Note: Descriptions are shown in the official language in which they were submitted.
WO 95/12416 PCT/US94/12621
21~57
~7~TM~NT FOR }~ JIu ARTlIRI~3:5
Fiela of the Invention
This invention generally relates to a method
5 of treating a patient suffering from a chronic inflam-
matory arthritides, and more particularly to the use of
corticotropin-releasing factor or its analogs in
reducing swelling during episodic acute responses of
chronic inflammatory conditions.
l0 Backqround of the Invention
Among the diseases that plague humans are the
chronic inflammatory arthritides, which include rheuma-
toid arthritis, Reiters Syndrome, ankylosing spondyitis,
psoriatic arthritis, and infl2 tory bowel disease. of
15 these, rheumatoid arthritis is the cause of an estimated
about 33, 000 deat1ls in the United States each year. As
yet, there is no cure for the disea6e, and the cause is
uncertain. Rheumatoid arthritis sufferers periodically
experience flare-ups, or episodic acute responses, of
20 from a week up to a month of duration.
Steinman, "Autoimmune Disease, " Scientific
~zeric~n, pp. 107-ll~ (September 1993) discusses several
possible new approaches to treating an autoimmune
disease, such as rheumatoid arthritis. In one, decoys
25 are being designed to fit into the HLA (human lymphocyte
antigen) cleft in an attempt to turn off T cells. In
another, a monoclonal antibody that binds with tumor
necrosis factor appears to increase ~oint mobility and
reduce the stiffness associated with rheumatoid
WO 95/12416 PCrNS9~/12621
2f 75457
arthritis. However, these possible new approache6 are
as yet not therapeutically established as to efficacy.
Assisting in the search for treatments is the
fact that animal models have been develo~?ed using
5 arthritis-susceptible rat strains. Among other possible
abnormalities, the "Lewis" arthritis-susceptible rats
have a deficient hypothalamic CRF response to various
stimuli so that researchers can experimentally induce
inflammatory arthritis and some other autoimmune
lO diseases. In the Piebald-Viral-Glaxo ("PVG") rat model,
unlike the Lewis strain rat, the animals do not appear
to have a hypothalmo-pituitary-adrenal ("HPA") axis
defect, but are nevertheless susceptible to arthritis.
U.S. Patent ~,006,330, issued April 9, l99l,
15 inventors Sternberg et al., describes use of the Lewis
rat animal model in which the experimental animals
developed an arthritis which mimics human rheumatoid
arthritis in response to exposure to Group A strepto-
coccal cell wall fragments. Sternberg et al. teach use
20 of the model for testing the susceptibility of mammals
to inflammatory diseases.- Sternberg et al. describe
measuring "CRH" (corticotropin-releasing hormone) after
injections of various agents. CRH is an alternative
term for corticotropin-releasing factor ~hereinafter
25 "CRF"), which is a 41 amino acid neuropeptide that is
present in brain and the peripheral nerve endings, and
stimulates ACTH release from pituitary cells in brain
and such diverse peripheral locations as nerve endings
and leukocytes. Sternberg et al. also describe
30 injections of CRH followed by measurements of ACTH and
corticosterone .
Antibodies directed against CRF have been
reported to partially abrogate the inf lammatory
response, which has led various researchers to believe
35 that CRF is a pro-inflammatory agent. Karalis et al.,
"Autocrine or paracrine Inflammatory Actions of
WO 95/12416 2 ~ 7 ~ 4 5 7 PCTIUS94/12621
Corticotropin-Releasing Hormone in Vivo," Science, 254,
421-423 (1991).
U.S. Patent 4,489,163, inventors Rivier et
al., issued December 18, 1984, discloses rat C~F and its
analogs. Human CRF has the same sequence as rat CRF.
There are a number of analogs of CRF known to the art.
U. S . Patent 4 , 415 , 558 , inventors Vale, Jr . et al .,
issued November 15, 1983, discloses the synthesis of
sheep CRF, analogs, and isolation of the oCRF from ovine
hypothalamic extracts. The synthetic oCRF was found to
lower blood pressure.
A generally similar peptide, sauvagine, was
described in Regulatory Peptides, 2, pp. 1-13 (1981).
Sauvagine is a 4 0 amino acid peptide and has been
reported to have biological activity in lowering blood
preseure in mammals and stimulating the secretion of
ACT~ and ,~-endorphin.
U . S . Patent 4, 528 ,189, inventors Lederis et
al., issued July 9, 1985, and U.S. Patent 4,533,654,
inventors Lederis et al., issued August 6, 1985,
disclose peptides similar to the rat and sheep CRF and
analogs thereof, and found this white sucker and carp
urotensin respectively to stimulate ACTH and to lower
blood pressure. The CRF-related peptide, white sucker
urotensin, has an amino acid sequence the same as the
carp urotensin, except the amino acid at the 24 position
is Isoleucine and the amino acid at the 27 position is
Glutamic Acid.
Ling et al., BBRC, 122, pp. 1218-1224 (1984),
disclose the structure of goat CRF, which is the same as
that for sheep CRF. Esch et al., BBRC, 122, pp. 899-905
(1984), disclose the structure of bovine CRF which
differs from sheep and goat CRF only by one amino acid
residue (number 33 which is Asparagine rather than the
number 33 Serine of goat and sheep CRF). Porcine CRF
has been isolated and characterized by Patthy et al.,
WO 95/12416 PCr/US9~/12621
2175457
Proc. Nat~. Acad. sci., 82, pp. 8762-8766 (1985). Por-
cine CRF shares a common amino acid sequence (residues
1-39) with rat/human CRF and differs from these only in
position 40 and 41. Residue 40 can be either asparagine
5 or isoleucine and residue 41 is phenylalanine-amide.
U.S. Patent 4,801,612, inventor Wei, issued
January 31, 1989, discloses the Use of inhibiting an
inflammatory response (acute) in the skin or mucosal
membranes of a patient by administering Corticotropin-
10 Releasing Factor, or its analogs, and U.S. Patent5,137,871, issued August 11, 1992, inventor Wei,
describes the use of CRF (or a salt ~or analog thereof )
in treating a patient for injury to or disease of the
brain, nervous system, or musculature in which edema is
15 a factor.
S~ rY of the Invention
Administration of corticotropin-releasing
factor to patients suffering from one of the chronic
inflammatory arthritides, such as rheumatoid arthritis,
20 reduces episodic acute inflammatory responses to the
disease. Doses of CRF in accordance with the invention
give anti-inflammatory results in a dose responsive
manner even in well-established disease. Thus, practice
of the invention provides a therapeutic treatment f or
25 these unremitting diseases that are refractory to
current therapies.
Therapeutic treatment in accordance with the
invention will be under the supervision of a health care
professional, usually a physician, and may be in
30 conjunction with other drugs and treatments. Because
anti-inflammatory effects and other ameliorating results
are experienced within several days when treating
episodic acute inflammatory responses that typically
would otherwise last for one week up to months, patients
35 receive effective relief ~ of uncomfortable or even
WO 95/12416 PCT/US94/12621
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in-~Ara-~itating symptoms in an inventive course of
treatment, although practice of the invention is not a
cure .
B~ief DescriDtion of the Drawinqs
Figure 1 is a perspective view illustrating
the paws of two mycobacterium induced arthritis-
susceptible male Lewis "LEW/N" rats where paw 10 is a
control and paw 12 has been injected with Mycobacterium
to induce arthritis;
Figure 2 graphically illustrates foot pad
swelling (with one unit equivalent to 0. 001 inches) in
an animal model of arthritis-susceptible rats as a
function of time ~or control animals and animals treated
in accordance with the invention at two different doses
(50 and 100 ~g CRF; injected subcutaneously, per kg of
body weight), with the foot pad swelling measurement
being made of mycobacterium injected paws (left hind);
Figure 3 is similar to Fig. 2, but illustrates
measurements of the paws opposite to the inj ected paws
in the transverse plane (right hind);
Figure 4 graphically illustrates the plot of
arthritis scores as a function of time for the right
hind paw of Fig. 3;
Figure 5 is similar to Fig. 4, but is of the
other pair of paws (left and right front);
Figure 6, including panels A-E, is similar to
Fig. 2 but plots the larger dose (100 ~g/kg body weight)
administered subcutaneously in the mycobacterium
injected foot pad (left hind) in accordance with the
invention beginning at times delayed up to 40 days a~ter
induction of disease;
Figure 7, including panels A-E, is similar to
Fig . 6 but is of the opposite f oot pad in the traverse
plane (right hind); and
WO 95112416 PCrlUS94/12621
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Figure 8 graphically illustrates plots of
arthritis scores in a manner similar to that of Fig. 4,
but in the format similar to that of Fig. 6.
De~; 1 ed DescriPtion of the Pref erred ~hr,~ i r ' c
It is possible that earlier reports, which
found the presence of CRF in the joints of mammals with
inf lammatory arthritis and suggested CRF was a pro-
inf lammatory agent, may be correct because it is
possible that the peptide may be either pro-inflammatory
or anti-inflammatory, depending upon the amount of
endogenous peptide produced, how the "inflammatory" cell
responds to CRF or when CRF appears during the course of
the inflammatory response. However, the following
description of the work demonstrates that administration
of CRF can be therapeutically effective in inhibiting
episodic acute and chronic inf lammatory responses in a
dose-responsive manner, and therefore this invention is
a therapeutic method for treating patients with chronic
inflammatory arthritides, such as rheumatoid arthritis.
Practice of this invention uses CRF. By "CRF"
are meant to be included the analogs and CRF-related
peptides known to the art . Therapeutic f ormations of
CRF may be prepared for storage by mixing CRF having the
desired degree of purity with optional physiologically
acceptable carriers, excipients or stabilizers, in the
form of lyophilized cake or aqueous solutions.
Acceptable carriers, excipients or stabilizers are
nontoxic to recipients at the dosages and concentratiOns
employed when administered, and include buffers such as
phosphate, citrate, and other organic acids; anti-
oxidants including ascorbic acid; low molecular weight
(less than about lO residues) polypeptides; proteins,
such as serum albumin, gelatin or immunoglobulins.
other components can include glycine, glutamine,
35 asparagine, arginine, or lysine; monosaccharides,
f ~ u~
IPE~U~ "Y4J/~1 1995~
~ ~7~
disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol: salt-
forming counterions such as sodium; and/or nonionic
5 surfactants such as TWEEN, PLURONICS, or PEG.
Administration may be by any mode of admini-
stration known in the art, including, but not limited
to, intra-articular, intravenous, intrathecal, subcu-
taneous, injection, intranasal, oral, or via an
10 implanted device. Suitable implants include, for
example, gel foam, wax, or microparticle-based implants.
Topical administration preferably includes agents or
apparatus to facilitate skin delivery, such as
solubilizing agents or directed delivery systems as are
15 known and being developed in the rapidly emerging
technologies of skin delivery systems.
Therapeutically effective doses of CRF or its
analogs in practicing this invention preferably are at
least about O . 01 ,ugJkg in humans . For systemic
20 administration (e.g. subcutaneous and intravenous), more
preferred doses are in the range of about 0.1 to about
50 ,ug/kg, and most preferred about 1 to about 30 ug/kg.
For local administration (e.g. intra-articular), the
preferred dose is in a range of about 1 to about 100
25 ug/kg. Administration of CRF doses may be infused
510wly, such as subcutaneously or intradermally, or may
be injected directly into an afflicted body part.
Patients treated will be under the care of a
health care professional, such as a physician, who may
30 herself administer the therapeutic composition including
CRF or may prescribe such for the patient s self
administration (such as, for example, preloaded
syringes). Systemic forms of administration are
particularly preferred due to the frequent multiple
35 sites of episodic situations, or flares. Unfortunately,
although treatments in accordance with the invention, as
A~jjEl\~r-G 'U-'T
. , . , . , . . . . , .. , . , . _ _ _ _ _ _ _ _ _ . _ ,
WO 95/12416 PCT~S94/12621
2~7~
further illustrated hereinafter, are shown efficacious,
when treatment is stopped then efficacy also ceases. As
with many other dif f erent types of therapies, the
therapeutic method of this invention may ber combined
5 with other pharmaceuticals and other therapeutic
approaches to the disease being treated.
Administration of CRF in accordance with this
invention may potentially be continued for up to about
three weeks, but side effects (e.g. Cushing's Syndrome)
lO may tend to develop after prolonged intake. Thus,
practice of this invention is primarily contemplated for
treating acute, episodic flares, and CRF administration
can be undertaken up to forty days after an episodic
acute inf lammation occurs .
Although the peptides are generally water
soluble as typically synthesized, they may be admini-
stered in the form of pharmaceutically acceptable
non-toxic salts, such as acid addition salts. Illustra-
tive acid addition salts are hydrochloride, hydrobro-
20 mide, sulfate, sulphate, acetate, citrate, benzoate,
succinate, malate, ascorbate, tartrate, or the like. In
addition to the f act that CRF may be administered in any
pharmacologically acceptable carrier, depending upon the
desired mode of administration it may be formulated
25 along with liquid carrier into liposomes, microcapsules,
polymers, or wax-based and controlled release prepara-
tions, or be formulated into tablet, pill, or capsule
f orms .
A method f or treating a patient in accordance
30 with this invention involves patients suffering from one
of the chronic inflammatory arthritides. By chronic
inflammatory arthritides are meant to include rheumatoid
arthritis, Reiters Syndrome, ankylosing spondylitis,
psoriatic arthritis, and inf lammatory bowel ~isease .
35 Treatments are particularly useful for episodic acute
inf lammations and provide anti-inf lammatory results in
_~J~ rt.~ t~
WO95112416 PCrlUS94112621
2i 7~7
a do6e respon6ive manner. Experiments demonstrate that
regardless of the time administered (e.g. up to about
forty days after an episodic acute inflammation),
practice of the invention reduces inf lammatio~ even in
5 well established disease; however, when treatment is
discontinued, then the inf lammatory symptoms have been
f ound to reoccur . Thus, the present invention is most
likely a treatment rather than a cure.
~ KlM~l~ ~ AL
Therapeutic efficacy was demonstrated with a
rat adjuvant arthritis model in which arthritis-
susceptible male Lewis "LEW/N" rats had killed Myco-
bacterium injected into the left-hind foot paw to induce
arthritis .
With reference generally to Fig. 1, approxi-
mately sixteen days post-induction of the disease
animals ( i . e . animals treated with saline) had paws
illustrated by paw 10; however, shortly after disease is
induced, the mycobacterium in3ected paws 12 (and later,
20 as will be described, other paws) experience foot pad
swelling. The foot pad swelling is a signal of
inflammation. As is known, inflammation is manifested
by redness, 6welling, heat and pain as a reaction of the
body against injury or assault. Five days after
25 induction of the disease in the rats, CRF was
administered subcutaneously twice a day at either a dose
per administration of 50 ,ILg/kg or 100 ~g/kg. The twice
a day administrations were continued for a period of 20
days. By approximately 16 days post-induction, the CRF
30 treated animals have comparatively little swelling or
inf lammation.
Starting with the first day of administration
(that is, day 5 after having injected the killed Myco-
bacterium), the amount of swelling was determined by the
WO 9S112416 PCTIUS9~/12621
57 ~I
use of calipers in mea6uring the thickness of left hind
(injected) paw. This measurement served as a
determination for the degree of "primary, " or acute,
inflammation. Subsequently (usually about an ~dditional
5 10-12 days) the right hind paw also swells and becomes
inf lamed . This represents a chronic inf lammatory
response .
Turning to Fig. 2, the primarily inflamed left
hind paw swelling data (measured through day 32) i6
10 shown for control rats, for rats receiving twice a day
administrations of CRF at 50 ~Lg/kg body weight, and for
rats receiving twice a day admini5trations of CRF at 100
,~g/kg body weight. The vertical axis is measured in
units where one unit is equivalent to 0 . 001 inches . As
15 is shown by the Fig. 2 data, administrations of CRF in
accordance with the invention gave anti-inf lammatory
re6ults and in a dose responsive manner. The three
graphs of Fig. 2 all begin five days after the left hind
paw was challenged wit1~ Mycobacterium. With twice a day
20 injection of 50 ,ug/kg CRF, there was reduced swelling
relative to control. Results were even of greater
signif icance with administrations of 100 ,ug/kg .
Turning to Fig. 3, data similar to that of
Fig. 2 are represented, but the degree of foot pad
25 swelling being measured is that of the right hind paw
and thus represents a chronic inflammatory response.
Administration of CRF at doses of either 50 ~g/kg or 100
g/kg were begun five days after challenge, as with Fig.
2, but the right hind paw has a swelling on-set later
30 than does the left hind paw illustrated by the Fig. 2
data. The right hind paw also experiences less swelling
than the left hind paw. Of course, the right hind paw
is not itself directly challenged as is the left hind
paw. Nevertheless, as seen, beginning about day 11, the
35 right hind paw begins to swell; however, for animals
receiving CRF in accordance with the invention, very
WO g5/12416 Pcr~S94/12621
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11
little swelling (for either the 50 ~g/kg body weight or
the 100 ,ug/kg body weight) is experienced.
Turning to Fig. 4, the 2nimals as in Fig. 3
were also assessed for the extent of arthritis by using
5 a scoring system in which "0" indicated no arthritis and
"4" represented severe arthritis. The arthritis scores
were made by evaluating a combination of factors
including the percent involvement of the foot pads plus
the overall symptoms of the disease (which include
lO swelling, redness, and disfigurement). The Fig. 4 data
shows that both doses of CRF (50 ~Lg/kg and 100 I~g/kg)
are active in reducing the arthritis scores, although
the 100 ~g/kg dose appears to show a greater ef f ect .
The right hind paw of the animals were used for the Fig.
15 4 scoring.
With Fig. 5, conducted similarly to the just
described Fig. 4, the two front paws were evaluated.
That is, Figs 4 and 5 both represent chronic, rather
than acute, situations. However, the front paws used
20 for t~ a Fig. 5 data experienced an on-set of swelling
and other symptoms even more delayed and less pronounced
than for the right hind paws of Fig. 4. As is seen
particularly with the Fig. 5 data, administrations of
CRF in accordance with this invention are very effective
25 in treating for the clinical manifestations of chronic
inf lammatory arthritis .
In the experiments summarized by the data of
Figs. 2-5, treatments began five days after induction of
the disease. Another set of experiments were undertaken
30 when the disease was induced as before, and the animals
were then treated in accordance with the invention (at
100 Lg/kg doses administered twice daily subcutaneously)
for a period of 20 days, except that the course of
treatment began five days, 10 days, 15 days, 25 days, or
35 40 days after disease was induced. These experiments
are summarized by the data of Figs. 6-8.
WO 95/12416 ~ PCr/US94112621
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12
Turning to Fig. 6, panels A-E illustrate
measurement6 taken of the left hind foot pad. Panel A
plots the foot pad thickness for the control rats and
for rats who received treatment beginning on dzy 5 after
5 induction of the disease ( indicated by the open arrow)
and ending on day 20 (indicated by the filled in, or
closed, arrow). Thus, beginning promptly after
treatment in accordance with the invention f ive days
after induction of disea8e, footpad thickness was
10 considerably reduced with respect to the control, and
the thickness reductions continued until treatment was
discontinued, at which time swelling began and continued
until by about day 60 there was substantially no
difference between the previously treated footpad and
15 the control (never treated) footpad. Similarly, panel
B illustrates data for the control and test rats with
treatment beginning on day 10 and ending on day 3 0,
panel C with treatment beginning on day lS and ending on
day 35, panel D with treatment beginning on day 25 and
20 ending on day 45, and panel E with treatment beginning
on day 40 and ending on day 60. These data demonstrate
that when treating with CRF it profoundly reduced foot
pad swelling, even in well established disease such as
illustrated by panel E. Thus, panel E of Fig. 6 had
25 treatment begun forty days after induction of disease,
yet promptly after treatment was begun, the treated
footpad experienced reduced swelling
Turning to Fig. 7, the data illustrated by
panels A-E are similar to those described for Fig. 6,
30 but the foot pad swelling was measured in the animals'
right hind foot pad. That is, as with Fig. 3, since the
degree of f oot pad swelling being measured is that of
the right hind paw (where the left hind paw is the site
of mycobacterium injections and may represent acute
35 flares), the right hind paw may represent chronic
inflammatory responses. Once again, these data
, ... . ...... _ . , _ , _ _ _ .. _ _ .
WO 9S/12416 PCTIUS94/12621
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13
demonstrate that administering CRF in accordance with
the invention profoundly reduces swelling, even in
established disease, for chronic inflammatory
conditions. r
In experiments 6imilar to Figs. 6 and 7, but
where the animals were assessed for the extent of
arthritis by using a scoring system such as is described
for Fig. 4, practice of the invention adds significantly
against overall arthritic condition, although perhaps
less than seen in reducing foot pad swelling. Turning
to Fig. 8, in each of panels A-E animals receiving
treatment are indicated for the times when treatment was
,~ ~n~rl by the open arrows. The times when treatments
stopped are indicated by the f illed in, or closed,
arrows.
Thus, with ref erence to panel A of Fig . 8,
five days after an animal's left hind footpad had been
challenged by the killed Mycobacterium, administrations
of CRF in accordance with the invention were begun in
the right hind footpad and were continued for a period
of twenty days. During the course of treatment, the
arthritis scores went down with respect to the control,
but when the treatment was discontinued, these scores
rose. Thus, practice of this invention is therapeutic
as opposed to prophylactic. However, it is important to
note, as illustrated by panel D of Fig. 8, that starting
treatment in accordance with the invention as late as 25
days into the disease was still efficacious.
-
It is to be understood that while the
invention has been described above in conjunction with
preferred specific ~rhQA;r~nts, the description and
examples are intended to illustrate and not limit the
WO 95/12416 " PCr/US9~112621
~1 ~5~5~
~:cope of the invention, which is defined by the scope ofthe ~rr~ d claims.
