Note: Descriptions are shown in the official language in which they were submitted.
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PROSTAGLANDIN ANALOGUES FOR PROMOTION OF HAIR GROWTH
FIELD OF THE INVENTION
s
This invention relates to the use of certain prostaglandin analogues to
promote the growth, thickness, and pigmentation of hair in mammals, including
man. The invention also relates to topical compositions for such use. More
specifically, the invention relates to the use of certain FP prostaglandin
to analogues for the promotion of hair growth.
BACKGROUND OF THE INVENTION
Although hair loss has plagued mankind for centuries, its cause is still not
is completely understood and no adequate cure has yet been found. Two of the
more recent commercial products for alopecia or male pattern baldness are
minoxidil (Rogaine~) and finasteride (Propecia~). The active compounds of both
these products were initially developed for different therapies: minoxidil for
hypertension, and finasteride for benign prostatic hypertrophy. See U.S.
Patent
2o Nos. 4,139,619 and 4,968,812 directed to the use of minoxidil, and U.S.
Patent
No. 5,981,543 for finasteride. To the extent U.S. Patent Nos. 4,139,619 and
4,968,812 disclose topical formulations for hair growth promotion, those
disclosures are by this reference incorporated herein.
2s More recently, it has been discovered that prostaglandin analogues,
originally developed as therapy for glaucoma, may also promote hair growth.
See U.S. Patent Nos. 6,262,105 B1 and U.S. Patent Application Serial No.
09/774,555 (U.S. Patent Application Publication No. US 2002/0037914A1 ), the
entire contents of both of which are by this reference incorporated herein.
The claimed invention in U.S. Patent No. 6,262,105 (the '105 patent) was
based in part on the observation that in some instances glaucoma patients
receiving the prostaglandin analogue, latanoprost, experienced increased
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eyelash growth. The patent broadly discloses the use of prostaglandins and
prostaglandin analogues for enhancing hair growth, generically disclosing
countless prostaglandin derivatives and analogues, as well as over a hundred
specific compounds. Characterized as preferred among the prostaglandin
s analogues disclosed in the '105 patent were those of the A, F, and E types;
and
particularly preferred was 13,14-dihydro-15-dehydro-17-phenyl-18,19,20-trinor-
PGF2a and its carboxylic acid esters. The compounds useful in the methods and
compositions of the present invention, however, are neither specifically
disclosed nor suggested in the '105 patent.
to
U.S. Patent Application Serial No. 09/774,555 (the '555 application)
discloses 5,6-13,14-tetrahydro PGF2a analogues (also characterized as 13,14-
dihydro PGF~a analogues) for treating hair loss. Once again, the compounds
useful in the methods and compositions of the present invention are neither
Is disclosed or suggested in the '555 application, nor are they encompassed
within
the scope of that application's broadest claims.
Cloprostenol and fluprostenol, both known compounds, are synthetic
analogues of PGF2a, a naturally-occurring F-series prostaglandin (PG).
2o Structures for PGF2a(I), cloprostenol (II), and fluprostenol (III), are
shown below:
HO
7 4 2 alpha chain
,s s,.vv 1
1o s' s 3 COOH
11 ~ 16 18
12 15 20
H~ 1g _ 17 1s omega chain
OH (I)
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HO
C02H
HO = O
OH
CI (II)
HO
,,~'~~ C02H
HO = O
OH
cF3 (III)
s The chemical name for cloprostenol is 16-(3-chlorophenoxy)-17,18,19,20-
tetranor PGF2a. Monograph No. 2461 (page 407) of The Merck Index, 12th
Edition (1996) is incorporated herein by reference to the extent that it
describes
the preparation and known pharmacological profiles of cloprostenol.
Fluprostenol has the chemical name 16-(3-trifluoromethylphenoxy)-17,18,19,20-
to tetranor PGF2a. Monograph No. 4231 (page 711) of The Merck Index, 12th
Edition (1996) is incorporated herein by reference to the extent that it
describes
the preparation and known pharmacological profiles of fluprostenol.
Cloprostenol and fluprostenol are 16-aryloxy PGs differing from the natural
product PGF2a by the substitution of a substituted phenoxy moiety for the last
4
is carbons of the lower (omega) chain of the compound.
The use of salts and esters of cloprostenol and fluprostenol and various
analogues thereof for the treatment of glaucoma and ocular hypertension are
described in U.S. Patent Nos. 5,510,383 and 5,889,052, the entire contents of
2o which are by this reference incorporated herein.
It has now been discovered that particular PGF2a analogues are
surprisingly effective agents for the promotion of hair growth when topically
applied to mammals including man.
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SUMMARY OF THE INVENTION
The present invention relates to methods and compositions for the
s promotion of hair growth comprising certain PGF2a analogues and their
pharmaceutically acceptable salts, esters, and amides. Most preferred in such
compositions and methods are potent and selective FP agonists selected from
the group consisting of 9-deoxy, 11-oxa, 13-oxa, and 15-fluoro analogues of
PGFZa, and the analogues described in U.S. Patent Nos. 5,510,383 and
l0 5,889,052, previously incorporated by reference. Encompassed within the
scope of the invention are various pharmaceutically acceptable carriers
suitable
for topical administration.
1s DETAILED DESCRIPTION OF THE INVENTION
The compounds useful in the present invention are described in U.S.
Patent Nos. 5,510,383, 5,889,052, 5,698,733, 6,025,392, and 6,232,344; and
U.S. Patent Application Serial Nos. 09/284,432 and 10/100,399; the entire
2o contents of each of the foregoing being incorporated herein by this
reference.
Among those compounds are those selected from the group consisting of the
compounds of formula:
R90s, ,~~'X~R1
Rs R2
O
R~~O Y
(IV)
wherein:
R~ = OR, where R = H; C~-C~2 straight-chain or branched alkyl; C~-C~2
straight-chain or branched acyl; C3-C$ cycloalkyl; or a cationic salt
moiety; or R = NR4R5, where R4 and R5 are the same or different
_4_
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and are H; C~-C~2 straight-chain or branched alkyl; C~-C~2 straight-
chain or branched acyl; or C3-C$ cycloalkyl;
R2, R3 = H, or C~-C5 straight-chain or branched alkyl; or R2 and R3 taken
together may represent O;
s X = O, S, or CH2;
-- represents any combination of a single bond, or a cis or traps double
bond for the alpha (upper) chain; and a single bond or traps double
bond for the omega (lower) chain;
R9 = H, C~-Coo straight-chain or branched alkyl, or C~-Coo straight-chain or
to branched acyl;
R~~ = H, C~-Coo straight-chain or branched alkyl, or C~-Coo straight-chain
or branched acyl;
Y = O; or H and OR~5 in either configuration wherein R~5 = H, C~-Coo
straight-chain or branched alkyl, or C~-Coo straight-chain or
Is branched acyl; and
Z = CI or CF3;
and compounds 1-80 identified in Table 1 below.
-s-
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TABLE 1: FP Prostaglandin Analogues
IMOLSTRUCTURE. FP binding IFP functional
jlCs°(nM) ;ECS°nM (°f°)
._._..~. . .._:____.___- ...~
I Hd~ I ~ I
1 ~ "~ - 9619.6(87:6)
..",
I
I Hs' t
2 ' ~ 310 76.6(60) .
°
H°
3 I
OH
1 I
I \
I i I
j HO O I
4 ~H
..._..____ o ~
.."
; H
0
..., off
~~ I
j Ho~o~a
6 off 10 2.6(100)
° l
°-~ I.
j ..,~ CFi~ i
Ho~a ~ ~
7 i ~ a
~°H I i
~( ~ ~ ~ I
i ~u I ° a
8 . H~ ~ 47 6.6(84)
_.._..__ .""\~°~°,.,3 .
. o '
y ~
9
_ I . :.~° ~ i
4
,,
i ~H °"
i ,,
1
11 ~ oI, °" 1, 900 108(60)
-6-
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FP Prostaglandin Analogues
~MOLSTRUCTURE FP binding FP functional
ICso (nM) ECSO nM (%)
~c~ ~ a
.... ~~ j _.
12 f ° i
OH
OH '...
O
O ~ ~ a
13 ~H
off
HO
a
14 ~ ~, ~H 7,300194(67)
~OH
HO_ .~ ' ~r\
f a
o . o
15 off 890 58(88)
__
~a i
16 OH
i OH i
1 ~o ~ i a
17 ~H F 26 9(64)
1 8 1 off
~ ~cH' 1
I o
,.
i
19 ~ ~ ~H
~ I.
y i
/~ ~ r
20 ' ~ «, 9,70010,000(67)
i~ _
21 ~ o" 78,000 > 10,000
~~ I
. : ~--"'' I
H
sv
22 I 'o~v y~, ' F F
_ 7_
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FP Prostaglandin Analogues
I,MOLSTRUCTURE FP binding FP functional
150 (nM) ECSO nM t°l°)
._.
° CHI
j HO
i \
° ~ ° I ~ F _ ~ _. .
23 °H
___._ ° i
o~ s I
! HO
24 H~° F F 230'72.5(85)
H°
I
I ~o
25 I HO F F F
f
off
j
Is F
26 ~ o ~H ° F F 1,800187(74)
____ o
~oN
H '\,J/~~Jo
I / F ; i
27 j o ~ o F F 15,000~889(43>
._. ~ °" - ' I
... , . I
' ° '
28 aH 1,900 80(100)
! : ',. ~ '
29 i °
~~
i «~
...
30 . _ o
°--~'~ ~ i
~' .... ° ~ i
(~ I \
31 ; o" °
__.. ~ ~ .
off
I .... o
I~
32 off 573 48.6(100)
off
o
....
w
l~
33 ' aH 640 293(67)
_8_
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FP Prostaglandin Analogues
MOLSTRUCTURE FP binding FP functional
ICso(nM) jECSOnM(%)
° ~ L
I Ho
34 ; ° ~, _.
__ ; °
HO , ' ,--J O' \ I
i
35 '<~° \ f ' I
O
Ho .., OH
i F
36 ~ -° ~ a~ ° 4,600 1240(75)
_- I °
HO "'~OH
F
37 ( .-~ 1 400 437(78)
I HO
°
y, ~,~, ~ r
38 ' °H j 36,000~> 10,000
___-
Ho 0
:.
j O ~ ~a
39 ' ~H ' 5,100 509(100)
~,
a
i :.~ ~ / a i
40 ~ ~H ° 430 2(82)
HO
,"
I a
; ° ~ o
41 I off
I ~° a5
Ha o YcH,
i
; ~o ~ f a
42 ' off .
._
- i -
I HO
v
1 \ ~ ,~ a
off
43 I
44 ' ~H
-9-
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FP Prostaglandin Analogues
MOLSTRUCTURE FP binding FP functional
' ICSO (nM) ECSO nM (%)
OH
i
O
j HO
~ %
_.
45 I HO F 52 9.5(79)
. , ~
~° I
46 . off
°
°
~H
47 I - ° ~ /
0
H° ° a~
I
a
48 ° ~ °
i ,~ ~°Y
~~ .
° o
49 ai
I
=.
i
. s ~ / . i
50 °H ' 5,100 252(59)
~oH
~~1
°
I / F I
51 1 ° OH ° F 9, 900 781 (61 )
'~ ~ i
52 ~H ° 330 57(83)
~OH
w
53 ~ ° OH ° F F 350 96(92) .
OH
;. .
~~ ~
s ~ / a
54 ° a~ ° 818 249(71)
; HO ~O~ ~ _..
oI~ F
55 OH F F
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FP Prostaglandin Analogues
MOLSTRUCTURE FP binding FP functional
I
IC5° (nM) ECso nM (%)
HO
a _ _.
56 aH °
°
HO ,~OH
'~~ ° ~' 1
57 ~ ~H F 267 3.8(104)
I '',~ ~
~~~s
_ ° ~' , I
OH F I
_ .. .._._.._ 58 ~ I
~a I
~i
~O \ 1
59 I ~ ,F a
/~/\/ I
HO .,~,~OH I
i
1 0 \ 1
60 ~ OH F 1120 96(98)
°Y~ I
I HO c
oIs
61 I
:.
I
62 ' off F a ~ 182I 81 (7.6)
//°
HO .: ~OH I
I j, i~
I ~o \ 1 a
63 I ~H F 238 2.2(84)
o~a~,
0
~o~o I ~ a
64
i . a~
° i i
65 I ~°~°' / a
H~ 540 57.8(91)
as
°
66 ~ ~° 4340 503(56)
-11-
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FP Prostaglandin Analogues
IMOLSTRUCTURE FP binding ~FP functional
fCso (nM) ECso nM t%)
~o i ' _ _
67
i ~'' i t
I ',~~~ I
I
i
68 i ~°~°
.~ -
.., o
l ~o~o~
69 ~° ~ 277 56(57)
_.. _
:~~ ~ ' s
70 I ~~~~ , / 1070 166(57)
--.
71 ~ O \ ~ F FF
110 F
-.-,-
i
...,~o
i
/ ~ F
\
F
NO F
72 ~ F
I ' o
! ~ 1
F
73 v
~OH
~O
j H JQ
74 i H690 8.8167)
a
I . ' ~,~O ~ ~ F
75 ~yy ~v ~~F F 160 11.1 (83)
_"
y F
76 ~ HO~ F 4,200 131(44.5)
o «~
. /' F
77 F F
HO OH
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Preferred among the compounds of formula IV for use in the present
invention are those having the structure of formula V:
s
wherein:
R~ = OR or NR3R4, where
H Ose
CORD
O
HO HO
2
v R
to R = H, a cationic salt moiety, a pharmaceutically acceptable amine
moiety, or
C~-C~2 alkyl, cycloalkyl, or aryl;
is R3 and R4 = same or different = H, alkyl, cycloalkyl, aryl, or ORS, with
the
proviso that R3 and R4 cannot both = OR5, where
R5 = H, alkyl, acyl, cycloalkyl, or aryl; and
ao R2 = CI or CF3.
Particularly preferred among such compounds are esters of
cloprostenol and fluprostenol, which correspond to formula V wherein:
2s R~ = OR, where
i
R= C~-C~2 alkyl; and
R2 = CI or CF3.
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Most preferred are the isopropyl esters of cloprostenol and fluprostenol in
enantiomercially pure form, which correspond to compounds having the absolute
stereochemical structure of formula V wherein:
s R' = OR, where R is isopropyl; and
R2 = CI or CF3.
Also preferred are the 15-keto analogues of cloprostenol and fluprostenol.
to The generic name for the isopropyl ester of the preferred enantiomer of
fluprostenol is travoprost. Travoprost, as well as fluprostenol and
cloprostenol,
and their 15-keto analogues are commercially available from Cayman Chemical
Company, Ann Arbor, Michigan.
is As used herein:
The term "acyl" represents a group that is linked by a carbon atom that
has a double bond to an oxygen atom and single bond to another carbon atom.
2o The term "alkenyl" includes straight or branched chain hydrocarbon
groups having 1 to 15 carbon atoms with at least one carbon-carbon double
bond. The chain hydrogens may be substituted with other groups, such as
halogen. Preferred straight or branched alkenyl groups include, allyl, 1-
butenyl,
1-methyl-2-propenyl and 4-pentenyl.
The term "alkoxy" represents an alkyl group attached through an oxygen
linkage.
The term "alkyl" includes straight or branched chain, saturated or
3o unsaturated aliphatic hydrocarbon groups having 1 to 15 carbon atoms. The
alkyl groups may be substituted with other groups, such as halogen, hydroxyl
or
alkoxy. Preferred straight or branched alkyl groups include lower alkyl groups
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such as methyl, ethyl, propyl, isopropyl, butyl and t butyl. Also included,
however, are alkenyl and alkynyl groups.
The term "alkynyl" includes straight or branched chain hydrocarbon
s groups having 1 to 15 carbon atoms with at least one carbon-carbon triple
bond.
The chain hydrogens may be substituted with other groups, such as halogen.
Preferred straight or branched alkynyl groups include, 2-propynyl, 2-butynyl,
3-
butynyl, 1-methyl-2-propynyl and 2-pentynyl.
io The term "aryl" refers to carbon-based rings which are aromatic. The
rings may be isolated, such as phenyl, or fused, such as naphthyl. The ring
hydrogens may be substituted with other groups, such as lower alkyl, or
halogen. As used herein, "aryl" includes heteroaryl groups.
is The term "cationic salt moiety" includes alkali and alkaline earth metal
salts as well as ammonium salts.
The term "cycloalkyl" includes straight or branched chain, saturated or
unsaturated aliphatic hydrocarbon groups which connect to form one or more
2o rings, which can be fused or isolated. The rings may be substituted with
other
groups, such as halogen, hydroxyl or lower alkyl. Preferred cycloalkyl groups
include cyclopropyl, cyclobutyl, cylopentyl and cyclohexyl.
The term "heteroaryl" refers to aromatic hydrocarbon rings which contain
2s at least one heteroatom such as O, S, or N in the ring. Heteroaryl rings
may be
isolated, with 5 to 6 ring atoms, or fused, with 8 to 10 atoms. The heteroaryl
rings) hydrogens or heteroatoms with open valency may be substituted with
other groups, such as lower alkyl or halogen. Examples of heteroaryl groups
include imidazole, pyridine, indole, quinoline, furan, thiophene, pyrrole,
3o tetrahydroquinoline, dihydrobenzofuran, and dihydrobenzindole.
-IS-
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The term "lower alkyl" represents alkyl groups containing one to six
carbons (C~-Cs).
Preferred among the compounds of Table 1 are those which are potent
s and selective FP receptor agonists; specifically, those for which the free
acid
form exhibits an FP binding IC5o (based upon a travoprost 0.004% standard) of
less than 1000 nM and/or greater than 50% agonistic activity (as reflected in
Table 1 - travoprost acid representing the standard full agonist (100%); and
most preferably those with IC5os less than 500 and/or agonistic activity
greater
Io than 75%. Most preferred among the compounds of Table 1 are compounds 9,
18, 25, 42, 71 and 77, all of which are isopropyl esters.
The preferred isopropyl esters isopropyl esters will preferably be in
enantiomerically pure form, which corresponds to compounds having the
is absolute stereochemical structure of PGF2a (I).
Representative compounds of formula V are presented in Table 2, which
includes corresponding FP receptor binding and agonistic activity data.
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Table 2.
Compound Structure K;, nM ECso, nM (%
res onse
81 Ho< ' ~,~/~COZH 91 1.4 (100%)
0
HO HO
CI
82 H°~ w~~cozH 35 3.8 (96%)
0
HO HO
~ ( CFa
HO' '~\~COZH 50 4 (100%)
0
HO HO
CI
84 HO- :~~'~~~COzH 130 210 (76%)
o
HO HO
CI
85 Ho° ' \~COzH 220 23 (100%)
Y o
HO HO
\ I CI
HO' '~~\~--~COzH 93 4.92 (100°10)
0
HO HO
CI
87 Hoe ~ \-f~f~co H 730 38 (88%)
z
0
HO HO
CI
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Table 2 continued.
HO, ,~~~~~COaH 150 11.6 (99%)
~~o
HO HO
CF3
89 Ho. ..~~~~coZH 260 41 (96%)
0
HO HO
CF3
s Topical formulations containing the prostaglandin analogues of the
present invention for the treatment of glaucoma and ocular hypertension as
well
as methods of synthesizing and formulating the same are disclosed in U.S.
Patent Nos. 5,510,383; 5,698,733; 5,889,052; 6,025,392; and 6,232,344; U.S.
Patent Application Serial Nos. 09/284,432 and 10/100,399, the entire contents
io of which were previously incorporated by reference. Storage-stable
formulations
and packaging systems for the compounds of the present invention are also
described in U.S. Patent Nos. 5,631,287; 6,011,062; and 6,235,781, the entire
contents of each of which are by this reference incorporated herein.
Is The invention is also related to dermatological compositions for topical
treatment for the stimulation of hair growth which comprise an effective hair
growth stimulating amount of one or more prostaglandin analogues as defined
above and a dermatologically compatible carrier. Effective amounts of the
active analogues will vary analogues on the derivative employed, frequency of
2o application and desired result, but will generally range from about
0.0000001 to
about 50% by weight of the dermatological composition; preferably from about
0.00001 to about 5% by weight; and most preferably from about 0.0001 to about
0.1 % by weight. Representative compositions may thus comprise from about
0.001 to about 50 pg of the analogues in about 1 to about 100 pg of total
2s dermatological composition, more preferably from about 0.01 to about 5 pg
in
about 10 to about 50 pg of the composition.
_ ~s_
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In forming compositions for topical administration, the compounds of the
present invention are generally formulated as between about 0.00003 to about 3
percent by weight (wt%) solutions in water at a pH between 4.5 to 8Ø The
compounds are preferably formulated as between about 0.0003 to about 0.3
s wt% and, most preferably, between about 0.003 and about 0.03 wt%. While the
precise regimen is left to the discretion of the clinician, it is recommended
that
the resulting solution be topically applied by spray, roll-on or dropper and
massaged into the affected area, for example the scalp, once a day.
io Other methods of administration include "brushing in," especially as in the
conventional application of mascara to eye lashes. Those skilled in the art
will
appreciate that the previously described amounts and concentrations of the
PGF2a analogues of the present invention may be added to conventional
mascara formulations. Examples of mascara formulations may be found in U.S.
is Patent No. 6,274,131, the contents of which are by this reference
incorporated
herein.
Other ingredients which may be desirable to use in the dermatalogical
preparations of the present invention include preservatives, co-solvents and
ao viscosity building agents.
Antimicrobial Preservatives:
Dermatological products are typically packaged in multidose form, which
generally require the addition of preservatives to prevent microbial
as contamination during use. Suitable preservatives include: benzalkonium
chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben,
phenylethyl
alcohol, edetate disodium, sorbic acid, ONAMER M~, or other agents known to
those skilled in the art. Such preservatives are typically employed at a
concentration between about 0.001 % and about 1.0% by weight.
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Co-Solvents:
Prostaglandins, and particularly ester derivatives, typically have limited
solubility in water and therefore may require a surfactant or other
appropriate co-
solvent in the composition. Such co-solvents include: Polysorbate 20, 60 and
s 80; Pluronic F-68, F-84 and P-103; Tyloxapol O; Cremophor~ EL; sodium
dodecyl sulfate; glycerol; PEG 400; propylene glycol; cyclodextrins; or other
agents known to those skilled in the art. Such co-solvents are typically
employed at a concentration between about 0.01 % and about 2% by weight.
lo Viscosity Agents:
Viscosity greater than that of simple aqueous solutions may be desirable
to increase ocular absorption of the active compound, to decrease variability
in
dispensing the formulations, to decrease physical separation of components of
a
suspension or emulsion of formulation and/or otherwise to improve the
is ophthalmic formulation. Such viscosity building agents include, for
example,
polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl
methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy
propyl
cellulose or other agents known to those skilled in the art. Such agents are
typically employed at a concentration between about 0.01 % and about 2% by
2o weight.
Included within the scope of the present invention are the individual
enantiomers of the title compounds, as well as their racemic and non-racemic
mixtures. The individual enantiomers can be enantioselectively synthesized
2s from the appropriate enantiomerically pure or enriched starting material by
means such as those described below. Alternatively, they may be
enantioselectively synthesized from racemic/non-racemic or achiral starting
materials. (Asymmetric Synthesis by J. D. Morrison and J. W. Scott, Ed.,
Academic Press Publishers: New York, 1983-1985 (five volumes published over
3o a three year span with chapters contributed by about two dozen authors) and
Principles ofAsymmetric Synthesis by R.E. Gawley and J. Aube, Ed., Elsevier
Publishers: Amsterdam, 1996). They may also be isolated from racemic and
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non-racemic mixtures by a number of known methods, e.g. by purification of a
sample by chiral HPLC (A Practical Guide to Chiral Separations by HPLC, G.
Subramanian, Ed., VCH Publishers: New York, 1994; Chiral Separations by
HPLC, A.M. Krstulovic, Ed., Ellis Horwood Ltd. Publishers, 1989), or by
s enantioselective hydrolysis of a carboxylic acid ester sample by an enzyme
(Ohno, M.; Otsuka, M. Organic Reactions, volume 37, page 1 (1989)). Those
skilled in the art will appreciate that racemic and non-racemic mixtures may
be
obtained by several means, including without limitation, nonenantioselective
synthesis, partial resolution or even mixing samples having different
to enantiomeric ratios. Also included within the scope of the present
invention are
the individual isomers substantially free of their respective enantiomers.
EXAMPLE 1
is
Data for Tables 1 and 2 were generated using the following
methodologies.
FP receptof~ binding assay: The bovine corpus luteum has been shown to express
high-
2o affinity [3H]PGFZa binding sites, in addition to [3H]PGE2 binding, which
appear to have
pharmacological characteristics of FP receptors. ~ Washed total particulate
bovine corpus
luteum membranes (20 mg/ml final) were incubated with [3H]PGF2a (0.9-1.5 nM)
in
0
I~rebs buffer (pH 7.4) for 2 h at 23 C in a total volume of 500 ml. Non-
specific binding
was defined with 1-10 pM unlabeled PGF2a or fluprostenol. The assays were
terminated
2s by vacuum filtration (using Whatman GFB glass fiber filter previously
soaked in 0.3%
polyethyleneimine) and the data analyzed by a non-linear, iterative, curve-
fitting
computer program.
FP receptor-mediated phosphoinositide tur ~ove~ assay: [3H]Inositol phosphates
([3H]-
3o IPs) produced by agonist-mediated activation of phospholipase C in Swiss
3T3 cells
expressing FP receptors were quantified as follows. Confluent 3T3 cells were
exposed to
1.0 - 1.5 p,Ci [3H]-myo-inositol (18.3 Ci/mmol) in 0.5 ml DMEM for 24-30 hours
at
37 C. Then cells were rinsed once with DMEM/F-12 containing 10 mM LiCI, and
the
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agonist stimulation experiment was performed in 0.5 ml of the same medium to
facilitate
accumulation of [3H]-IPs. Cells were exposed to the agonist or solvent for 60
min at
37~C (triplicate determinations), followed by aspiration of the medium and
immediate
addition of 1 ml of ice-cold O.1M formic acid. The plates were kept cold and
then
frozen. Samples frozen up to one week were thawed prior to chromatographic
separation
of radiolabeled components. The cell lysates (0.9 ml) were loaded on columns
packed
with approximately 1 ml AG 1-X8 anion exchange resin. The elution procedure
consisted of a wash with 10 ml of H20, then 8 ml of 50 mM ammonium formate,
and
finally 4 ml of 1.2M ammonium formate with 0.1 M formic acid, which was
collected in
to a scintillation vial. To this eluate was added 15 ml of scintillation fluid
and the total
[3H]-IPs determined by scintillation counting on a beta-counter. Data were
analyzed by
the sigmoidal fit function of the Origin Scientific Graphics software
(Microcal Software,
Northampton, MA) to determine agonist potency (ECSO value) and efficacy,
relative to
the standard cloprostenol.
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EXAMPLE 2
The following Formulations 1-8 are representative pharmaceutical
compositions of the invention for topical use in promoting hair growth. Each
of
Formulations 1 through 8 may be formulated in accordance with procedures
s known to those skilled in the art.
FORMULATION 1
Ingredient Amount (wt%)
to
Travoprost 0.004
Dextran 70 0.1
Hydroxypropyl methylcellulose0.3
Sodium chloride 0.77
Potassium chloride 0.12
Disodium EDTA 0.05
Benzalkonium chloride 0.01
HCI and/or NaOH pH 7.2 - 7.5
Purified water q.s. to 100%
FORMULATION 2
Ingredient Amount (wt%)
Travoprost 0.004
Monobasic sodium phosphate 0.05
Dibasic sodium phosphate 0.15
(anhydrous)
Sodium chloride 0.75
Disodium EDTA 0.01
Benzalkonium chloride 0.02
Polysorbate 80 0.15
HCI and/or NaOH ~ pH 7.3 - 7.4
Purified water q.s. to 100%
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FORMULATION 3
Ingredient Amount (wt%)
Travoprost 0.004
Dextran 70 0.1
Hydroxypropyl methylcellulose0.5
Monobasic sodium phosphate 0.05
Dibasic sodium phosphate 0.15
(anhydrous)
Sodium chloride 0.75
Disodium EDTA 0.05
Benzalkonium chloride 0.01
NaOH and/or HCI pH 7.3 - 7.4
Purified water q.s. to 100%
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FORMULATION 4
Ingredient Amount (wt%)
Travoprost 0.004
Monobasic sodium phosphate ' 0.05
Dibasic sodium phosphate 0.15
(anhydrous)
Sodium chloride 0.75
Disodium EDTA 0.05
Benzalkonium chloride 0.01
HCI and/or NaOH pH 7.3 - 7.4
Purified water q.s. to 100%
s FORMULATION 5
Ingredient Amount (wt%)
Compound 42 0.004
Dextran 70 0.1
Hydroxypropyl methylcellulose0.3
Sodium chloride 0.77
Potassium chloride 0.12
Disodium EDTA 0.05
Benzalkonium chloride 0.01
HCI and/or NaOH pH 7.2 - 7.5
Purified water q.s. to 100%
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FORMULATION 6
Ingredient Amount (wt%)
Compound 42 0.004
Monobasic sodium phosphate 0.05
Dibasic sodium phosphate 0.15
(anhydrous)
Sodium chloride 0.75
Disodium EDTA 0.01
Benzalkonium chloride 0.02
Polysorbate 30 0.15
HCI and/or NaOH pH 7.3 - 7.4
Purified water q.s. to 100%
FORMULATION 7
Ingredient Amount (wt%)
Compound 42 0.004
Dextran 70 0.1
Hydroxypropyl methylcellulose 0.5
Monobasic sodium phosphate 0.05
Dibasic sodium phosphate 0.15
(anhydrous)
Sodium chloride 0.75
Disodium EDTA 0.05
Benzalkonium chloride 0.01
NaOH and/or HCI pH 7.3 - 7.4
Purified water ~ q.s. to 100%
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FORMULATION 8
Ingredient Amount (wt%)
Compound 42 0.004
Monobasic sodium phosphate 0.05
Dibasic sodium phosphate 0.15
(anhydrous)
Sodium chloride 0.75
Disodium EDTA 0.05
Benzalkonium chloride 0.01
HCI and/or NaOH pH 7.3 - 7.4
Purified water q.s. to 100%
s EXAMPLE 3
Eyelash photographs of each eye were taken at determination of patient
eligibility 8AM (baseline) and Months 1.5, 3, 4.5 and 6 in four clinical
studies.
Additional photographs were taken at Months 9 and 12 in two clinical studies.
Eyelash change was classified using the following categories: change in
eyelash
to color, increase in eyelash length, increase in eyelash density and increase
in
eyelash thickness. Two independent readers evaluated the photographs
subjectively for any change from baseline in eyelash characteristics. If the
two
readers did not agree, the photographs were evaluated by a third reader. In
the
case when all three readers had a different assessment, a meeting was held to
Is come to a consensus. Eyelash changes from baseline evaluated by
photographs were reported and are summarized in. the following Table 3.
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Table 3
Percent of Patients with Eyelash
Change
s Treatment N % Total
N
Travoprost 0.0015% 285 48.1 592
Travoprost 0.004% 361 61.3 589
Latanoprost 0.005% 50 25.8 194
to Total 835 35.4 2360
The majority of eyelash changes associated with travoprost (0.0015% and
0.004%) includes changes in color, and increases in length, density and/or
thickness (Table 4). No clinically significant difference within a treatment
group
is was observed for eyelash color, length, density and/or thickness. A
concentration-related change in eyelash color, length, density and/or
thickness
was observed between patients receiving Travoprost 0.0015% and 0.004%.
Patients receiving latanoprost 0.005% experienced similar types of changes in
eyelash color, length, density, and/or thickness, but at a greatly reduced
2o frequency compared to that of travoprost.
Table 4
Percent of Patients with Eyelash Change by Category in Study
2s
Change Color ChangeLength Density Thickness
Treatment Reported N % Change Change Change
N % N % N % N
Travoprost285 48.1 221 37.3 285 48.1 264 44.6 200 33.8
0.0015%
N=592
Travoprost361 61.3 282 47.9 360 61.1 343 58.2 301 51.1
0.004%
N-589
Latanoprost50 25.8 32 16.5 50 25.8 43 22.2 34 17.5
0.005%
N-194
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The compounds of the present invention may be formulated and applied
as topical creams, ointments, solutions or suspensions, lotions, aerosols,
dusting powders and the like. For topical use on the skin and the scalp, the
prostaglandin analogues of the present invention can be advantageously
s formulated using ointments, creams, liniments or patches as a carrier of the
active ingredient. Also, these formulations may or may not contain
preservatives, depending on the dispenser and nature of use. Such
preservatives include those mentioned above, and methyl-, propyl-, or butyl-
parahydroxybenzoic acid, betain, chlorhexidine, benzalkonium chloride, and the
to like. Various matrices for slow release delivery may also be used.
Typically, the
dose to be applied on the scalp is in the range of about 0.1 ng to about 100
mg
per day, more preferably about 1 ng to about 10 mg per day, and most
preferably about 10 ng to about 1 mg per day dependingn on the prostaglandin
analogue and the formulation. To achieve the daily amount of medication
is depending on the formulation, the prostaglandin analogue may be
administered
once or several times daily with or without antioxidants. Those skilled in the
art
will appreciate that the compounds of the present invention, and especially
travoprost or compound 42 may be substituted for the compounds described in
examples 2-11 of the '105 patent.
Moreover, the compounds of the present invention may be combined with
one or more known agents for the promotion of hair growth. While bound by no
theories, the inventors suspect that the mechanism of action by which the
compounds of the present invention promote hair growth may be distinct from
2s those of the presently available commercial hair growth products.
Specifically,
the compounds of the present invention may be combined with: i) minoxidil
(Pharmacia) and minoxidil-type compounds; ii) finasteride (Merck) and
finasteride-type compounds (dihydrotestosterone (DHT) blockers); and iii)
copper-peptides or retinoic acid related compounds. These three types of
3o conventional hair growth products are described at
http://www.skinbiology.com/hairregrowth.html (as published 07/24/2001), the
contents of which are by this reference incorporated herein. Examples of
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minoxidil-type compounds include: aminexil (Dercap) (L'Oreal); cromakalim/BRL
34915 (Pharmacia); diazoxide (Hyperstat IV, Proglycem); pinacidil; and its
analogue PC-1075. An example of a finasteride-type compound would be the
herbal product Saw palmetto (serenoa repens), which acts as a DHT blocker.
The invention has been described by reference to certain preferred
embodiments; however, it should be understood that it may be embodied in
other specific forms or variations thereof without departing from its spirit
or
essential characteristics. The embodiments described above are therefore
io considered to be illustrative in all respects and not restrictive, the
scope of the
invention being indicated by the appended claims rather than by the foregoing
description.
Is
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