter, may
also be used. Cloning of the OvIFN.tau. synthetic polynucleotide into a
modified pIN III omp-A expression vector is described in the Materials and
For the experiments described herein, the OvIFN.tau. coding sequence
present in SEQ ID NO:1 was cloned into a vector, suitable for
transformation of yeast cells, containing the methanol-regulated alcohol
oxidase (AOX) promoter and a Pho1 signal sequence. The vector was used to
transform P. pastoris host cells and transformed cells were used to
express the protein according to the manufacturer's instructions.
Other yeast vectors suitable for expressing IFN.tau. for use with methods
of the present invention include 2 micron plasmid vectors (Ludwig, et al.,
1993), yeast integrating plasmids (YIps; e.g., Shaw, et al., 1988), YEP
vectors (Shen, et al., 1986), yeast centromere plasmids (YCps; e.g.,
Ernst, 1986), and other vectors with regulatable expression (Hitzeman, et
al., 1988; Rutter, et al., 1988; Oeda, et al., 1988). Preferably, the
vectors include an expression cassette containing an effective yeast
promoter, such as the MF.alpha.1 promoter (Ernst, 1986; Bayne, et al.,
1988, GADPH promoter (glyceraldehyde-3-phosphate-dehydrogenase; Wu, et
al., 1991) or the galactose-inducible GAL10 promoter (Ludwig, et al.,
1993; Feher, et al., 1989; Shen, et al., 1986). The yeast transformation
host is typically Saccharomyces cerevisiae, however, as illustrated above,
other yeast suitable for transformation can be used as well (e.g.,
Schizosaccharomyces pombe, Pichia pastoris and the like).
Further, a DNA encoding an IFN.tau. polypeptide can be cloned into any
number of commercially available vectors to generate expression of the
polypeptide in the appropriate host system. These systems include the
above described bacterial and yeast expression systems as well as the
following: baculovirus expression (Reilly, et al., 1992; Beames, et al.,
1991; Clontech, Palo Alto Calif.); plant cell expression, transgenic plant
expression (e.g., Gelvin and Schilperoot, 1988), and expression in
mammalian cells (Clontech, Palo Alto Calif.; Gibco-BRL, Gaithersburg Md.).
These recombinant polypeptides can be expressed as fusion proteins or as
native proteins. A number of features can be engineered into the
expression vectors, such as leader sequences which promote the secretion
of the expressed sequences into culture medium. The recombinantly produced
polypeptides are typically isolated from lysed cells or culture media.
Purification can be carried out by methods known in the art including salt
fractionation, ion exchange chromatography, and affinity chromatography.
Immunoaffinity chromatography can be employed, as described above, using
antibodies generated based on the IFN.tau. polypeptides.
In addition to recombinant methods, IFN.tau. proteins or polypeptides can
be isolated from selected cells by affinity-based methods, such as by
using appropriate antibodies. Further, IFN.tau. peptides may be chemically
synthesized using methods known to those skilled in the art.
III. Effectiveness of Orally-Administered IFN.tau.
Experiments performed in support of the present invention and detailed
below demonstrate that orally-administered IFN.tau. polypeptide
compositions are comparable in efficacy to injected IFN.tau. compositions
with respect to the treatment of diseases or disease conditions which
benefit from treatment with IFN.tau..
Not only was orally-administered IFN.tau. effective at treating a disease
benefiting from IFN.tau. treatment (EAE), but the oral route of
administration resulted in unexpected advantages relative to treatment
with injected IFN.tau. compositions. For example, orally-administered
IFN.tau. resulted in a significantly lower level of anti-IFN.tau.
antibodies in the serum of treated individuals (see Example 7). This is
beneficial because the orally-administered IFN.tau. is therefore less
likely to be rendered ineffective by a host immune response (i.e.,
desensitization to the treatment and/or dose level is significantly
decreased), and the individual receiving the treatment is less likely to
suffer adverse side effects as a result of such an immune response.
Results of experiments demonstrating these and related findings are
A. Orally-Administered IFN.tau. Inhibits Development of EAE
The efficacy of IFN.tau. in treating autoimmune disorders may be evaluated
in rodents with experimental allergic encephalomyelitis (EAE; Zamvil and
Steinman, 1990), an animal model of antigen-induced autoimmunity. EAE
resembles human multiple sclerosis (MS) both in its clinical and
pathological manifestations and can thus be used to assess treatments for
human autoimmune diseases such as MS. EAE is a T-cell-mediated
inflammatory autoimmune demyelinating disease induced by immunizing
susceptible mouse, rat or guinea pig strains with myelin basic protein (MBP)
or with encephalitogenic peptide fragments. Genetic susceptibility in the
model animal strains is based in part on the capacity of encephalitogenic
peptides to bind to particular class II major histocompatibility complex (MHC-II)
molecules (Fritz, et al., 1983; Wraith, et al., 1989). In particular, mice
having the H-2.sup.u haplotype are susceptible to EAE. Susceptible mouse
strains include PL/J mice (Klein, et al., 1983), (PL/J.times.SJL)F.sub.1
mice (Zamvil and Steinman, 1990; Wraith, et al., 1989), B10.PL mice (Figuero,
et al., 1982), NZW mice (Kotzin, et al., 1987), and (NZB.times.NZW)F1 (Kotzin,
et al., 1987) mice.
Gamma-interferon (IFN.gamma.) and beta-interferon (IFN.beta.) have been
demonstrated to be effective in treating multiple sclerosis (Johnson, et
al., 1994; IFN.beta. Multiple Sclerosis Study Group, 1993). In fact,
IFN.beta. has been approved by the FDA as a therapeutic for multiple
sclerosis. Although .beta.-IFN is effective against MS, it has relatively
high toxicity, and as a result, has a variety of undesirable side effects.
As described herein, however, IFN.tau. has significantly lower toxicity
that other interferons and may therefore exhibit fewer undesirable side
In experiments performed in support of the present invention and detailed
in Example 1, orally-administered and injected IFN-.tau. was tested for
its ability to prevent the induction of EAE. EAE was induced in New
Zealand White (NZW) mice by immunization with bovine myelin basic protein
(bMBP). Recipient NZW mice received OvIFN.tau. by either i.p. injection or
oral feeding 48 hours prior to, on the day of, and 48 hours after
immunization with bovine myelin basic protein (bMBP) for induction of
experimental allergic encephalomyelitis (EAE).
Both oral feeding and i.p. injection of OvIFN.tau. protected against EAE
(Example 1, Table 3). All animals that received IFN.tau. via i.p.
injection, and 7 of 9 animals that received IFN.tau. orally, were
protected from symptoms of EAE. Furthermore, anti-OvIFN.tau. monoclonal
antibody HL127 was effective at partially neutralizing the ability of the
OvIFN.tau. to block EAE. These experiments demonstrate that
orally-administered IFN.tau. is effective in treating symptoms of EAE, an
animal model of multiple sclerosis.
B. OvIFN.tau. is Present in Sera Following Oral Administration
To confirm that orally-administered IFN.tau. enters the circulation, the
sera of mice that received IFN.tau. by i.p injection or by oral
administration were tested for the presence of IFN.tau. using a cytopathic
effect (antiviral) assay (Familetti, et al., 1981) as described in Example
The results are shown in FIG. 1 (see Original Patent). Specific activities
are expressed in antiviral units/mg protein obtained from antiviral assays
using MDBK cells. OvIFN.tau. was detected for up to two 2 hours following
oral feeding (filled bars) at levels of 200 U/ml. These data indicate that
orally-administered IFN.tau. enters the circulation and remains in serum
for about two hours after being administered.
C. Lack of Toxicity From Orally-administered OvIFN.tau.
It has been previously demonstrated that the type I IFNs IFN.alpha. and
IFN.beta. induced toxic side effects manifested as flu like symptoms,
fever, nausea and malaise when used as therapeutics in humans (Degre,
1974; Fent and Zbinden, 1987). In contrast, OvIFN.tau. exhibits a
remarkable lack of toxicity both in vitro and in vivo. Experiments
performed in support of the present invention compared OvIFN.tau. with
IFNs .alpha. and .beta. for induction of toxicity as measured by
lymphocyte depression in peripheral blood when given via oral feeding.
Blood was obtained from the tail and white blood cells (WBC) counts were
enumerated using a hemocytometer. Differential WBC counts were performed
on Wright-Giemsa-stained blood smears.
The results are shown in Tables 2a, 2b and 2c (see Original Patent).
Significant levels of toxicity were detected in mice fed either IFN
.alpha. and .beta. while no significant lymphocyte depression was detected
in mice fed 10.sup.5, 2.times.10.sup.5 or 5.times.10.sup.5 U of OvIFN.tau.
or PBS alone. These data suggest that orally-administered OvIFN.tau. has
significantly-reduced toxicity with respect to other type I IFNs.
D. OvIFN.tau. Prevents Chronic Relapse of EAE
In addition to preventing the onset of symptoms associated with EAE,
orally-administered OvIFN.tau. prevents paralysis in a chronic-relapsing
model of EAE, as detailed in Example 3. Whereas 5/5 mice immunized with
MBP (to induce EAE) which did not receive OvIFN.tau. treatment developed
chronic relapsing paralysis, 4/5 animals treated with OvIFN.tau. (either
i.p. injection or oral feeding, administered every 48 hours) were fully
protected from the disease (FIGS. 2B and 2C). These data further support
the results described above, and indicate that oral administration of
IFN.tau. can block the development of chronic relapsing EAE. The
experiments also suggest that oral-administration of IFN.tau. as
infrequently as once every 48 hours, over an extended period of time, is
as effective as i.p. injection at treating a disease condition responsive
to treatment by interferon-tau.
E. Histological Analyses of Spinal Chord from EAE Mice following Oral
Administration of IFN.tau..
The ability of OvIFN.tau. to prevent EAE was also assayed by analyzing the
effect of OvIFN.tau. treatment on cellular consequences of the disease,
manifested in the central nervous system (CNS) as lymphocytic lesions in
spinal cord white matter. The lesions are indicative of the extent of
lymphocyte infiltration into the CNS. MBP-immunized mice were either not
treated (control) or treated with OvIFN.tau. by oral or i.p. routes, and
sections of the spinal cord lumbar region were stained and evaluated for
lymphocytes as described in Example 4. Lymphocytic lesions were present in
spinal cord white matter of control animals (FIG. 3A), but not in mice
treated with OvIFN.tau. by i.p. injection (FIG. 3B) or oral feeding (FIG.
3C). These data indicate that the protective effect of IFN.tau. is
associated with inhibition of lymphocyte infiltration of the CNS. Further,
the data demonstrate that IFN.tau. treatment inhibits cellular
manifestation of the autoimmune disease, rather than simply masking
F. Cessation of Treatment with OvIFN.tau. Results in Relapsing Paralysis
Experiments detailed in Example 6 were performed to determine the type and
duration of treatment effective to prevent EAE in mice injected with MBP.
The mice were protected from EAE by OvIFN.tau. treatment via i.p.
injection or oral feeding (every 48 hours) as long as the treatment
persisted (58 days in Example 6), but developed symptoms of the disease
after OvIFN.tau. treatment was stopped (FIG. 5). These results suggest
that while IFN.tau. may not cure an autoimmune condition like EAE (e.g.,
MS), it is an effective treatment that inhibits the pathological
manifestations of the condition so long as treatment is continued.
G. Oral Administration of OvIFN.tau. Reduces Anti-OvIFN.tau. Antibody
As detailed in Example 7, one advantage of orally-administered (as opposed
to injected) IFN.tau. treatment is a reduction in the anti-IFN.tau.
antibody titer in individuals receiving the oral treatment. After removal
of OvIFN.tau. treatment, mice from each treatment group were bled and sera
were examined for the presence of anti-OvIFN.tau. antibodies by ELISA.
Whereas mice receiving IFN.tau. by i.p. injection exhibited elevated
levels of anti-IFN.tau. antibodies, animals receiving IFN.tau. by oral
feeding exhibited much lower anti-IFN.tau. antibody titers (typically 3 to
5-fold lower). As expected mice which received no OvIFN.tau. treatment
displayed no anti-OvIFN.tau. antibodies.
The sera were also examined for their ability to neutralize OvIFN.tau.
antiviral activity on the MDBK cell line. None of the sera from either i.p.
injected or orally fed mice possessed neutralizing activity (Table 4 (see
Original Patent)). These results suggest that oral feeding of OvIFN.tau.
largely circumvents an antibody response directed against the OvIFN.tau.
protein. Such a reduced antibody response in orally-treated subjects
reduces the chance of undesirable immune system-related side effects of
A. IFN.tau. as a Treatment for Immune System Disorders
Diseases which may be treated using methods of the present invention
include autoimmune, inflammatory, proliferative and hyperproliferative
diseases, as well as cutaneous manifestations of immunologically mediated
diseases. In particular, methods of the present invention are advantageous
for treating conditions relating to immune system hypersensitivity. There
are four types of immune system hypersensitivity (Clayman, 1991). Type I,
or immediate/anaphylactic hypersensitivity, is due to mast cell
degranulation in response to an allergen (e.g., pollen), and includes
asthma, allergic rhinitis (hay fever), urticaria (hives), anaphylactic
shock, and other illnesses of an allergic nature. Type II, or autoimmune
hypersensitivity, is due to antibodies that are directed against perceived
"antigens" on the body's own cells. Type III hypersensitivity is due to
the formation of antigen/antibody immune complexes which lodge in various
tissues and activate further immune responses, and is responsible for
conditions such as serum sickness, allergic alveolitis, and the large
swellings that sometimes form after booster vaccinations. Type IV
hypersensitivity is due to the release of lymphokines from sensitized
T-cells, which results in an inflammatory reaction. Examples include
contact dermatitis, the rash of measles, and "allergic" reactions to
The mechanisms by which certain conditions may result in hypersensitivity
in some individuals are generally not well understood, but may involve
both genetic and extrinsic factors. For example, bacteria, viruses or
drugs may play a role in triggering an autoimmune response in an
individual who already has a genetic predisposition to the autoimmune
disorder. It has been suggested that the incidence of some types of
hypersensitivity may be correlated with others. For example, it has been
proposed that individuals with certain common allergies are more
susceptible to autoimmune disorders.
Autoimmune disorders may be loosely grouped into those primarily
restricted to specific organs or tissues and those that affect the entire
body. Examples of organ-specific disorders (with the organ affected)
include multiple sclerosis (myelin coating on nerve processes), type I
diabetes mellitus (pancreas), Hashimotos thyroiditis (thyroid gland),
pernicious anemia (stomach), Addison's disease (adrenal glands),
myasthenia gravis (acetylcholine receptors at neuromuscularjunction),
rheumatoid arthritis (joint lining), uveitis (eye), psoriasis (skin),
Guillain-Barre Syndrome (nerve cells) and Grave's disease (thyroid).
Systemic autoimmune diseases include systemic lupus erythematosus and
Other examples of hypersensitivity disorders include asthma, eczema,
atopical dermatitis, contact dermatitis, other eczematous dermatitides,
seborrheic dermatitis, rhinitis, Lichen planus, Pemplugus, bullous
Pemphigoid, Epidermolysis bullosa, uritcaris, angioedemas, vasculitides,
erythemas, cutaneous eosinophilias, Alopecia areata, atherosclerosis,
primary biliary cirrhosis and nephrotic syndrome. Related diseases include
intestinal inflammations, such as Coeliac disease, proctitis, eosinophilia
gastroenteritis, mastocytosis, inflammatory bowel disease, Chrohn's
disease and ulcerative colitis, as well as food-related allergies.
Autoimmune diseases particularly amenable for treatment using the methods
of the present invention include multiple sclerosis, type I (insulin
dependent) diabetes mellitus, lupus erythematosus, amyotrophic lateral
sclerosis, Crohn's disease, rheumatoid arthritis, stomatitis, asthma,
uveitis, allergies and psoriasis.
Methods of the present invention may be used to therapeutically treat and
thereby alleviate autoimmune disorders such as those discussed above.
These treatments are exemplified herein with respect to the treatment of
EAE, an animal model for multiple sclerosis.
B. IFN.tau. as Treatment for Reproductive Disorders
Although IFN.tau. bears some similarity to the IFN.alpha. family based on
structure and its potent antiviral properties, the IFN.alpha.s do not
possess the reproductive properties associated with IFN.tau.. For example,
recombinant human IFN.alpha. had no effect on interestrous interval
compared to IFN.tau., even when administered at twice the dose (Davis, et
Therefore, although IFN.tau. has some structural similarities to other
interferons, it has very distinctive properties of its own: for example,
the capability of significantly influencing the biochemical events of the
The IFN.tau. compositions of the present invention can be used in methods
of enhancing fertility and prolonging the life span of the corpus luteum
in female mammals as generally described in Hansen, et al. (1991), herein
incorporated by reference. According to the teachings herein, such methods
of enhancing fertility include oral administration of IFN.tau. in a
therapeutically-effective amount. Further, the compositions may be
similarly employed to regulate growth and development of uterine and/or
fetal-placental tissues. Compositions containing human IFN.tau. are
particularly useful for treatment of humans, since potential antigenic
responses are less likely using a same-species protein.
C. IFN.tau. as an Antiviral Treatment
The antiviral activity of IFN.tau. has broad therapeutic applications
without the toxic effects that are usually associated with IFN.alpha.s. As
described above, IFN.tau. exerts its therapeutic activity without adverse
effects on the cells. The relative lack of cytotoxicity of IFN.tau. makes
it extremely valuable as an in vivo therapeutic agent and sets IFN.tau.
apart from most other known antiviral agents and all other known
Formulations containing IFN.tau. can be orally-administered to inhibit
viral replication. Further, the compositions can be employed in methods
for affecting the immune relationship between fetus and mother, for
example, in preventing transmission of maternal viruses (e.g., HIV) to the
developing fetus. Compositions containing a human interferon-.tau. are
particularly useful for treatment of humans, since potential antigenic
responses are less likely using a homologous protein.
Examples of specific viral diseases which may be treated by
orally-administered IFN.tau. include, but are not limited to, hepatitis A,
hepatitis B, hepatitis C, non-A, non-B, non-C hepatitis, Epstein-Barr
viral infection, HIV infection, herpes virus (EB, CML, herpes simplex),
papilloma, poxvirus, picorna virus, adeno virus, rhino virus, HTLV I, HTLV
II, and human rotavirus.
D. IFN.tau. as an Antiproliferative Treatment
IFN.tau. exhibits potent anticellular proliferation activity. Accordingly,
pharmaceutical compositions containing IFN.tau., suitable for oral
administration, can be used to inhibit cellular growth without the
negative side effects associated with other interferons which are
currently known. Such compositions or formulations can be used to inhibit,
prevent, or slow tumor growth.
Examples of specific cell proliferation disorders which may be treated by
orally-administered IFN.tau. include, but are not limited to, hairy cell
leukemia, Kaposi's Sarcoma, chronic myelogenous leukemia, multiple myeloma,
superficial bladder cancer, skin cancer (basal cell carcinoma and
malignant melanoma), renal cell carcinoma, ovarian cancer, low grade
lymphocytic and cutaneous T cell lymphoma, and glioma.
Furthermore, the development of certain tumors is mediated by estrogen.
Experiments performed in support of the present invention indicate that
IFN.tau. can suppress estrogen receptor numbers. Therefore, the IFN.tau.-containing
compositions may be particularly useful in the treatment or prevention of
E. Veterinary Applications
In addition to the uses of the methods of the present invention detailed
above, it will be appreciated that the methods may be applied to the
treatment of a variety of immune system disorders suffered by domesticated
and wild animals. For example, hypothyroidism in dogs typically results
from a progressive destruction of the thyroid, which may be associated
with Lymphocytic thyroiditis (Kemppainen and Clark, 1994). Lymphocytic
thyroiditis, which resembles Hashimoto's thyroiditis in humans, is thought
to be an autoimmune disorder. According to the guidance presented herein,
hypothyroidism due to Lymphocytic thyroiditis in dogs may be treated with
IFN.tau. as described above.
Another type of autoimmune disorder in dogs that may be alleviated by
treatment with IFN.tau. is characterized by antinuclear antibody (ANA)
positivity, pyrexia and seronegative arthritis (Day, et al., 1985).
Immune-mediated thrombocytopenia (ITP; Kristensen, et al., 1994; Werner,
et al., 1985), systemic lupus erythematosus (Kristensen, et al., 1994),
and leukopenia and Coomb's positive hemolytic anemia (Werner, et al.,
1985), may also be amenable to treatment using methods of the present
V. IFN Pharmaceutical Composition Useful for Oral Administration
Therapeutic preparations containing IFN.tau. or related polypeptides or
proteins can be formulated according to known methods for preparing
pharmaceutically useful compositions. Formulations comprising polypeptides
like interferons have been previously described (e.g., Martin, 1976). In
general., the IFN.tau. therapeutic compositions are formulated such that
an effective amount of the IFN.tau. is combined with a suitable additive,
carrier and/or excipient in order to facilitate effective oral
administration of the composition. For example, tablets and capsules
containing IFN.tau. may be prepared by combining IFN.tau. (e.g.,
lyophilized IFN.tau. protein) with additives such as pharmaceutically
acceptable carriers (e.g., lactose, corn starch, light silicic anhydride,
microcrystalline cellulose, sucrose), binders (e.g., alpha-form starch,
methylcellulose, carboxymethylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose, polyvinylpyrrolidone), disintegrating agents
(e.g., carboxymethylcellulose calcium, starch, low substituted
hydroxy-propylcellulose), surfactants (e.g., Tween 80,
polyoxyethylene-polyoxypropylene copolymer), antioxidants (e.g., L-cysteine,
sodium sulfite, sodium ascorbate), lubricants (e.g., magnesium stearate,
talc), or the like.
Further, IFN.tau. polypeptides of the present invention can be mixed with
a solid, pulverulent or other carrier, for example lactose, saccharose,
sorbitol, mannitol, starch, such as potato starch, corn starch,
millopectine, cellulose derivative or gelatine, and may also include
lubricants, such as magnesium or calcium stearate, or polyethylene glycol
waxes compressed to the formation of tablets. By using several layers of
the carrier or diluent, tablets operating with slow release can be
Liquid preparations for oral administration can be made in the form of
elixirs, syrups or suspensions, for example solutions containing from
about 0.1% to about 30% by weight of IFN.tau., sugar and a mixture of
ethanol, water, glycerol, propylene, glycol and possibly other additives
of a conventional nature.
An orally-active IFN.tau. pharmaceutical composition is administered in a
therapeutically-effective amount to an individual in need of treatment.
The dose may vary considerably and is dependent on factors such as the
seriousness of the disorder, the age and the weight of the patient, other
medications that the patient may be taking and the like. This amount or
dosage is typically determined by the attending physician. The dosage will
typically be between about 1.times.10.sup.5 and 1.times.10.sup.8
units/day, preferably between about 1.times.10.sup.6 and 1.times.10.sup.7
units/day. It will be appreciated that because of its lower toxicity,
IFN.tau. can be administered at higher doses than, for example, IFN.beta..
By way of comparison, patients with multiple sclerosis (MS) were treated
with 10.sup.6 U and 8.times.10.sup.6 U of IFN.beta.. Patients receiving
8.times.10.sup.6 U suffered fewer relapses of disease than did patients
receiving 10.sup.6 U. However, patients receiving the higher dose of
IFN.beta. (8.times.10.sup.6 U) also exhibited more side-effects associated
with IFN.beta.'s toxicity. In view of the lower toxicity of IFN.tau.,
these higher effective dosages could be administered without the
associated toxic side-effects.
Disorders requiring a steady elevated level of IFN.tau. in plasma will
benefit from administration as often as about every two to four hours,
while other disorders, such as MS, may be effectively treated by
administering a therapeutically-effective dose at less frequent intervals,
e.g., once every 48 hours. The rate of administration of individual doses
is typically adjusted by an attending physician to enable administration
of the lowest total dosage while alleviating the severity of the disease
Once improvement of a patient's condition has occurred, a maintenance dose
is administered if necessary. Subsequently, the dosage or the frequency of
administration, or both, may be reduced, as a function of the symptoms, to
a level at which the improved condition is retained.
C. Combination Therapies
It will, of course, be understood that the compositions and methods of
this invention may be used in combination with other therapies. For
example, in view of IFN.tau.'s relative lack of toxicity at high dosages,
MS patients that do not show improvement at IFN.beta.1b's low dosage or
could not tolerate IFN.beta.1b due to toxicity may benefit from subsequent
or simultaneous treatment with higher dosages of IFN.tau. or peptides
derived therefrom. Further, development of neutralizing antibodies has
been demonstrated in IFN.beta.1b treated patients (Weinstock-Guttman, et
al., 1995). In cases where such neutralizing antibodies prove to impede
the effectiveness of IFN.beta.1b, IFN.tau. may be an important alternative
therapy, since antibody cross-reactivity is unlikely to occur, and IFN.tau.
is unlikely to generate neutralizing antibodies (see Example 7).
Orally-administered IFN.tau. is particularly advantageous in this respect,
since it causes a significantly lower anti-IFN.tau. antibody response than
Another type of combination therapy enabled by the present invention is
the oral administration of an antigen against which an autoimmune response
is directed in combination with IFN.tau.. Oral administration of such an
antigen can result in tolerization, reducing the severity of the
autoimmune disease (for review, see, e.g., Weiner, et al., 1994). It is
contemplated that the IFN.tau. has a synergistic effect with the
tolerization induced by the antigen, thereby alleviating the severity of
the autoimmune disease. For example, MBP has been shown to suppress EAE (Lider,
et al., 1989). According to the methods of the present invention, MBP may
be administered in combination with IFN.tau. to treat multiple sclerosis.
Other examples include administration of IFN.tau. with collagen to treat
rheumatoid arthritis, and with acetylcholine receptor polypeptides to
treat myasthenia gravis.
Furthermore, IFN.tau. may be orally administered with known
immunosuppressants, such as steroids, to treat autoimmune diseases such a
multiple sclerosis. The immunosuppressants may act synergistically with
IFN.tau. and result in a more effective treatment that could be obtained
with an equivalent dose of IFN.tau. or the immunosuppressant alone.
Similarly, in a treatment for a cancer or viral disease, IFN.tau. may be
administered in conjunction with, e.g., a therapeutically effective amount
of one or more chemotherapy agents such as busulfan, 5-fluoro-uracil
(5-FU), zidovudine (AZT), leucovorin, melphalan, prednisone,
cyclophosphamide, dacarbazine, cisplatin, and dipyridamole.
Claim 1 of 5 Claims
1. A method of reducing probability of
relapse in a mammal with chronic, relapsing-remitting multiple sclerosis,
comprising: orally administering an interferon tau (IFN.tau.) to said
mammal in an amount effective to reduce probability of relapse of the
chronic relapsing-remitting multiple sclerosis, and continuing to
administer the IFN.tau. over an extended period of time.
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