Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
NEUROTROPHIC TACROLIMUS ANALOGS
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to a tacrolimus derivative having a high level of
neurotrophic
activity and a low level of immunosuppressive activity.
Discussion of the Back rg. ound
Certain macrolide compounds, e.g. tacrolimus and related compounds, are known
to
help prevent or treat cerebral ischemia (W094/14443). Particular pipecolic
acid derivatives
that have affinity for FKBP-type irmnunophilins, such as tacrolimus, are known
to stimulate
growth of damaged peripheral nerves or promote neuronal regeneration
(W096/40140).
Certain non-irmnunosuppressive compounds, i.e., geldanamycin and its analogs,
are shown to
disrupt the steroid receptor complex and promote nerve growth (W099/21552).
SUMMARY OF THE INVENTION
The present inventors have found that a particular tacrolimus analogue, i.e.
Compound
(I), mentioned below, has an excellent neurotrophic activity but, unlilce
tacrolimus, has little
or no immunosuppressive activity. As shown below, Compound (I) exerts superior
levels of
neurotropic activity compared to tacrolimus, for instance, as measured by its
ability to
increase neurite length. Similarly, the administration of Compound (I) is
shown to induce
axonal regeneration and speed recovery from nerve crush or spinal cord
injuries. Moreover,
Compound (I) exerts these advantageous neurotropic effects with little or no
immunosuppressive activity compared to tacrolimus.
Accordingly, the present invention provides new uses for Compound (I) as a
superior
neurotrophic agent, as well as a neurotropic agent with little or no
immunosuppressive
activity.
Further, the invention provides a neurotrophic agent or composition that
comprises
Compound (I).
Still further, this invention provides a method for preventing or treating
neuronal
injury/dysfunction that comprises administering Compound (I) to a mammal.
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
DETAILED DESCRIPTION OF THE INVENTION
Unexpectedly, the present inventors have discovered that Compound (I) is
useful for
ameliorating, preventing, or treating neurological injury or dysfunction
caused by damage or
injury to, deterioration of, or disease of the nervous system, while
advantageously having
little or no immunosuppressive effect.
Compound (I) is useful for treating damage, deterioration or dysfunction
caused by
physical injury, nutritional disorders, ischemia, degenerative diseases,
malignant diseases,
infectious diseases, and by drug interactions, toxins or poisons. For
instance, Compound (I)
is useful for treating neurological damage or dysfunction caused by
neurosurgery, peripheral
nerve injury, burns, encephalomyelitis, HIV, herpes, cancer, radiation
treatment, drug
interaction, folic acid or Vitamin B-12 deficiency, and by exposure to
neurotoxins or
chemicals such us lead.
Accordingly, Compound (I) is useful for preventing or treating neuronal injury
and
dysfunction, such as polymyositis (multiple myositis), Guillan-Barre syndrome,
Meniere's
disease, polyneuritis (multiple neuritis), mononeuritis (solitary neuritis),
Alzheimer's disease,
Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's
disease, radiculopathy,
neuropathy (such as diabetic neuropathy, chemotherapy-induced neuropathy,
etc.), spinal
cord injury, senile dementia, vascular dementia, multiple sclerosis, physical
palsy, etc.
Compound (I), the tacrolimus analog used in the present invention, has the
following
chemical formula:
-2-
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
HO
CH30' ~ I CH3
CH3 O
O
-O OH
o
O O _ CH3
OH
CH3 O CH3
(I)
OCH3 OCH3
This compound may be produced as described by U.S. Patent 5,376,663, Example
29.
With respect to Compound (I) used in the present invention, it is to be
understood that there
may be conformers and one or more stereoisomers, such as optical and
geometrical isomers
due to asymmetric carbon atoms) or double bond(s), and such conformers and
isomers are
also included within the scope of the compound in the present invention.
Compound (I) may also be in the form of a pharmaceutically acceptable salt,
derivative, solvate or pro-drug, all of which are included within the scope of
the present
invention. The solvate preferably includes a hydrate and an ethanolate.
A preferable form of Compound (I) is the following one:
-3-
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
HO
CH30' ~ I CH3
CH3 O
O
O OH
N
O
O ~~O CH3
OH ~''''sCH
CH3 3
O
- (Ia)
OCH3 OCH3
Compound (I) in the present invention may be administered as a pure compound
or as
a mixture with another compound or other ingredients, preferably, in a
pharmaceutical vehicle
or carrier. When Compound (I) is used in the form of a pharmaceutical
preparation or
composition it may be admixed with an organic or inorganic carrier, vehicle or
excipient
suitable for external (topical), oral, enteral, subcutaneous, intravenous,
intramuscular, or
parenteral applications. For example, it may be present in solid, semisolid or
liquid
composition, which contains Compound (I) as an active ingredient and one or
more carriers,
vehicles or excipients. Typical carriers, vehicles or excipients include, but
are not limited to
conventional pharmaceutical carriers, medicinal or pharmaceutical agents,
buffers,
dispersants, emulsifying agents and adjuvants.
Compound (I) may also be compounded with the usual non-toxic, pharmaceutically
acceptable carriers for tablets, pellets, capsules, eye drops, suppositories,
solutions (saline, for
example), emulsions, suspensions (olive oil, for example), ointments, aerosol
sprays, creams,
slcin plasters, patches and any other form suitable for use.
Suitable carriers include water, aqueous saline and dextrose solutions, oils,
including
animal, vegetable and synthetic oils, and petroleum products. Other useful
carriers include
-4-
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium
trisilicate, talc, corn
starch, keratin, colloidal silica, potato starch, urea, and other carriers
suitable for use in
manufacturing preparations, in solid, semisolid, or liquid form. In addition
auxiliary,
stabilizing, emulsifying, thickening, coloring agents, flavoring agents, and
perfumes may be
used.
Compound (I) is included in the pharmaceutical composition in an amount
effective to
produce the desired effect upon a particular disease process or condition.
Preferably,
Compound (I) is included in an amount sufficient to provide a neurotropic
effect or stimulate
nerve cell growth.
Mammals which may be treated using the method of the present invention include
livestock mammals such as cows, horses, pigs, etc., domestic animals such as
dogs, cats, rats,
mice, rabbits, hamsters, etc., primates, and humans.
Preferable modes for the administration or application of products or
compositions
containing Compound 1 to humans include injection or oral administration.
While the therapeutically effective amount or dosage of Compound (I) may vary
among individual patients and also depends upon the age and condition of each
individual
patient to be treated, a daily dose ranging from about 0.0001-1000 mg,
preferably 0.001-500
mg and more preferably 0.01-100 mg of the active ingredient is generally given
for treating
diseases, and an average single dose of about 0.001- O.Olmg, 0.2-0. 5 mg, 1
mg, 5 mg, 10 mg,
50 mg, 100 mg, 250 mg and 500 mg is generally administered. Daily doses for
chronic
administration in humans will be in the range of about 0.1-30 mg/lcg/day.
Compound (I) may
also be administered or applied simultaneously, separately or sequentially
with other agents
having neurotrophic or nerve cell growth stimulating activity.
Pharmaceutical compositions according to the invention can be periodically
administered to a mammalian subject (e.g., a human patient), in need of such
treatment, to
promote neuronal regeneration and functional recovery and to stimulate neurite
outgrowth
and thereby to treat various neuropathological states, including damage to
peripheral nerves
and the central nervous system caused by physical injury (e.g., spinal cord
injury and trauma,
sciatic or facial nerve lesion or injury, limb transplantation following
amputation); disease
(e.g., diabetic neuropathy); cancer chemotherapy (e.g., neuropathy induced by
acrylamide,
taxol, vinca alkaloids and doxorubicin); sequela--e.g. allophasis (such as
articulation
-5-
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
disorders), clouding of consciousness, dyskinesia, etc. associated with
cerebral infarction,
hemorrhage infarct, etc.; and neurological disorders including, but not
limited to, various
peripheral neuropathic and neurological disorders including, but not limited
to: trigeminal
neuralgia, glosspharyngeal neuralgia, Bell's palsy, myasthenia gravis,
muscular dystrophy,
amyotrophic lateral sclerosis, progressive muscular atrophy, progressive
bulbar inherited
muscular atrophy, herniated, ruptured or prolapsed vertebral disk syndromes,
cervical
spondylosis, plexus disorders, thoracic outlet destruction syndromes,
peripheral neuropathles
such as those caused by lead, acrylamides, gamma-diketones (glue-sniffer's
neuropathy),
carbon disulfide, dapsone, ticks, porphyria, Gullain-Barre syndrome,
Alzheimer's disease,
Parkinson's disease, and Huntington's chorea.
A transsection of a peripheral nerve or a spinal cord injury can be treated by
administering a nerve growth stimulating amount of the agent to the mammal and
grafting to
the peripheral nerve or spinal cord a nerve graft such as an allograft (Osawa
et al., J.
Neurocytol. 19:833-849, 1990; Buttemeyer et al., Anh. Plastic Sm°ge~y
35:396-401, 1995) or
an artificial nerve graft (Madison and Archibald, Exp. Neur~ol. 128:266-275,
1994; Wells et
al., Exp. Neu~ol. 146:395-402, 1997). The space between the transected ends of
the
peripheral nerve or spinal cord is preferably filled with a non-cellular gap-
filling material
such as collagen, methyl cellulose, etc., or cell suspensions that promote
nerve cell growth,
such as Schwann cells (Xu et al., J. Neu~ocytol. 26:1-16, 1997), olfactory
cells and sheathing
cells (Li et al. Science 277:2000-2002, 1997). The nerve gxowth promoting
agent can be
included together with such cellular or non-cellular gap-filling materials, or
administered
systemically before, during or after the nerve graft procedure.
Particularly, compound (I) is useful for treating or preventing the neuronal
injury/dysfunction polymyositis (multiple myositis), Guillain-Barre syndrome,
Meniere's
disease, polyneuritis (multiple neuritis), mononeuritis (solitary neuritis),
Alzheimer's disease,
Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's
disease, radiculopathy,
diabetic neuropathy, chemotherapy-induced neuropathy, senile dementia,
vascular dementia,
multiple sclerosis, physical palsy, or spinal cord injury.
The following examples illustrate the present invention in further detail. It
should be
understood that these examples describe certain aspects or embodiments of the
invention, but
are not intended to limit the scope of the invention.
-6-
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
Example 1: Treatment with Compound (I1 Significantly Increases Neurite Len tg-
hshs of
Hippocampal Neurons
Preparation of cell cultures:
Embryonic hippocampal neurons were obtained from rat pups on embryonic day
18.5
("E18.5"), according to Banker and Cowan (Brain Research, 1977, 126: 397-425).
Briefly,
the hippocampal regions were removed, minced, and incubated in 100 LU. papain
at 37°C for
45 min, and the cells were resuspended in complete neuronal medium: minimal
essential
medium without L-glutainine (GIBCO, Grand Island, NY), 1.5m1/100m1 medium of
high
glucose minimal essential medium (GIBCO), O.lml/100m1 medium of serum extender
(Hito
+ Tm; Collaborative Research Inc, Lexington, MA), glutamine (GIBCO), 5% fetal
calf serum
(GIBCO).
Cells were seeded onto coverslips (500 cells/coverslip) coated with poly-L-
lysine.
The coverslips were inverted onto dishes that had been precoated with a
monolayer of cortical
astrocytes.
Analysis of axonal lengths in hippoocampal neurons:
Hippocampal neurons (identified by their characteristic polarity and
dendrites) were
examined daily and randomly photographed (9-12 frames/coverslip) at 72 h. Axon
(defined
as the longest process) lengths were measured on photographic prints using a
Houston
Instrument HI-PAD digitizing tablet connected to an IBM XT computer with
appropriate
software (Bioquant IV, R & M Biometrics, Nashville, TN); only processes more
than three-
fold of the cell body length were measured. Data from identically treated
coverslips (three or
four per group) were not different and therefore were combined. Mean values
were
calculated and compared using a one-way (groups treated with Compound (Ia) or
tacrolimus
versus an entreated control group) ANOVA followed by Newman-Kuels multiple
comparisons test (WINKS 4.62 professional edition).
Results:
_7_
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
At 72 hours, there was no significant difference between the untreated control
group
and the group treated with 10 nM tacrolimus. However, the group treated with a
10 nM
concentration of Compound (Ia) elicited a statistically significant increase
in length of
neurites. See results in Table I below:
Table 1: Effects of Compound (Ial and Tacrolimus on Mean Neurite Lengths in
PrimarX
Hippocampal Cell Cultures in Rats at 72 hr
Neurite Lengths
(,um)
No Treatment 372.318.23
Tacrolimus ( 10 nM) 423.4 19.50
Compound (Ia) (10 nM) 502.223.61
P<0.05
versus
No
Treatment
(one-way
ANOVA
followed
by
Newman-Kuels
multiple
comparisons test)
Example 2: Treatment with Compound (I1 Increases Mean Neurite Lengths in SH
SYSY
Human Neuroblastoma Cells
Preparation of SH-SYSY neuroblastoma cell cultures:
SH-SYSY human neuroblastoma cells were maintained in DMEM medium (GIBCO)
supplemented with 10% fetal calf serum (SIGMA), 50 LU./ml of penicillin, and
50 ,ug/ml
streptomycin (GIBCO) at 37°C in 7% CO~. Cells were plated in six-well
plates at 15,000
cells/well and treated with 0.4 ~,M aphidicolin (SIGMA). At 5 days, cells were
washed and
treated with nerve growth factor (NGF) at 10 ng/ml (to induce process
outgrowth) in the
presence or absence of tacrolimus (10 nM) or Compound (Ia) (1 nM). Medium was
changed
at 96 h and replaced with fresh medium for an additional 72 h (total time, 168
h). Duplicate
wells were run in all experiments and the data were averaged for each
treatment group.
Analysis of neurite lengths in SH-SYSY neuroblastoma cells:
For analysis of process length, cells (20 fields per well) were randomly
photographed
at 168 h. Neurite lengths were measured on photographic prints using a Houston
Instrument
HI-PAD digitizing tablet connected to an IBM XT computer with appropriate
software
_g_
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
(Bioquant IV, R & M Biometrics, Nashville, TN); only those processes greater
than two-fold
of the cell body length were measured. Data from identically treated wells
were not different
and were therefore combined. Mean values were calculated and compared using a
one-way
(Compound (Ia) or tacrolimus treated samples versus samples treated with NGF
alone)
ANOVA followed by Newman-Kuels multiple comparisons test (WINKS 4.62
professional
edition).
Results:
Measurement of the lengths of neurite processes demonstrated that both
Compound
(Ia) (1 nM) and tacrolimus (10 nM) significantly increased the length of
neurite processes at
168 h compared to NGF (10 ng/ml) alone. However, the effects of 1 nM Compound
(Ia) in
combination with NGF were higher than the effects of 10 nM tacrolimus in
combination with
NGF.
Table 2: Effect of Compound Ial and Tacrolimus on Mean Neurite Lengths in SH
SYSY
Human Neuroblastoma Cells at 168 hr
Neurite Lengths
(,um)
NT 94.753.734
NGF (10 ng/ml) 198.88.991
tacrolimus (10 nM) + NGF (10 ng/ml)227.69.130 *
Compound (Ia) (1 nM) +NGF (10 ng/ml)256.09.067
P<0.05 versus NGF (one-way ANOVA
followed by Newman-Kuels multiple
comparisons
test)
Examble 3: Treatment with Compound~Il Promotes Functional Recovery in the Rat
Sciatic
Nerve Crush Model
Animals and surgical procedure:
Nine 6-week-old male Sprague-Dawley rats were anesthetized with 2% halothane,
the
right sciatic nerve was exposed, and the nerve was crushed twice (for a total
of 60 s using a
-9-
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
No.7 Dumont jeweler's forceps) at the level of the hip. The crush site was
marked by tying a
sterile 9-O suture through the epineurial sheath.
Preparation of Compound (Ia) and administration:
Compound (Ia) was dissolved in vehicle comprising 30% dimethylsulfoxide
(DMSO):70% saline. Three axotomized rats received subcutaneous daily
injections either
Compound (Ia) (1 or 5 mg/lcg) or an equivalent volume of vehicle (30% DMSO in
saline)
(Sml/kg)
Behavioral assessment:
Animals were examined daily until the day of perfusion (18 days). The
following
semi-quantitative scale was used to evaluate the functional recovery of the
animals:
0: paralysis with the foot turned-out upon walking and the toes curved;
1: ability to right the foot and move the toes;
2: ability to constantly walk on the foot;
3 : demonstrates tae spread during walking;
4: wallcs off of heel and shows near normal toe spread.
Animals demonstrating intermediate abilities were given partial scores: +,
0.25; ++, 0.5; +++,
0.75.
Tissue fixation and preparation:
At 18 days after nerve crush, the rats were deeply anesthetized with 4%
halothane,
heparinized, and perfused with 4% paraformaldehyde in 0.1 M sodium phosphate
buffer (pH
7.4) for lOs followed by 5% glutaraldehyde (1L) in 0.1 M sodium phosphate
buffer (pH 7.4)
and fixed at 4 ° C for 24 h. Tissues were sampled from the sciatic
nerve at a known (5 mm)
distance from the crush site. In the present study, only the data from the
branch of the
posterior tibial nerve supplying the soleus muscle are reported. Tissues were
placed in 0.1 M
sodium phosphate buffer (pH 7.4), postfixed with 1 % osmium tetroxide (in 0.1
M phosphate
buffer) for 2.5 h, dehydrated in ethanol and embedded in plastic. Semithin
sections were
stained with uranyl acetate and lead citrate, mounted on film-supported 75
mesh grids, and
-10-
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
examined in a JEOL 100 CX electron microscope.
Morphornetric analysis:
Analysis of axonal calibers was performed in the soleus nerve. The numbers of
regenerating myelinated axons were counted using electron microscopy. Mean
values and
standard errors were calculated for the vehicle-treated group, Compound (Ia)
(1 mg/kg)-
treated group, and Compound (Ia) (5 mg/kg)-treated group.
Statistical analysis:
For the behavioral analysis, mean values for recovery of function were
compared
using one-way ANOVA followed by the Newman-Keuls multiple comparisons test for
comparison of individual values. For the morphometric analysis, mean values
for the number
of axons were compared using one-way ANOVA followed by the Newman Keuls
multiple
comparisons test for comparison of individual values.
Results:
Functional recovery:
Functional recovery was observed on days 15-17, and occurred earlier in both 1
mg/lcg-treated rats and 5 mg/kg-treated rats than in vehicle-treated rats. See
Table 3 below.
Table 3: Effect of Compound (Ial on Functional Recovery of Sciatic Nerve
Injiury in Rats
Functional Score
Vehicle (s.c.) Compound (Ia)
(30% DMSO i
n
1 mg/kg (s.c.) 5 mg/kg (s.c.)
saline)
Day 15 1.670.08 2.580.17 * 3.170.08 *
Day 16 1.830.08 2.830.17 * 3.500.00
Day 17 2.500.00 3.08~0.2~ * 3.500.00
*: P<0.05 versus Vehicle (one-way ANOVA followed by Newman-Kuels multiple
comparisons test)
-11-
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
Electron microscopy
Morphological examination of the animals was conducted at 18 days following
axotomy.
The numbers of regenerating myelinated axons per nerve area (5,000 ,u m2) were
dramatically increased from 5.5 X2.7 (mean~SEM) in vehicle-treated rats to 19
~ 2.4 and 20 ~
2.9 in l and 5 mg/kg-treated rats, respectively (P<0.05). See Table 4 below.
Table 4~ Effect of Compound (Ial on Numbers of Regenerating Myelinated Axons
per Nerve
Area (5,000 ,umzl in the Soleus Nerve 18 Days after Sciatic Nerve Crush in
Rats
Vehicle (s.c.) Compound (Ia)
(30%
DMSO in saline) 1 mg/lcg (s.c.) 5 mg/lcg (s.c.)
Regenerating 5.5 ~ 2.7 19 ~ 2.4 * 20 ~ 2.9
myelinated
axons
~: Y<U.US versus Vehicle (one-way ANOVA followed by Newman-Kuels multiple
comparisons test)
Examt~le 4: Treatment with Compound (I1 Promotes Functional Recovery in the
Rat Spinal
Cord Iniury Model
(1) Methods
Animals and surgical procedure
Twenty-eight 6-week-old male Sprague-Dawley rats were anesthetized with 2%
halothane, a laminectomy at T10/Tl 1 was performed and a hemisection lesion of
spinal cord
was performed at the level of T10/T11 spinal cord.
Preparation of Compound (Ial and administration
Compound (Ia) was dissolved in vehicle comprising 30% dimethylsulfoxide
(DMSO):
70% saline. The spinal cord lesioned rats received subcutaneous daily
injections the
Compound (Ia) (2 mg/kg) or an equivalent volume of vehicle (30% DMSO in
saline)
(Sml/kg) for seven weeks following the surgery.
-12-
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
Eyaluation of functional recovery
Functional recovery was assessed using a modified Tarlov/Klinger scale, narrow
beam
test and footprint test at 2 weeks postlesion.
A. modified Tarlov/Klin er scale
Rats were allowed to move freely in an open field for 1 min and rated 0-6
according to
the scale presented below.
0: No movement of the lesioned hind limb
1: Barely perceptible movement in the lesioned hind limb
2: Bride movements at the lesioned hind limb joints (Hip, knee or anlcle) but
no
coordination, no weight support
3: Alternate stepping and propulsive movements of the lesioned hind limb, no
weight
support
4: Can support weight on the injured hind limb
5: Walk with only mild deficit
6: Normal walking
B. Narrow beam test
Rats were tested on wooden beams (1.5m long) with decreasing width: 7.7cm,
4.7cm,
2.7cm and 1.7cm. Rats were allowed to walk on the bars, and the narrowest bar
they could
walk on without any slips in at least two trails was recorded.
0: No walking on any beam
1: Can walls on the 7.7cm beam
2: Can walk on the 4.7cm beam
3: Can walls on the 2.7cm beam
4: Can walls on the 1.7cm beam
C. Footprint test
The hind limbs of rats were inked and footprint were made on paper covering a
narrow runway of 60cm length and 7.Scm width. A series of at least six
sequential steps was
used to determine the 5-point footprint score.
-13-
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
0: Constant dorsal stepping or hind limb dragging, i.e. no footprint is
visible
1: Has visible toe prints of at least three toes in at least three footprints
2: Shows exo- or endo-rotation of the feet of more than double values as
compared to
its own baseline values
3: Shows no signs of toe dragging but foot rotation
4: Shows no signs of exo- or endo-rotation (less than twice the angle of the
baseline
values), but more than one heel print are visible
5: No heel prints are visible
Statistical anal,
For the behavioral analysis, mean values for score of each functional test
were
compared using one-way ANOVA followed lay the Newman-Keuls multiple
comparisons
test for comparison of individual values.
(2) Results
Functional recovery
In all three functional recovery measurements carried out using a modified
Tarlov/Klinger scale (Table 5), Beam walking test (Table 6) and footprint test
(Table 7)
Compound (Ia) improved motor functional impairment in modified Tarlov/Klinger
scale
(Table 5), Beam walking test (Table 6) and footprint test (Table 7).
Table 5: Effect of Compound (Ial on modified Tarlov/Klinger score of spinal
cord iniur~in
rats
Modified Tarlov/Klinger
score
Vehicle (s.c.) Compound (Ia)
(30% DMSO in saline) 2mg/lcg (s.c.)
Weelc 2 2.1 ~ 0.1 3.7 ~ 0.2*
~: r~u.u~ versus Velucle (one-way ANOVA followed by Newman-Kuels multiple
comparisons test)
Table 6: Effect of Compound (Ial on beam walking score of spinal cord injury
in rats
-14-
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
Beam walking score
Vehicle (s.c.) Compound (Ia)
(30% DMSO in saline) 2mg/lcg (s.c.)
Weelc2 0.90.1 2.0~0.3*
. rw.~~ versus vemcle (one-way ANUVA followed by Newman-Kuels multiple
comparisons test)
Table 7: Effect of Combound (Ial on footprint score of spinal cord inj~urv in
rats
Footprint score
Vehicle (s.c.) Compound (Ia)
(30% DMSO in saline) 2mg/lcg (s.c.)
Week2 1.70.4 3.6~0.3*
*: P<0.05 versus Vehicle (one-way ANOVA followed by Newman-Kuels multiple
comparisons test)
Examble 5: Compound (Ial Binds to FKBP12 but Unlike Tacrolimus Exerts Little
or No
Immunosupnessive Effect
(1) Binding Assay to FKBP12
The binding assay was performed according to a similar manner to that of
Tamura, K.,
et al (Biochemical arid Biophysical Research Communications, Vol. 202, No. l,
437-499,
1994). The results are shown in Table 8.
(2) Mixed lymphocyte reaction (MLR)
MLR test was performed according to a similar manner to that of U.S. Patent
4,929,611.
The Results are shown in the Table 8.
Table 8: Pharmacolo ig cal profiles of Compound ,Ial and tacrolimus i~ vitro
-15-
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
FKBP12 binding MLR ICSo
ICSO
(nM) (nM)
Compound (Ia) <5 > 100
Tacrolimus
The above results indicate that the compound (Ia) does not have
immunosuppressive
activity though it can bind to FKBP 12.
The results shown above illustrate the potent neurotrophic effects of Compound
(I)
using both ih vitro and ih vivo models. In two cell culture models, Compound
(I) even at low
concentrations increased neurite outgrowth. Moreover, systemic administration
of
Compound (I) at low doses speeded functional recovery following a nerve crush
lesion by
increasing the rate of axonal regeneration in the sciatic nerve and promoted
functional
recovery from spinal cord injury.
Moreover, as shown above, Compound (I) provides a potent neurotrophic or nerve
cell
growth stimulating activity, though it has no immunosuppressive activity.
Accordingly, the
present invention provides a useful neurotrophic agent for stimulating or
promoting neuronal
growth or regeneration, particularly when an immunosuppressive effect is not
advantageous
or desired.
Other aspects of the present invention include:
An article of manufacture, comprising packaging material and Compound (I)
identified in the above contained within said packaging material, wherein said
Compound (I)
is therapeutically effective for preventing or treating neuronal dysfunction,
and wherein said
packaging material comprises a label or a written material which indicates
that Compound (I)
can or should be used for preventing or treating neuronal injury/dysfunction.
A commercial package comprising the pharmaceutical composition containing
Compound (I) identified in the above and a written matter associated
therewith, wherein the
written matter states that Compound (I) can or should be used for preventing
or treating
neuronal injury/dysfunction.
A composition, such as a cell suspension, tissue, or graft comprising a cell
treated
with Compound (I). Such compositions are useful for repairing damage to the
nervous
system. Such compositions may also include other nerve cell growth stimulating
agents, such
-16-
CA 02433384 2003-06-27
WO 02/053159 PCT/USO1/50419
as other types of cell suspensions that promote or assist nerve cell growth,
such as myelin-
producing cells such as Schwann cells or oligodendrocytes, glial cells and
sheathing cells;
extracellular matrix material, such as collagen; or other specific
neuroregulators such as
cytolcines, mitogenic factors, immunophilins, and neurotrophins, such as NGF-
1, BDNF,
CNTF, NT-3, NT-4 and NT-5.
Grafts, such as homografts, allografts or xenografts may also be treated with
Compound (I) in order to facilitate neuronal outgrowth and their use as
transplants and for
other applications.
Incorporation by Reference
The content of each document, patent application or patent publication cited
by or
referred to in this disclosure is incorporated by reference in its entirety.
The content of any
patent document to which this application claims priority is also incorporated
by reference in
its entirety. Specifically, the content of U.S. Provisional Application No.
60/258,500 is
incorporated by reference.
Modifications and other embodiments
Obviously, numerous modifications and variations of the present invention are
possible in light of the above teachings. It is therefore to be understood
that within the scope
of the appended claims, the invention may be practiced otherwise than as
specifically
described herein. Various modifications and variations of the described
compositions and
methods, as well as the concept of the invention, will be apparent to those
skilled in the art
without departing from the scope and spirit of the invention. Although the
invention has been
described in connection with specific preferred embodiments, it should be
understood that the
invention as claimed is not intended to be limited to such specific
embodiments. Various
modifications of the described modes for carrying out the invention which are
obvious to
those skilled in the medical, biological, chemical or pharmacological arts or
related fields are
intended to be within the scope of the present invention.
-17-
