Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF TREATING HAIR LOSS USING
NON-IMMUNOSUPPRESSIVE COMPOUNDS
FIELD OF THE INVENTION
The present invention relates to methods for treating hair loss in mammals,
including
arresting and / or reversing hair loss and promoting hair growth.
BACKGROUND OF THE INVENTION
Hair loss is a common problem which occurs, for example, through natural
processes or
is often chemically promoted through the use of certain therapeutic drugs
designed to alleviate
conditions such as cancer. Often such hair loss is accompanied by lack of hair
regrowth which
causes partial or full baldness.
As is well-known in the art, hair growth occurs by a cycle of activity which
involves
alternating periods of growth and rest. This cycle is often divided into three
main stages which
are known as anagen, catagen, and telogen. Anagen is the growth phase of the
cycle and may be
characterized by penetration of the hair follicle deep into the dermis with
rapid proliferation of
cells which are differentiating to form hair. The next phase is catagen, which
is a transitional
stage marked by the cessation of cell division, and during which the hair
follicle regresses
through the dermis and hair growth is ceased. The next phase, telogen, is
often characterized as
the resting stage during which the regressed follicle contains a germ with
tightly packed dermal
papilla cells. At telogen, the initiation of a new anagen phase is caused by
rapid cell proliferation
in the germ, expansion of the dermal papilla, and elaboration of basement
membrane
components. Wherein hair growth ceases, most of the hair follicles reside in
teiogen and anagen
is not engaged, thus causing the onset of full or partial baldness.
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There have been many attempts in the literature to invoke the regrowth of hair
by, for
example, the promotion or prolongation of anagen. Currently, there are two
drugs approved by
the United States Food and Drug Administration for the treatment of male
pattern baldness:
topical minoxidil (marketed as Rogaine by Pharmacia & Upjohn), and oral
fmasteride (marketed
as Propecia by Merck & Co., Inc.). For several reasons, however, including
safety concerns and
/ or lack of efficacy, the search for efficacious hair growth inducers is
ongoing.
Interestingly, cyclosporin A is known to invoke a prominent hair induction
side effect.
Unfortunately, however, cyclosporin A is not practical for use as a hair
growth agent due to its
strongly immunosuppressive activity. See Yamamoto et al., "Hair Growth-
Stimulating Effects of
Cyclosporin A and FK506, Potent Immunosuppressants", Journal of Dermatological
Science,
Vol. 7 (suppl.), pp. S47 - S54 (1994); Maurer et al., "Hair Growth Modulation
by Topical
Immunophilin Ligands", American Journal of Pathology, Vol. 150, No. 4, pp.
1433 - 1441
(1997); Paus et al., "Hair Growth Control by Immunosuppression", Archives of
Dermatological
Research, Vol. 288, pp. 408 - 410 (1996); Paus et al., "Cyclosporin A, PSC 833
and FK 506, but
not Cyclosporin H and Rapamycin, Induce Anagen and Inhibit Catagen in Murine
Skin", The
Journal of Investigative Dermatology, Vol. 101, p. 420 (1994); Paus et al.,
"Cyclosporin A,
FK506 and Related Drugs as Tools for Hair Research", Archives of
Dermatological Research,
Vol. 285, p. 80 (1993); and Traber et al., "Cyclosporins - New Analogues by
Precursor Directed
Biosynthesis", The Journal ofAntibiotics, Vol. 42, No. 4, pp. 591 - 597
(1988).
It has been reported that PSC833, which is a non-immunosuppressive analog of
cyclosporin D, induces anagen in mice at a weaker level than cyclosporin A.
See Paus et al.,
"Cyclosporin A, PSC 833 and FK 506, but not Cyclosporin H and Rapamycin,
Induce Anagen
and Inhibit Catagen in Murine Skin", The Journal of Investigative Dermatology,
Vol. 101, p. 420
(1994); Paus et al., "Cyclosporin A, FK506 and Related Drugs as Tools for Hair
Research",
Archives ofDermatological Research, Vol. 285, p. 80 (1993).
Based on the weaker effect of PSC833 as compared to cyclosporin A, the
challenge has
been to provide non-immunosuppressive analogs of cyclosporin A which exhibit
hair growth
induction approaching that of cyclosporin A itself. Surprisingly, the present
inventors have
discovered analogs of cyclosporin A which are devoid of immunosuppressive
activity yet induce
hair growth at levels comparable to cyclosporin A. Accordingly, potent methods
of treating hair
loss using cyclosporin A analogs which are practical for use are provided
herein.
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SUMMARY OF THE INVENTION
The present invention relates to methods for treating hair loss comprising
administering
to a mammal a non-immunosuppressive compound which has been found by the
present inventors
to be particularly useful for treating hair loss in mammals, including
arresting and / or reversing
hair loss and promoting hair growth. The compounds utilized in the present
method have the
structure:
R1
R"
1
O RI~ CH3
I L-1 O
N~N N~N/
O N ~I = I I = R
O ~ O RZ
O
N N
O N ~ O Ns.
O.
~N~
H R~ IOI I Rs O
and pharmaceutically acceptable salts, hydrates, and biohydrolyzable amides,
esters, and imides
thereof, wherein R~, R,', R,", R2, R3, R4, R5, RS', R6, R~, and R8 are as
defined herein.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods of using compounds and compositions
which
are particularly useful for treating hair loss in mammals, preferably humans,
including arresting
and / or reversing hair loss and promoting hair growth.
In addition to discovering that the present compounds are useful for treating
hair loss, the
present inventors have also surprisingly discovered that immunosuppression is
not required for
hair growth stimulation. The present inventors have further discovered
compounds that are
useful for treating hair loss but are surprisingly non-immunosuppressive. The
compounds useful
in the method of the present invention are therefore, as defined herein, non-
immunosuppressive.
Publications and patents are referred to throughout this disclosure. All
references cited
herein are hereby incorporated by reference.
All percentages, ratios, and proportions used herein are by weight unless
otherwise
specified.
Definition and Usage of Terms
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The following is a list of definitions for terms used herein:
As used herein "salt" is a cationic salt formed at any acidic (e.g., carboxyl)
group, or an
anionic salt formed at any basic (e.g., amino) group. Many such salts are
known in the art.
Preferred cationic salts include the alkali metal salts (such as, for example,
sodium and
potassium), alkaline earth metal salts (such as, for example, magnesium and
calcium), and
organic salts. Preferred anionic salts include the halides (such as, for
example, chloride salts).
Such acceptable salts must, when administered, be appropriate for mammalian
use.
As used herein, "alkenyl" is an unsaturated hydrocarbon chain radical.
Alkenyls have at
least one double bond. Unless otherwise specified, alkenyls have from 2 to
about 15 carbon
atoms (Cz - C,5); preferably from 2 to about 10 carbon atoms (CZ - C,o); more
preferably from 2 to
about 8 carbon atoms (CZ - C8), and most preferably from 2 to about 6 carbon
atoms (CZ - C6).
Non-limiting examples of alkenyls include vinyl, allyl, and butenyl.
As used herein, "alkoxy" is an oxygen radical having an alkyl, alkenyl, or
alkynyl,
preferably an alkyl or alkenyl, and most preferably an alkyl substituent.
Examples of alkoxy
radicals include -O-methyl and -O-ethyl.
As used herein, "alkyl" is a saturated hydrocarbon chain radical. Unless
otherwise
specified, alkyls have from 1 to about 15 carbon atoms (C, - C,5); preferably
from 1 to about 10
carbon atoms (C, - C,o); more preferably from 1 to about 6 carbon atoms (C, -
C6); and most
preferably from 1 to about 4 carbon atoms (C, - CQ). Preferred alkyls include,
for example,
methyl, ethyl, propyl, iso-propyl, and butyl.
As used herein, "alkynyl" is an unsaturated hydrocarbon chain radical.
Alkynyls have at
least one triple bond. Unless otherwise specified, alkynyls have from 2 to
about 15 carbon atoms
(CZ - C,5); preferably from 2 to about 10 carbon atoms (CZ - C,o); more
preferably from 2 to about
8 carbon atoms (CZ - C8), and most preferably from 2 to about 6 carbon atoms
(Cz - C6).
As used herein, "biohydrolyzable amides" are amides of the compounds used in
the
present invention which do not interfere with the activity of the compound, or
that are readily
converted in vivo by a mammalian subject to yield an active compound.
As used herein, "biohydrolyzable esters" are esters of the compounds used in
the present
invention which do not interfere with the activity of the compound, or that
are readily converted
in vivo by a mammalian subject to yield an active compound.
As used herein, "biohydrolyzable imides" are imides of the compounds used in
the
present invention which do not interfere with the activity of the compound, or
that are readily
converted in vivo by a mammalian subject to yield an active compound.
As used herein, a "lower" moiety (e.g., "lower" alkyl) is a moiety having 1 to
about 6,
preferably 1 to about 4, carbon atoms.
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As used herein, "pharmaceutically acceptable" means suitable for use in a
human or other
mammal.
As used herein, "safe and effective amount of a compound" (or composition, or
the like)
means an amount that is effective to exhibit biological activity, preferably
wherein the biological
activity is arresting and / or reversing hair loss or promoting hair growth,
at the sites) of activity
in a mammalian subject, without undue adverse side effects (such as toxicity,
irritation, or allergic
response), commensurate with a reasonable benefit / risk ratio when used in
the manner of this
invention.
As used herein, substituent groups may themselves be substituted. Such
substitution may
be with one or more substituents. Such substituents include those listed in C.
Hansch and A. Leo,
Substituent Constants for Correlation Analysis in Chemistry and Biology
(1979). As used herein
unless otherwise specified, the term "substituted" in reference to a group,
moiety, or the like,
preferably means having one or more substituent groups each independently
selected from
hydrogen, alkyl, alkenyl, alkoxy, hydroxy, oxo, nitro, amino, alkylamino,
cyano, halo, carboxy,
alkoxyacyl (e.g., carboethyoxy), thiol, aryl, cycloalkyl, heteroaryl,
heterocycloalkyl (e.g.,
piperidinyl, morpholinyl, pyrrolidinyl), imino, thioxo, hydroxyalkyl, aryloxy,
and arylalkyl, more
preferably hydrogen, alkyl, alkenyl, alkoxy, hydroxy, oxo, nitro, amino,
alkylamino, halo, thiol,
and aryloxy, even more preferably hydrogen, alkyl, alkenyl, alkoxy, hydroxy,
nitro, amino,
alkylamino, and halo, still more preferably hydrogen, alkyl, and alkoxy, and
most preferably
alkoxy.
As used herein, wherein any variable, moiety, group, or the like occurs more
than one
time in any variable or structure, its definition at each occurrence is
independent of its definition
at every other occurrence.
Methods of the Present Invention
The present invention relates to methods of treating hair loss comprising
administering to
a mammal a composition comprising a compound having the structure:
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Rt
R"
t
O Rt~ CH3
H O
N~N N~N
O N ~I = ~ ~ = R
O ~ O RZ
O
N N_
O N ~ O Ns.
O. ~
N~I II N
H R~ O ~ Rs O
and pharmaceutically acceptable salts, hydrates, and biohydrolyzable amides,
esters, and imides
thereof, wherein:
(a) Rt is selected from the group consisting of 1-propenyl, propyl, 3-hydroxy-
1-propenyl,
-O-methyl, -O p-benzoyl benzyl, and hydroxy;
(b) R,' is selected from hydroxy, oxo, and acyloxy;
(c) Rt" is selected from hydrogen and methyl;
(d) Rz is selected from ethyl, n-butyl, 2-hydroxypropyl, 2-methoxypropyl, 1-
methylpropyl, and 2-acyloxy-propyl;
(e) R3 is selected from hydrogen, methyl, benzyl, 1-propenyl, and 2-methyl-3-
hydroxy-
propyl;
(~ R4 is substituted or unsubstituted C1 - C9 straight or branched alkyl;
(g) RS is substituted or unsubstituted C1 - C6 straight or branched alkyl;
(h) RS' is selected from hydrogen, methyl, benzyl, p-fluorobenzyl, 1-propenyl,
and 1-
phenyl-1-propenyl;
(i) R6 is selected from 2-methylpropyl, 2-methyl-3-hydroxypropyl, methyl, and
ethyl;
(j) R~ is selected from methyl and phenyl; and
(k) R8 is selected from methyl and hydroxymethyl.
The R, Moie
The R, moiety is selected from 1-propenyl, propyl, 3-hydroxy-1-propenyl, -O-
methyl, -O-
p-benzoyl benzyl, and hydroxy. More preferably, R, is selected from 1-
propenyl, propyl, and 3-
hydroxy-1-propenyl, most preferably R, is 1-propenyl.
The R,' Moie
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The R~' moiety is selected from hydroxy, oxo, and acyloxy. As used herein, the
term
"oxo" means a doubly bonded oxygen radical. As used herein, the term "acyloxy"
is an oxygen
radical having an acyl substituent. "Acyl" means a radical which could be
formed by removal of
the hydroxy from a carboxylic acid (i.e., X-C(=O)-), wherein X is any
substituent but preferably
selected from alkyl, alkenyl, alkynyl, and aryl, most preferably alkyl. Thus,
for example,
"acyloxy" is illustrated by -O-C(=O)-alkyl.
While both hydroxy and oxo are both highly preferred, the most preferred R,'
moiety is
oxo.
The R," Moie
The R," moiety is selected from hydrogen and methyl. Most preferably, R," is
hydrogen.
These R,, R~', and R," moieties are further described in, for example, U.S.
Patent No.
5,767,069, Ko et al., assigned to Novartis, issued June 16, 1998; WO 97/18828,
Steiner et al.,
assigned to Guilford Pharmaceuticals, Inc., published May 29, 1997; and Bartz
et al., "Inhibition
of Human Immunodeficiency Virus Replication by Nonimmunosuppressive Analogs of
Cyclosporin A", Proceedings of the National Academy of Sciences U.S.A., Vol.
92, pp. 5381
5385 (1995).
The R~ Moie
The RZ moiety is selected from ethyl, n-butyl, 2-hydroxypropyl, 2-
methoxypropyl, 1-
methylpropyl, and 2-acyloxy-propyl. As used herein, the term "acyloxy" is an
oxygen radical
having an acyl substituent. "Acyl" means a radical which could be formed by
removal of the
hydroxy from a carboxylic acid (i.e., X-C(=O)-), wherein X is preferably
selected from alkyl,
alkenyl, alkynyl, and aryl. Thus, 2-acyloxy-propyl is exemplified by:
point of attachment ~ ~ O
O" X
H3C
wherein X is any substituent but preferably selected from alkyl, alkenyl,
alkynyl, and aryl,
most preferably alkyl.
RZ is preferably selected from ethyl, n-butyl, 2-hydroxypropyl, and 2-
methoxypropyl.
Most preferably, R~ is ethyl.
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These RZ moieties are further described in, for example, U.S. Patent No.
5,767,069, Ko et
al., assigned to Novartis, issued June 16, 1998; and WO 97/18828, Steiner et
al., assigned to
Guilford Pharmaceuticals, Inc., published May 29, 1997.
The R~ Moie
The R3 moiety is selected from hydrogen, methyl, benzyl, 1-propenyl, and 2-
methyl-3-
hydroxy-propyl. Preferably, R3 is selected from hydrogen, methyl and benzyl,.
Most preferably
R3 is hydrogen or benzyl.
These R3 moieties are further described in, for example, U.S. Patent No.
5,767,069, Ko et
al., assigned to Novartis, issued June 16, 1998; WO 97/18828, Steiner et al.,
assigned to Guilford
Pharmaceuticals, Inc., published May 29, 1997; and Hu et al., "Cyclosporin
Analogs Modified in
the 3,7,8-Positions: Substituent Effects on Peptidylprolyl Isomerase
Inhibition and
Immunosuppressive Activity Are Nonadditive", Journal of Medicinal Chemistry,
Vol. 38, pp.
4164 - 4170 (1995).
With respect to stereochemistry at the R3 position, the R3 moiety may be in
the L or D
configuration, but is preferably in the D configuration.
The R4 Moie
The R4 moiety is substituted or unsubstituted C~ - C9 straight or branched
alkyl,
preferably substituted or unsubstituted CI - C6 straight or branched alkyl,
and more preferably
substituted or unsubstituted C~ - C4 straight or branched alkyl.
Preferably, R4 is selected from 2-methylpropyl, 2-methyl-3-hydroxypropyl, 2-
methylbutyl, iso-propyl, 2-hydroxypropyl, and methyl. Most preferably, R4 is 2-
methylpropyl.
These R4 moieties are further described in, for example, U.S. Patent No.
5,767,069, Ko et
al., assigned to Novartis, issued June 16, 1998; WO 97/18828, Steiner et al.,
assigned to Guilford
Pharmaceuticals, Inc., published May 29, 1997; and Bartz et al., "Inhibition
of Human
Immunodeficiency Virus Replication by Nonimmunosuppressive Analogs of
Cyclosporin A",
Proceedings of the National Academy of Sciences U.S.A., Vol. 92, pp. 5381 -
5385 (1995).
The RS Moie
The RS moiety is substituted or unsubstituted C1 - C6 straight or branched
alkyl, and more
preferably substituted or unsubstituted C, - C4 straight or branched alkyl.
Preferably RS is selected from 2-methylpropyl, n-butyl and iso-propyl. Most
preferably,
RS is iso-propyl.
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These RS moieties are further described in, for example, U.S. Patent No.
5,767,069, Ko et
al., assigned to Novartis, issued June 16, 1998; and WO 97/18828, Steiner et
al., assigned to
Guilford Pharmaceuticals, Inc., published May 29, 1997.
The RS' Moie
The RS' moiety is selected from hydrogen, methyl, benzyl, p-fluorobenzyl, 1-
propenyl,
and 1-phenyl-1-propenyl. For clarity, 1-propenyl is exemplified as:
point of attachment
to nitrogen
For further clarity, 1-phenyl-1-propenyl is exemplified as:
point of attachment ~
to nitrogen
See also, Papa~eor~iou et al., "Conformational Control of Cyclosporin Through
Substitution of
the N-5 Position. A New Class of Cyclosporin Antagonists", Bioorganic &
Medicinal Chemistry,
Vol. S, No. 1, pp. 187 - 192 (1997).
Preferably, RS' is selected from hydrogen, methyl, and 1-propenyl, more
preferably
hydrogen and 1-propenyl. Most preferably, RS' is hydrogen.
The R6 Moie
The R6 moiety is selected from 2-methylpropyl, 2-methyl-3-hydroxypropyl,
methyl, and
ethyl, more preferably, 2-methylpropyl and 2-methyl-3-hydroxypropyl. Most
preferably, R6 is 2-
methylpropyl.
These R6 moieties are further described in, for example, U.S. Patent No.
5,767,069, Ko et
al., assigned to Novartis, issued June 16, 1998; and WO 97/18828, Steiner et
al., assigned to
Guilford Pharmaceuticals, Inc., published May 29, 1997.
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The R~ Moie
The R~ moiety is selected from methyl and phenyl. Preferably, R, is methyl.
These R~ moieties are further described in, for example, WO 97/18828, Steiner
et al.,
assigned to Guilford Pharmaceuticals, Inc., published May 29, 1997 and Hu et
al., "Cyclosporin
Analogs Modified in the 3,7,8-Positions: Substituent Effects on Peptidylprolyl
Isomerase
Inhibition and Immunosuppressive Activity Are Nonadditive", Journal of
Medicinal Chemistry,
Vol. 38, pp. 4164 - 4170 (1995).
The R8 Moie
The R8 moiety is selected from methyl and hydroxymethyl. Preferably, R8 is
methyl.
These R$ moieties are further described in, for example, WO 97/18828, Steiner
et al.,
assigned to Guilford Pharmaceuticals, Inc., published May 29, 1997 and Hu et
al., "Cyclosporin
Analogs Modified in the 3,7,8-Positions: Substituent Effects on Peptidylprolyl
Isomerase
Inhibition and Immunosuppressive Activity Are Nonadditive", Journal of
Medicinal Chemistry,
Vol. 38, pp. 4164 - 4170 (1995).
Preferred compounds useful in the methods of the present invention are shown
below:
1
CH3 O O H O CH3 O HO", O
\ Nv _N N~N/ \ N~N 1 N~N/
O N O ~ I O ~ ~ O N
O ~~ O ~ ~ O ~ ~O
N-
'N O H ~ O H 'N O H ~ O N-
~ I~
O~N~N~N N O~N N~N N
H O I O _ H IO I O
HO"
CH3 OII H O CH3 OII O H O
\ N~N ". N~N/ \ N~N N~N/
o N o /~ I o ~ ~o / \ o N o /~ I o W ~o ~ 1
N- N-
~N\ O H ~ O H ~N\ O H ~ O H
/~ I = I /~ II I I
O' 'N N~N, N O~N~N~N N
_ I II I ~ O - H IOI I O
H O
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0 0 0 ~,"
CH3 H O ~3 H O
\ Nv ' i N~N/ O \N N i N
O ; _ _ N
O ~ O \ ~O ~~ O ~ O ~ ~O
~N N- N N-
O H ~ O O H O H
O' 'N 1'1~N N O~N N~N N
H~ O I O H~ IOI I O
\ \
O" HO.,
~ H O ~3 O H O
N
O \ N ,.. N N \N N N
O ~ I O ~ O O O ~ I O
O
N- //~ _
'N O H ~O H 'N O H ~ O H N
~ I I ~
O ' 'N N N N~ p' \N N N N
O _ I I O
H O
\ ,,o l
~o
CH3 O HO'' H O CH3 O~ H O
\ N N N N/ \ N~N ,,'' N~N/
O = = N
N ~ ~ ~ O ~ O O ~ I O
O ~O
N- ~N N
'N O H ~ O H ~ O H ~ O H
~ O~N N~N N
O "N IIN
_ I N N - H O I O
H~ O ~ O
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O HO~~ HO"
O N N ,,.. N N O N ".. /
CH3 H O CI-I~ O H O
I
O ~~ O
O ~ I O \ ~ OI ~ I
N- N-
'N O H ~ O ~N O H ~ O
O~ N~ N I I N N O~ N N I I N N
H I IO I O H~ O I O
\ I I
OH OH
O HO,,
CH3 H O ~ O O
O \N N ~,.. N N N N N N
O ~ I O ~ ~ O O ~ I O
O ~O
N- /~
'N O H ~ O H 'N O H ~ O H N
~ I I -
O' \N N II N N O~N N II N N
- H O I O _ I I O
H O
Analytical Methods
The present invention relates to methods of treating hair loss in mammals by
administering to a mammal a non-immunosuppressive compound having a structure
as described
herein. Compounds (test compounds) may be tested for their ability to induce
anagen and their
immunosuppressive activity (or lack thereof) using the following methods.
Alternatively, other
methods well-known in the art may be used (but with the term "non-
immunosuppressive" being
defined according to the method disclosed herein below).
Teloeen Conversion Assay:
The Telogen Conversion Assay measures the potential of a test compound to
convert
mice in the resting stage of the hair growth cycle ("telogen"), to the growth
stage of the hair
growth cycle ("anagen").
Without intending to be limited by theory, there are three principal phases of
the hair
growth cycle: anagen, catagen, and telogen. It is believed that there is a
longer telogen period in
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C3H mice (Harlan Sprague Dawley, Inc., Indianapolis, IN) from approximately 40
days of age
until about 75 days of age, when hair growth is synchronized. It is believed
that after 75 days of
age, hair growth is no longer synchronized. Wherein about 40 day-old mice with
dark fur (brown
or black) are used in hair growth experiments, melanogenesis occurs along with
hair (fur) growth
wherein the topical application of hair growth inducers are evaluated. The
Telogen Conversion
Assay herein below is used to screen compounds for potential hair growth by
measuring
melanogenesis.
Three groups of 44 day-old C3H mice are utilized: a vehicle control group, a
positive
control group, and a test compound group, wherein the test compound group is
administered a
compound used in the method of the present invention. The length of the assay
is at least 19 days
with 15 treatment days (wherein the treatment days occur Mondays through
Fridays). Day 1 is
the first day of treatment. Most studies will end on Day 19, but a few may be
carried out to Day
24 if the melanogenesis response looks positive, but occurs slowly. A typical
study design is
shown in Table 1 below:
Table 1
GroupAnimal Compound ConcentrationApplication Length of
volume Study
# #
1 1 - Test 5% in vehicle**400 ~L topical19 or 24 days
Compound
2 11 - Cyclosporin0.19% in 400 ~,L topical19 or 24 days
A vehicle**
3 21 - Vehicle**N/A 400 ~L topical19 or 24 days
**The vehicle is 60% ethanol, 20% propylene glycol, and 20% dimethyl
isosorbide
(commercially available from Sigma Chemical Co., St. Louis, MO).
The mice are treated topically Monday through Friday on their lower back (base
of tail to
the lower rib). A pipettor and tip are used to deliver 400 ~L to each mouse's
back. The 400 ~L
application is applied slowly while moving hair on the mouse to allow the
application to reach the
skin.
While each treatment is being applied to the mouse topically, a visual grade
of from 0 to
4 will be given to the skin color in the application area of each animal. As a
mouse converts from
telogen to anagen, its skin color will become more bluish-black. As indicated
in Table 2, the
grades 0 to 4 represent the following visual observations as the skin
progresses from white to
bluish-black.
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Table 2
Visual Observation Grade
Whitish Skin Color 0
Skin is light gray (indication of initiation of 1
anagen)
Appearance of Blue Spots 2
Blue Spots are aggregating to form one large blue 3
area
Skin is dark blue (almost black) with color covering4
majority of treatment
area (indication of mouse in full anagen)
Immunosuppression Assay_:
The immunosuppression assay herein predicts the immunosuppressive activity (or
non-
immunosuppressive activity) of a compound used in the method of the present
invention. The
assay is performed as follows:
Spleens are excised from euthanized (CO, asphyxiation) adult male C3H mice
ranging in
age from seven to sixteen weeks old (live mice commercially available from
Harlan Sprague
Dawley, Inc., Indianapolis, IN). The spleens are placed immediately in cold
Hanks Balanced Salt
Solution (HBSS, commercially available from Gibco-BRL, Gaithersburg, MD). The
spleens are
then ground up between frosted glass slides and filtered through a sterile
screen to remove tissue
debris. The resulting cell suspension is underlayed with an equal volume of
Ficoll-Paque Plus
(commercially available from Pharmacia Biotech, Piscataway, NJ) and
centrifuged at 400 x g for
approximately forty minutes at 20 °C in order to collect the
splenocytes. The splenocytes are
collected from the interface using a disposable pipet and are washed twice
with HBSS, followed
by centrifugation at 100 x g for ten minutes at 20 °C. Splenocytes are
resuspended in five to ten
mL of cell culture media consisting of phenol red-free RPMI 1640 (culture
media commercially
available from Gibco-BRL) containing 10% heat-inactivated fetal bovine serum
(Gibco-BRL),
penicillin (50 U/mL), streptomycin (100 ~g/mL), L-glutamine (2 mM), 2-
mercaptoethanol (10-5
M), and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (10 mM).
The cells are
counted and checked for viability using, for example, trypan blue. Splenocytes
are resuspended
in medium at 106 cells/mL and pipetted into 96 well round bottom plates at 105
cells/well.
Splenocytes are activated by addition of 50 ~L/well of conconavalin A (final
assay concentration
= 5 pg/ml) in the presence or absence of a test compound. Test compounds are
made up as stock
solutions in methyl sulfoxide (DMSO), then diluted in medium and 50 ~.L/well
added, such that
the final concentration of DMSO in the assay is below 0.05%. The plates are
incubated at 37 °C
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with 5% COZ for 48 hours. After 48 hours, the cells are pulsed with 1 ~Ci/well
of methyl-3H-
thymidine (commercially available from Amersham, Buckinghamshire, England) and
incubated
an additional 24 hours.
After 24 hours, the cells are harvested onto GF/C filter plates (commercially
available
from Packard, Downers Grove, IL), solubilized in Microscint 20 (Packard), and
counted on a
TopCount microplate scintillation and luminescence plate counter (Packard).
Activity is
measured as a percentage of control activity in the absence of test compound
and plotted versus
test compound concentration. The data are fit to a 4-parameter curve fit
(Sigmaplot) and IC50
values are calculated. As used herein, test compounds are considered non-
immunosuppressive if,
by using this method, the ratio of (cyclosporin A ICS°/test compound
ICS°) x 100 is less than or
equal to 0.02, i.e., as defined herein, a non-immunosuppressive test compound
has 5 2% of the
immunosuppressive activity of cyclosporin A.
Cell viability is assessed using the MTT (3-[4,5-dimethyl-thiazoyl-2-yl]2,5-
diphenyl-
tetrazolium bromide) dye assay as described by Nelson et al., Journal of
Immunology, Vol. 150,
No. 6, pp. 2139 - 2147 (1993), with the exception that the assay is carried
out in serum-free,
phenol red-free RPMI 1640 and the dye is solubilized in 100 pL/well DMSO and
read at an OD
of 540 nm with a background correction at 650 nm on a SpectraMax Plus
microplate reader
(Molecular Devices, Menlo Park, CA).
Method of Makin
The compounds used in the methods of the present invention are prepared
according to
procedures which are well-known to those skilled in the art. The starting
materials used in
preparing the compounds are known, made by known methods, or are commercially
available as
a starting material.
It is recognized that the skilled artisan in the art of organic chemistry can
readily carry out
standard manipulations of organic compounds without further direction.
Examples of such
manipulations are discussed in standard texts such as J. March, Advanced
Organic Chemistry,
John Wiley & Sons (1992).
The skilled artisan will readily appreciate that certain reactions are best
carried out when
other functionalities are masked or protected in the compound, thus increasing
the yield of the
reaction and / or avoiding any undesirable side reactions. Often, the skilled
artisan utilizes
protecting groups to accomplish such increased yields or to avoid the
undesired reactions. These
reactions are found in the literature and are also well within the scope of
the skilled artisan.
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16
Examples of many such manipulations can be found in, for example, T. Greene,
Protecting
Groups in Organic Synthesis, John Wiley & Sons (1981).
The compounds of the present invention may have one or more chiral centers. As
a
result, one may selectively prepare one optical isomer, including
diastereomers and enantiomers,
over another, for example by chiral starting materials, catalysts or solvents,
or may prepare both
stereoisomers or both optical isomers, including diastereomers and enantiomers
at once (a
racemic mixture). Since the compounds of the invention may exist as racemic
mixtures, mixtures
of optical isomers, including diastereomers and enantiomers, may be separated
using known
methods, such as through the use of, for example, chiral salts and chiral
chromatography.
In addition, it is recognized that one optical isomer, including a
diastereomer and
enantiomer, or a stereoisomer, may have favorable properties over the other.
Thus, when
disclosing and claiming the invention, when one racemic mixture is disclosed,
it is clearly
contemplated that both optical isomers, including diastereomers and
enantiomers, or
stereoisomers substantially free of the other are disclosed and claimed as
well.
The syntheses of the compounds useful in the present invention are described
in the art.
Accordingly, the ordinarily skilled artisan will be able to prepare the
compounds described
herein. For further guidance, the following references describe syntheses of
the compounds
utilized in the present method: Bartz et al., "Inhibition of Human
Immunodeficiency Virus
Replication by Nonimmunosuppressive Analogs of Cyclosporin A", Proceedings of
the National
Academy of Sciences U.S.A., Vol. 92, pp. 5381 - 5385 (1995); WO 98/28328,
Barriere et al.,
assigned to Rhone-Poulenc, published July 2, 1998; U.S. Patent No. 5,643,870,
Boelsterli et al.,
assigned to Sandoz Ltd., issued July 1, 1997; U.S. Patent No. 5,525,590,
Bollinger et al., assigned
to Sandoz Ltd., issued June 11, 1996; U.S. Patent No. 5,116,816, Drevfuss et
al., assigned to
Sandoz Ltd., issued May 26, 1992; U.S. Patent No. 5,284,826, Eberle, assigned
to Sandoz Ltd.,
issued February 8, 1994; U.S. Patent No. 5,807,820, Elias, assigned to
Novartis, issued
September 15, 1998; Hu et al., "Cyclosporin Analogs Modified in the 3,7,8-
Positions:
Substituent Effects on Peptidylprolyl Isomerase Inhibition and
Immunosuppressive Activity Are
Nonadditive", Journal ofMedicinal Chemistry, Vol. 38, pp. 4164 - 4170 (1995);
U.S. Patent No.
5,767,069, Ko et al., assigned to Novartis, issued June 16, 1998; Papageorgiou
et al.,
"Conformational Control of Cyclosporin through Substitution of the N-5
Position. A New Class
of Cyclosporin Antagonists", Bioorganic & Medicinal Chemistry, Vol. 5, pp. 187
- 192 (1997);
Rich et al., "Synthesis, Biological Activity, and Conformational Analysis of
(2S, 3R, 4S)-
MeBmt'-cyclosporin, a Novel 1-Position Epimer of Cyclosporin A", Journal of
Medicinal
Chemistry, Vol. 32, pp. 1982 - 1987 (1989); EP 0,194,972, Seebach, assigned to
Sandoz Ltd.,
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17
published September 17, 1986; U.S. Patent No. 4,703,033, Seebach, assigned to
Sandoz Ltd.,
issued October 27, 1987; U.S. Patent No. 4,771,122, Seebach, assigned to
Sandoz Ltd., issued
September 13, 1988; WO 97/18828, Steiner et al., assigned to Guilford
Pharmaceuticals, Inc.,
published May 29, 1997; Sun et al., "Synthesis, Conformation, and
Immunosuppressive Activity
of Cyclosporins That Contain s-Oxygen (4R)-4-[(E)-Butenyl]-4,N-dimethyl-L-
threonine Analogs
in the 1-Position", Journal ofMedicinal Chemistry, Vol. 33, pp. 1443 - 1452
(1990); U.S. Patent
No. 4,289,851, Traber et al., assigned to Sandoz Ltd., issued September 15,
1981; Traber et al.,
"Cyclosporins - New Analogues by Precursor Directed Biosynthesis", The Journal
of Antibiotics,
Vol. 42, No. 4, pp. 591 - 597 (1988); U.S. Patent No. 4,764,503, Wen er,
assigned to Sandoz
Ltd., issued August 16, 1988; and WO 93/17039, Weneer, assigned to Sandoz
Ltd., published
September 2, 1993.
As even further guidance, the following non-limiting examples illustrate
methods of
making preferred compounds used in the present invention:
Example 1
O HO" O O
CH3 H O CH3 H OII
O \N N~ I " N~N/ O \N N~ I N~N/
O ~ O \ ~O --i ~ O \ ~O
N- N
'N~ O H ~ O H 'N O H ~ O H
O'' \N N = N N O~N N = N N
_ H~ ~ I O _ H~ ~ I O
(3'-keto)cyclosporin A: Dess-Martin periodinane (2.12 g, 5 mmol; prepared
according to the
procedure reported in Ireland et al., "An Improved Procedure for the
Preparation of the Dess-
Martin Periodinane", Journal of Organic Chemistry, Vol. 58, p. 2899 (1993)) is
combined with
dichloromethane (20 mL) and stirred for 10 minutes at ambient temperature. The
solution is
chilled to 5 °C and a solution of cyclosporin A (2 g, 1.66 mmol;
commercially available from
Alexis Corporation, San Diego, CA) in dichloromethane (40 mL) is added
dropwise over 10
minutes. The mixture is stirred at 5 °C for 1 hour followed by addition
of a solution of sodium
thiosulfate (5.53 g, 35 mmol), sodium bicarbonate (5.03 g, 60 mmol) and water
(100 mL). The
resulting mixture is stirred rapidly for 20 minutes while warming from 5
°C to ambient
temperature. The mixture is extracted with dichloromethane (3 x 75 mL), then
the combined
organic extracts are dried (MgS04), filtered, and concentrated under reduced
pressure. The
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18
resulting residue is purified via preparative chromatography (silica gel;
water saturated ethyl
acetate) to afford the desired (3'-keto)cyclosporin A.
Example 2
CH O HO" CH O HO",,
~ H O 3 ~ H OII
O \N N~ I ,. N~N/ O \N N~ I N~N/
O ~ O \ ~O -.~ O ~ O \ ( ~O
'N O H ~ O H N ~N~ O H ~ O ~ N
O~N N ~ N N O' \N N '-_ N N
_ H~ ~ I O _ H~ ~ I O
(NS-1-propenyl) Cyclosporin A: Cyclosporin A (2 g, 1.66 mmol; commercially
available from
Alexis Corporation, San Diego, CA) is dissolved in tetrahydrofuran (40 mL) at
ambient
temperature under inert atmosphere. The reaction solution is cooled to -78
°C and 1N
phosphazene base P4-t-butyl solution (8.4 mL, 8.32 mmol; commercially
available from Fluka
Chemika AG, Buchs, Switzerland) is added dropwise over 5 minutes. The solution
is stirred an
additional 5 minutes at -78 °C followed by the dropwise addition of
allyl bromide (1.16 mL, 13.3
mmol) over 1 minute. The solution is stirred for 90 minutes at -78 °C
followed by the addition of
1N aqueous citric acid (20 mL). The mixture is poured into a solution of 1N
aqueous citric acid
(80 mL) and brine (20 mL), then extracted with ethyl acetate (3 x SOmL). The
combined organic
extracts are dried (MgS04), Eltered, and concentrated under reduced pressure.
The residue is
purified via preparative chromatography (silica gel; gradient elution with 1:1
to 1:0 (ethyl acetate
dichloromethane)) to afford the desired (NS-allyl)cyclosporin A.
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Example 3
CH3 0 HO" H O ~H3 p HO" H O
O \N N N ,.. N N \N N N ... N N/
O~I O \ ~0--~ O O~I
~N N- N-
~ O H ~ O H ~ OII H ~ O H
O~N~N~N N O~N N '--_ N
I I N
H O I O H~ ~ I O
(N-Me-D-Phe)3-CsA: Cyclosporin A (5.0 g, 4.16 mmol; commercially available
from Alexis
Corporation, San Diego, CA) is dissolved in tetrahydrofuran (250 mL) at
ambient temperature
under inert atmosphere. The reaction is cooled to -78 °C and 1N lithium
bis(trimethylsilyl)amide
in tetrahydrofuran (41.6 mL, 41.6 mmol; commercially available from Aldrich
Chemical Co.,
Milwaukee, WI) is added dropwise over S minutes. After stirring at -78
°C for about 1.5 hours,
benzyl bromide ( 14.2 g, 83.2 mmol; commercially available from Aldrich
Chemical Co.,
Milwaukee, WI) is added dropwise over 5 minutes. The reaction mixture is
stirred at -78 °C for
about 1 hour, then at 0 °C for about 1.5 hours, and finally at ambient
temperature for about 45
minutes. Water (50 mL) is added and the reaction mixture is concentrated under
reduced
pressure to remove the tetrahydrofuran. The mixture is then poured onto water
(150 mL) and
extracted with diethyl ether (3 x 200 mL). The combined ether extracts are
washed with 1:1
water : brine (100 mL), dried over MgS04, filtered, and concentrated under
reduced pressure.
The resulting crude product is purified via preparative chromatography (silica
gel; gradient
elution 99:1 to 96:4 dichloromethane : methanol) to afford the desired (N-Me-D-
Phe)3-CsA.
Example 4
CH3 0 HO" H O ~ ~ ~H O O H 0II
IIIII 7 II
O\ \ N~N .. N~Ni \N N~N N~N~
\ N \ \
I 0 ~ ~0 ~ O ~ I
~N N_ ~N N-
~ OII H ~ O H OII H ~ O H
O~N~N~N N O~N~N~N N
H O I ~ 0 H 0 I ~ O
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WO 00/51558 PCT/LTS00/05300
(3'-Keto)'(N-Me-D-Phe)3-CsA: Dess-Martin periodinane (6.50 g, 15.33 mmol;
prepared
according to the procedure reported in Ireland et al., "An Improved Procedure
for the Preparation
of the Dess-Martin Periodinane", Journal of Organic Chemistry, Vol. 58, p.
2899 (1993)) is
combined with dichloromethane (20 mL) and stirred for 10 minutes at ambient
temperature. The
solution is chilled to 0 °C followed by the dropwise addition of (N-Me-
D-Phe)3-CsA (2.20 g, 1.70
mmol; prepared according to Example 3 herein) in dichloromethane (40 mL) over
10 minutes.
After the mixture is stirred at 0 °C for about 2.5 hours, additional
Dess-Martin periodinane (2.16
g, 5.1 mmol) is added to the reaction. The reaction mixture is stirred for an
additional 5 hours at 0
°C. A solution of sodium thiosulfate (17 g, 107.3 mmol), sodium
bicarbonate (15.45 g, 183.9
mmol), and water (300 mL) is added. The resulting mixture is stirred rapidly
for about 20
minutes at ambient temperature. The mixture is extracted with dichloromethane
(3 x 100 mL),
then the combined organic extracts are dried (MgS04), Eltered, and
concentrated under reduced
pressure. The resulting residue is puriEed via preparative chromatography
(silica gel; gradient
elution 99:1 to 96:4 dichloromethane : methanol) to afford the desired (3'-
Keto)'(N-Me-D-Phe)3-
CsA.
Use of the Present Compounds
The methods of the present invention are performed by administration of a
compound
having a structure as described herein and, preferably, a pharmaceutically-
acceptable or
cosmetically-acceptable carrier.
The compounds herein may be used for the treatment of such conditions as
treating hair
loss in mammals, including arresting and / or reversing hair loss and
promoting hair growth.
Such conditions may manifest themselves in, for example, alopecia, including
male pattern
baldness and female pattern baldness.
The compounds of the present invention are, as defined herein, non-
immunosuppressive.
Preferably, in the methods of the present invention, the compounds are
formulated into
pharmaceutical or cosmetic compositions for use in treatment or prophylaxis of
conditions such
as the foregoing. Standard pharmaceutical formulation techniques are used,
such as those
disclosed in Remin~ton's Pharmaceutical Sciences, Mack Publishing Company,
Easton, PA.
( 1990).
Typically, from about 5 mg to about 3000 mg, more preferably from about 5 mg
to
about 1000 mg, more preferably from about 10 mg to about 100 mg, of a compound
having a
structure as described herein is administered per day for systemic
administration. It is
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21
understood that these dosage ranges are by way of example only, and that daily
administration can be adjusted depending on various factors. The specific
dosage of the
compound to be administered, as well as the duration of treatment, and whether
the treatment
is topical or systemic are interdependent. The dosage and treatment regimen
will also
depend upon such factors as the specific compound used, the treatment
indication, the
efficacy of the compound, the personal attributes of the subject (such as, for
example,
weight, age, sex, and medical condition of the subject), compliance with the
treatment
regimen, and the presence and severity of any side effects of the treatment.
According to the present invention, the subject compounds are co-administered
with a
pharmaceutically-acceptable or cosmetically-acceptable carrier (herein
collectively described as
"carrier"). The term "carrier", as used herein, means one or more compatible
solid or liquid filler
diluents or encapsulating substances which are suitable for administration to
a mammal. The
term "compatible", as used herein, means that the components of the
composition are capable of
being commingled with a compound of the present invention, and with each
other, in a manner
such that there is no interaction which would substantially reduce the
efficacy of the composition
under ordinary use situations. Carriers must, of course, be of sufficiently
high purity and
sufficiently low toxicity to render them suitable for administration to the
animal, preferably
mammal (most preferably human), being treated. The carrier can itself be inert
or it can possess
pharmaceutical and / or cosmetic benefits of its own.
The compositions of this invention may be in any of a variety of forms,
suitable (for
example) for oral, rectal, topical, nasal, ocular or parenteral
administration. Of these, topical
and / or oral administration are especially preferred with topical being most
preferred.
Depending upon the particular route of administration desired, a variety of
carriers well-
known in the art may be used. These include solid or liquid fillers, diluents,
hydrotropes,
surface-active agents, and encapsulating substances. Optional pharmaceutically-
active or
cosmetically-active materials may be included which do not substantially
interfere with the
activity of the compound of the present invention. The amount of carrier
employed in
conjunction with the compound is sufficient to provide a practical quantity of
material for
administration per unit dose of the compound. Techniques and compositions for
making
dosage forms useful in the methods of this invention are described in the
following
references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds.
(1979);
Lieberman et al., Pharmaceutical Dosaee Forms: Tablets (1981); and Ansel,
Introduction to
Pharmaceutical Dosage Forms, 2"d Ed., (1976).
Some examples of substances which can serve as carriers or components thereof
are
sugars, such as lactose, glucose and sucrose; starches, such as corn starch
and potato starch;
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22
cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl
cellulose, and methyl
cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as
stearic acid and
magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil,
cottonseed oil, sesame oil,
olive oil, corn oil and oil of theobroma; polyols such as propylene glycol,
glycerine, sorbitol,
mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the
TWEENS; wetting
agents, such sodium lauryl sulfate; coloring agents; flavoring agents;
tableting agents, stabilizers;
antioxidants; preservatives; pyrogen-free water; isotonic saline; and
phosphate buffer solutions.
The choice of a carrier to be used in conjunction with the subject compound is
typically
determined by the way the compound is to be administered.
In particular, carriers for systemic administration include sugars, starches,
cellulose
and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils,
synthetic oils, polyols,
alginic acid, phosphate buffer solutions, emulsifiers, isotonic saline, and
pyrogen-free water.
Preferred carriers for parenteral administration include propylene glycol,
ethyl oleate,
pyrrolidone, ethanol, and sesame oil. Preferably, the carrier, in compositions
for parenteral
administration, comprises at least about 90% by weight of the total
composition.
Various oral dosage forms can be used, including such solid forms as tablets,
capsules, granules and bulk powders. These oral forms comprise a safe and
effective
amount, usually at least about 5%, and preferably from about 25% to about 50%,
of a
compound used in the present invention. Tablets can be compressed, tablet
triturates,
enteric-coated, sugar-coated, film-coated, or multiple-compressed, containing
suitable
binders, lubricants, diluents, disintegrating agents, coloring agents,
flavoring agents, flow-
inducing agents, and melting agents. Liquid oral dosage forms include aqueous
solutions,
emulsions, suspensions, solutions and/or suspensions reconstituted from non-
effervescent
granules, and effervescent preparations reconstituted from effervescent
granules, containing
suitable solvents, preservatives, emulsifying agents, suspending agents,
diluents, sweeteners,
melting agents, coloring agents and flavoring agents.
The carriers suitable for the preparation of unit dosage forms for oral
administration are
well-known in the art. Tablets typically comprise conventional
pharmaceutically-compatible
adjuvants as inert diluents, such as calcium carbonate, sodium carbonate,
mannitol, lactose and
cellulose; binders such as starch, gelatin and sucrose; disintegrants such as
starch, alginic acid and
croscarmelose; lubricants such as magnesium stearate, stearic acid and talc.
Glidants such as
silicon dioxide can be used to improve flow characteristics of the powder
mixture. Coloring
agents, such as the FD&C dyes, can be added for appearance. Sweeteners and
flavoring agents,
such as aspartame, saccharin, menthol, peppermint, and fruit flavors, are
useful adjuvants for
chewable tablets. Capsules (including time release and sustained release
formulations) typically
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23
comprise one or more solid diluents disclosed above. The selection of carrier
components
depends on secondary considerations like taste, cost, and shelf stability,
which are not critical for
the purposes of the subject invention, and can be readily made by a person
skilled in the art.
Orally administered compositions also include liquid solutions, emulsions,
suspensions,
powders, granules, elixirs, tinctures, syrups, and the like. The carriers
suitable for preparation of
such compositions are well known in the art. Typical components of carriers
for syrups, elixirs,
emulsions and suspensions include ethanol, glycerol, propylene glycol,
polyethylene glycol,
liquid sucrose, sorbitol and water. For a suspension, typical suspending
agents include methyl
cellulose, sodium carboxymethyl cellulose, AVICEL RC-591, tragacanth and
sodium alginate;
typical wetting agents include lecithin and polysorbate 80; and typical
preservatives include
methyl paraben and sodium benzoate. Peroral liquid compositions may also
contain one or more
components such as sweeteners, flavoring agents and colorants disclosed above.
Such compositions may also be coated by conventional methods, typically with
pH or
time-dependent coatings, such that the subject compound is released in the
gastrointestinal tract in
the vicinity of the desired topical application, or at various times to extend
the desired action.
Such dosage forms typically include, but are not limited to, one or more of
cellulose acetate
phthalate, polyvinylacetate phthalate, hydroxypropyl methyl cellulose
phthalate, ethyl cellulose,
Eudragit coatings, waxes and shellac.
Other compositions useful for attaining systemic delivery of the subject
compounds
include sublingual, buccal and nasal dosage forms. Such compositions typically
comprise one or
more of soluble filler substances such as sucrose, sorbitol and mannitol; and
binders such as
acacia, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl
methyl cellulose.
Glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents
disclosed above may
also be included.
The compounds of the present invention may also be topically administered. The
carrier
of the topical composition preferably aids penetration of the present
compounds into the skin to
reach the environment of the hair follicle. Topical compositions of the
present invention may be
in any form including, for example, solutions, oils, creams, ointments, gels,
lotions, shampoos,
leave-on and rinse-out hair conditioners, milks, cleansers, moisturizers,
sprays, skin patches, and
the like.
Topical compositions containing the active compound can be admixed with a
variety of
carrier materials well known in the art, such as, for example, water,
alcohols, aloe vera gel,
allantoin, glycerine, vitamin A and E oils, mineral oil, propylene glycol, PPG-
2 myristyl
propionate, and the like.
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Other materials suitable for use in topical carriers include, for example,
emollients,
solvents, humectants, thickeners and powders. Examples of each of these types
of materials,
which can be used singly or as mixtures of one or more materials, are as
follows:
Emollients, such as stearyl alcohol, glyceryl monoricinoleate, glyceryl
monostearate,
propane-1,2-diol, butane-1,3-diol, mink oil, cetyl alcohol, iso-propyl
isostearate, stearic acid, iso-
butyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl
laurate, decyl oleate,
octadecan-2-ol, isocetyl alcohol, cetyl palmitate, dimethylpolysiloxane, di-n-
butyl sebacate, iso-
propyl myristate, iso-propyl palmitate, iso-propyl stearate, butyl stearate,
polythylene glycol,
triethylene glycol, lanolin, sesame oil, coconut oil, arachis oil, castor oil,
acetylated lanolin
alcohols, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic
acid, isopropyl linoleate,
lauryl lactate, myristyl lactate, decyl oleate, and myristyl myristate;
propellants, such as propane,
butane, iso-butane, dimethyl ether, carbon dioxide, and nitrous oxide;
solvents, such as ethyl
alcohol, methylene chloride, iso-propanol, castor oil, ethylene glycol
monoethyl ether, diethylene
glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl
sulphoxide, dimethyl
formamide, tetrahydrofuran; humectants, such as glycerin, sorbitol, sodium 2-
pyrrolidone-5-
carboxylate, soluble collagen, dibutyl phthalate, and gelatin; and powders,
such as chalk, talc,
fullers earth, kaolin, starch, gums, colloidal silicon dioxide, sodium
polyacrylate, tetra alkyl
ammonium smectites, trialkyl aryl ammonium smectites, chemically modified
magnesium
aluminium silicate, organically modified montmorillonite clay, hydrated
aluminium silicate,
fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose, and
ethylene glycol
monostearate.
The compounds used in the present invention may also be administered in the
form of
liposome delivery systems, such as small unilamellar vesicles, large
unilamellar vesicles, and
multilamellar vesicles. Liposomes can be formed from a variety of
phospholipids, such as
cholesterol, stearylamine or phosphatidylcholines. A preferred formulation for
topical delivery of
the present compounds utilizes liposomes such as described in Dowton et al.,
"Influence of
Liposomal Composition on Topical Delivery of Encapsulated Cyclosporin A: I. An
in vitro
Study Using Hairless Mouse Skin", S.T.P. Pharma Sciences, Vol. 3, pp. 404 -
407 (1993);
Wallach and Philippot, "New Type of Lipid Vesicle: Novasome~", Liposome
Technolo~y, Vol.
l, pp. 141 - 156 (1993); Wallach, U.S. Patent No. 4,911,928, assigned to Micro-
Pak, Inc., issued
March 27, 1990; and Weiner et al., U.S. Patent No. 5,834,014, assigned to The
University of
Michigan and Micro-Pak, Inc., issued November 10, 1998 (with respect to Weiner
et al., with a
compound as described herein administered in lieu of, or in addition to,
minoxidil).
The compounds of the present invention may also be administered by
iontophoresis. See3
e.g,,, Internet site www.unipr.it/area/dipfarm/erasmus/erasml4.html; Banua et
al., "Hydrogel-
CA 02366312 2001-09-04
WO 00/51558 PCT/US00/05300
based Iontotherapeutic Delivery Devices for Transdermal Delivery of
Peptide/Protein Drugs",
Pharm. Res., Vol. 10 (5), pp. 697-702 (1993); F~, "Theoretical Model of
Iontophoresis
Utilized in Transdermal Drug Delivery", Pharmaceutical Acta Helvetiae, Vol 70,
pp. 279-287
(1995); Gangarosa et al., "Modern Iontophoresis for Local Drug Delivery", Int.
J. Pharm, Vol.
123, pp. 159-171 (1995); Green et al., "Iontophoretic Delivery of a Series of
Tripeptides Across
the Skin in vitro", Pharm. Res., Vol 8, pp. 1121-1127 (1991); Jadoul et al.,
"Quantification and
Localization of Fentanyl and TRH Delivered by Iontophoresis in the Skin", Int.
J. Pharm., Vol.
120, pp. 221-8 (1995); O'Brien et al., "An Updated Review of its Antiviral
Activity,
Pharmacokinetic Properties and Therapeutic Efficacy", Drugs, Vol. 37, pp. 233-
309 (1989); P
et al., "Acyclovir Biovailability in Human Skin", J. Invest. Dermatol., Vol.
98 (6), pp. 856-63
(1992); Santi et al., "Drug Reservoir Composition and Transport of Salmon
Calcitonin in
Transdermal Iontophoresis", Pharm. Res., Vol 14 (1), pp. 63-66 (1997); Santi
et al., "Reverse
Iontophoresis - Parameters Determining Electroosmotic Flow: I. pH and Ionic
Strength", J.
Control. Release, Vol. 38, pp. 159-165 (1996); Santi et al., "Reverse
Iontophoresis - Parameters
Determining Electroosmotic Flow: II. Electrode Chamber Formulation", J.
Control. Release, Vol.
42, pp. 29-36 (1996); Rao et al., "Reverse Iontophoresis: Noninvasive Glucose
Monitoring in vivo
in Humans", Pharm. Res., Vol. 12 (12), pp. 1869-1873 (1995); Thvsman et al.,
"Human
Calcitonin Delivery in Rats by Iontophoresis", J. Pharm. Pharmacol., Vol. 46,
pp. 725-730
(1994); and Volpato et al., "Iontophoresis Enhances the Transport of Acyclovir
through Nude
Mouse Skin by Electrorepulsion and Electroosmosis", Pharm. Res., Vol. 12 (11),
pp. 1623-1627
(1995).
The compositions of the present invention may also optionally comprise an
activity
enhancer. The activity enhancer can be chosen from a wide variety of molecules
which can
function in different ways to enhance hair growth effects of a compound of the
present invention.
Particular classes of activity enhancers include other hair growth stimulants
and penetration
enhancers.
Additional hair growth stimulants can be chosen from a wide variety of
molecules which
can function in different ways to enhance the hair growth effects of a
compound of the present
invention. These optional other hair growth stimulants, when present, are
typically employed in
the compositions herein at a level ranging from about 0.01% to about 15%,
preferably from about
0.1% to about 10%, most preferably from about 0.5% to about 5% by weight of
the composition.
Vasodilators such as potassium channel agonists including, for example,
minoxidil and
minoxidil derivatives such as aminexil and such as those described in U.S.
Patent 3,382,247, U.S.
Patent 5,756,092, issued May 26, 1998, U.S. Patent 5,772,990, issued June 30,
1998, U.S. Patent
5,760,043, issued June 2, 1998, U.S. Patent 328,914, issued July 12, 1994,
U.S. Patent
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WO 00/51558 PCT/US00/05300
26
5,466,694, issued November 14, 1995, 5,438,058, issued August l, 1995, and
U.S. Patent
4,973,474, issued November 27, 1990, (all of which are herein incorporated by
reference), and
cromakalin and diazoxide can be used as an additional hair growth stimulant in
the compositions
herein.
One suitable class of additional hair growth stimulant for use herein are
antiandrogens.
Examples of suitable antiandrogens may include, but are not limited 5-a-
reductase inhibitors
such as flnasteride and those described in U.S. Patent 5,516,779, issued May
14, 1996 (herein
incorporated by reference) and in Nane et al., Cancer Research 58, "Effects of
Some Novel
Inhibitors of C17,20-Lyase and Sa-Reductase in vitro and in vivo and Their
Potential Role in the
Treatment of Prostate Cancer," as well as cyproterone acetate, azelaic acid
and its derivatives and
those compounds described in U.S. Patent 5,480,913, issued January 2, 1996,
flutamide, and
those described in U.S. Patents 5,411,981, issued May 2, 1995, U.S. Patent
5,565,467, issued
October 15, 1996 and U.S. Patent 4,910,226, issued March 20, 1990, all of
which are herein
incorporated by reference.
Another suitable class of optional hair growth stimulants are FK506 analogs
such as those
described in McIver et al., U.S. Patent Application Serial No. 09/400,681,
filed September 21,
1999; McIver et al., U.S. Patent Application Serial No. 09/400,682, filed
September 21, 1999;
McIver et al., U.S. Patent Application Serial No. 09/400,679, filed September
21, 1999; Tiesman
et al., U.S. Patent Application Serial No. 09/400,021, filed September 21,
1999; Fulmer et al.,
U.S. Patent Application Serial No. 09/400,425, filed September 21, 1999; U.S.
Provisional
Patent Application No. 60/147,279, Deeenhardt et al., filed August 5, 1999;
U.S. Provisional
Patent Application No. 60/147,313, De~enhardt et al., filed August 5, 1999;
U.S. Provisional
Patent Application No. 60/147,280, Deeenhardt et al., filed August 5, 1999;
U.S. Provisional
Patent Application No. 60/147,278, Degenhardt et al., filed August 5, 1999;
and U.S. Provisional
Patent Application No. 60/147,276, Eickhoff et al., filed August 5, 1999; all
of which are herein
incorporated by reference.
Another suitable class of optional hair growth stimulants are antimicrobials
such as
selenium sulfide, ketoconazole, triclocarbon, triclosan, zinc pyrithione,
itraconazole, asiatic acid,
hinokitiol, mipirocin and those described in EPA 0,680,745 (herein
incorporated by reference),
clinacycin hydrochloride, benzoyl peroxide, benzyl peroxide and minocyclin.
Anti-inflammatories can also be incorporated into the compositions herein as
an optional
hair growth stimulant. Examples of suitable anti-inflammatories may include
glucocorticoids
such as hydrocortisone, mometasone furoate and prednisolone, nonsteroidal anti-
inflammatories
including cyclooxygenase or lipoxygenase inhibitors such as those described in
U.S. Patent
CA 02366312 2001-09-04
WO 00/51558 PCT/L1S00/05300
27
5,756,092, and benzydamine, salicylic acid, and those compounds described in
EPA 0,770,399,
published May 2, 1997, WO 94/06434, published March 31, 1994, and FR
2,268,523, published
November 21, 1975, all of which are herein incorporated by reference.
Another suitable class of optional hair growth stimulants are thyroid hormones
and
derivatives and analogs thereof. Examples of suitable thyroid hormones for use
herein may
include triiodothyrionine. Examples of thyroid hormone analogs which may be
suitable for use
herein include those described in U.S. Provisional Patent Application No.
60/136,996, Zhang et
al., filed June 1, 1999, U.S. Provisional Patent Application No. 60/137,024,
Zhang et al., filed
June l, 1999, U.S. Provisional Patent Application No. 60/137,022, Zhang et
al., filed June l,
1999, U.S. Provisional Patent Application No. 60/137,023, Zhang et al., filed
June 1, 1999, U.S.
Provisional Patent Application No. 60/137,052, Youngquist et al., filed June
1, 1999, U.S.
Provisional Patent Application No. 60/137,063, Youngquist et al., filed June
1, 1999, and U.S.
Provisional Patent Application No. 60/136,958, Youngquist et al., filed June
1, 1999.
Prostaglandin agonists or antagonists can also be used as optional hair growth
stimulants
in the compositions herein. Examples of suitable prostaglandins agonists or
antagonists include
latanoprost and those described in WO 98/33497, Johnstone, published August 6,
1998, WO
95/11003, Stjernschantz, published April 27, 1995, JP 97-100091, Ueno and JP
96-134242,
Nakamura.
Another class of optional hair growth stimulants for use herein are retinoids.
Suitable
retinoids may include isotretinoin, acitretin, and tazarotene.
Another class of optional hair growth stimulants for use herein are
triterpenes such as, for
example, those disclosed in Bradbury et al., U.S. Patent Application Serial
No. 09/353,408,
"Method for Regulating Hair Growth", filed July 15, 1999 and Bradbury et al.,
U.S. Patent
Application Serial No. 09/353,409, "Compositions Which Contain Triterpenes for
Regulating
Hair Growth", filed July 15, 1999, each incorporated by reference in their
entirety.
Other classes of optional hair growth stimulants for use herein include
flavinoids,
ascomycin derivatives and analogs, histamine antagonists such as
diphenhydramine
hydrochloride, other triterpenes such as oleanolic acid and ursolic acid and
those described in
U.S. Patent 5,529,769, JP 10017431, WO 95/35103, U.S. Patent 5,468,888, JP
09067253, WO
92/09262, JP 62093215, U.S. Patent 5,631,282, U.S. Patent 5,679,705, JP
08193094, saponins
such as those described in EP 0,558,509 to Bonte et al., published September
8, 1993 and WO
97/01346 to Bonte et al, published January 16, 1997 (both of which are herein
incorporated by
reference in their entirety), proteoglycanase or glycosaminoglycanase
inhibitors such as those
described in U.S. Patents 5,015,470, issued May 14, 1991, U.S. Patent
5,300,284, issued April 5,
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28
1994 and U.S. Patent 5,185,325, issued February 9, 1993 (all of which are
herein incorporated in
their entirety by reference) estrogen agonists and antagonists, pseudoterins,
cytokine and growth
factor promotors, analogs or inhibitors such as interleukin 1 inhibitors,
interleukin-6 inhibitors,
interleukin-10 promotors, and tumor necrosis factor inhibitors, vitamins such
as vitamin D
analogs and parathyroid hormone antagonists, Vitamin B 12 analogs and
panthenol, interfuron
agonists and antagonists, hydroxyacids such as those described in U.S. Patent
5,550,158,
benzophenones, and hydantoin anticonvulsants such as phenytoin.
Other additional hair growth stimulants are described in detail in, for
example, JP 09-
157,139 to Tsuji et al., published June 17, 1997; EP 0277455 Al to Mirabeau,
published August
10, 1988; WO 97/05887 to Cabo Soler et al., published February 20, 1997; WO
92/16186 to
Bonte et al., published March 13, 1992; JP 62-93215 to Okazaki et al.,
published April 28, 1987;
U.S. Patent 4,987,150 to Kurono et al., issued January 22, 1991; JP 290811 to
Ohba et al.,
published October 15, 1992; JP OS-286,835 to Tanaka et al., published November
2, 1993, FR
2,723,313 to Greff, published August 2, 1994, U. S. Patent 5,015,470 to
Gibson, issued May 14,
1991, U.S. Patent 5,559,092, issued September 24, 1996, U.S. Patent 5,536,751,
issued July 16,
1996, U.S. Patent 5,714,515, issued February 3, 1998, EPA 0,319,991, published
June 14, 1989,
EPA 0,357,630, published October 6, 1988, EPA 0,573,253, published December 8,
1993, JP 61-
260010, published November 18, 1986, U.S. Patent 5,772,990, issued June 30,
1998, U.S. Patent
5,053, 410, issued October 1, 1991, and U.S. Patent 4,761,401, issued August
2, 1988, all of
which are herein incorporated by reference.
Non-limiting examples of penetration enhancers which may be used in the
compositions
herein include, for example, 2-methyl propan-2-ol, propan-2-ol, ethyl-2-
hydroxypropanoate,
hexan-2,5-diol, POE(2) ethyl ether, di(2-hydroxypropyl) ether, pentan-2,4-
diol, acetone, POE(2)
methyl ether, 2-hydroxypropionic acid, 2-hydroxyoctanoic acid, propan-1-ol,
1,4-dioxane,
tetrahydrofuran, butan-1,4-diol, propylene glycol dipelargonate,
polyoxypropylene 15 stearyl
ether, octyl alcohol, POE ester of oleyl alcohol, oleyl alcohol, lauryl
alcohol, dioctyl adipate,
dicapryl adipate, di-isopropyl adipate, di-isopropyl sebacate, dibutyl
sebacate, diethyl sebacate,
dimethyl sebacate, dioctyl sebacate, dibutyl suberate, dioctyl azelate,
dibenzyl sebacate, dibutyl
phthalate, dibutyl azelate, ethyl myristate, dimethyl azelate, butyl
myristate, dibutyl succinate,
didecyl phthalate, decyl oleate, ethyl caproate, ethyl salicylate, iso-propyl
palmitate, ethyl laurate,
2-ethyl-hexyl pelargonate, iso-propyl isostearate, butyl laurate, benzyl
benzoate, butyl benzoate,
hexyl laurate, ethyl caprate, ethyl caprylate, butyl stearate, benzyl
salicylate, 2-hydroxypropanoic
acid, 2-hyroxyoctanoic acid, dimethyl sulphoxide, N,N-dimethyl acetamide, N,N-
dimethyl
formamide, 2-pyrrolidone, 1-methyl-2-pyrrolidone, 5-methyl-2-pyrrolidone, 1,5-
dimethyl-2-
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29
pyrrolidone, 1-ethyl-2-pyrrolidone, phosphine oxides, sugar esters,
tetrahydrofurfural alcohol,
urea, diethyl-m-toluamide, and, 1-dodecylazacyloheptan-2-one.
In all of the foregoing, of course, the compounds of the invention can be
administered
alone or as mixtures, and the compositions may further include additional
drugs or excipients as
appropriate for the indication.
Examples of Composition Administration
The following examples do not limit the invention, but provide guidance to the
skilled
artisan to perform the methods of the present invention. In each example, a
compound other than
the one mentioned may be substituted by another having a structure as
described herein.
Example A
A composition for topical administration is made, comprising:
Component Amount
Compound of Example 2 5
Ethanol 57
Propylene Glycol 19
Dimethyl Isosorbide 19
A human male subject suffering from male pattern baldness is treated by a
method of this
invention. Specifically, for 6 weeks, the above composition is daily
administered topically to the
subject.
Example B
A composition for topical administration is made according to the method of
Dowton et
al., "Influence of Liposomal Composition on Topical Delivery of Encapsulated
Cyclosporin A: I.
An in vitro Study Using Hairless Mouse Skin", S.T.P. Pharma Sciences, Vol. 3,
pp. 404 - 407
(1993), using the compound of Example 1 in lieu of cyclosporin A and using the
Novasome 1 for
the non-ionic liposomal formulation.
A human male subject suffering from male pattern baldness is treated each day
with the
above composition. Specifically, for 6 weeks, the above composition is
administered topically to
the subject.
Example C
Four different shampoo compositions are made as follows:
Ex. C-1 Ex. C-2 Ex. C-3 Ex. C-4
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WO 00/51558 PCT/LTS00/05300
Com onent
Ammonium Lauryl 11.5 % 11.5 % 9.5 % 7.5
Sulfate
Ammonium Laureth 4 % 3 % 2 % 2
Sulfate
Cocamide MEA 2 % 2 % 2 % 2
Eth lene Glycol 2 % 2 % 2 % 2
Distearate
Cet 1 Alcohol 2 % 2 % 2 % 2
Stearyl Alcohol 1.2 % 1.2 % 1.2 % 1.2
Glycerin 1 % 1 % 1 % 1
Pol uaternium 10 0.5 % 0.25 % - -
Pol uaternium 24 - - 0.5 % 0.25
Sodium Chloride 0.1 % 0.1 % 0.1 % 0.1
Sucrose Polyesters 3 % 3 % - -
of
Cottonate Fatt Acid
Sucrose Polyesters 2 % 3 % -
of
Behenate Fatty Acid
Polydimethyl Siloxane- - 3 % 2
Cocamino ro 1 Betaine- 1 % 3 % 3
Lauryl Dimethyl 1.5 % 1.5 % 1.5 % 1.5
Amine
Oxide
Decyl Pol lucose - - 1 % 1
DMDM Hydantoin 0.15 % 0.15 % 0.15 % 0.1 S
Com ound of Exam 5 % - - -
le 1
Com ound of Exam - 2 % - -
le 2
Com ound of Exam - - 3 % -
le 3
Com ound of Exam - - - 6 %
le 4
Minoxidil - - - 2 %
Phenox ethanol 0.5 % 0.5 % 0.5 % 0.5
Fra ance 0.5% 0.5% 0.5% 0.5%
Water q.s. q.s. q.s. q.s.
A human subject suffering from male pattern baldness is treated by a method of
this
invention. Specifically, for 12 weeks, the above shampoo is used daily by the
subject.
