| United States Patent Application |
20020042425
|
| Kind Code |
A1 |
| Gormley, Glenn J. ;
et al. |
April 11, 2002 |
Transdermal treatment with 5-alpha reductase inhibitors
Abstract
The instant invention involves a method of treating and/or
reversing androgenic alopecia and promoting hair growth, and
methods of treating acne vulgaris, seborrhea, and female hirsutism,
by administering to a patient in need of such treatment a
5.alpha.-reductase 2 inhibitor, such as finasteride, in a
dosage amount under 5 mgs/day.
| Inventors: |
Gormley, Glenn J.; (Westfield,
NJ) ; Kaufman, Keith D.; (Westfield, NJ)
; Stoner, Elizabeth; (Westfield, NJ) ;
Waldstreicher, Joanne; (Scotch Plains, NJ)
|
| Correspondence Name
and Address: |
MERCK AND CO INC
P O BOX 2000
RAHWAY
NJ
070650907
|
| Serial No.:
|
010678 |
| Series Code: |
10 |
| Filed: |
December 7, 2001 |
| U.S. Current Class: |
514/290; 424/70.1
|
| U.S. Class at Publication: |
514/290; 424/70.1
|
| Intern'l Class:
|
A61K 031/473; A61K
007/06 |
Claims
1. A method of treating androgenic alopecia comprising administering
to a person in need of such treatment a 5.alpha.-reductase 2
inhibitor in a dosage amount under 5.0 mgs/day.
2. The method of claim 1 wherein the dosage amount is from about
0.01 to 3.0 mgs/day.
3. The method of claim I wherein the dosage amount is from about
0.05 to 1.0 mg/day.
4. The method of claim 1 wherein the dosage amount is about
0.05 mg/day.
5. The method of claim 1 wherein the dosage amount is about
0.2 mgs/day.
6. The method of claim 1 wherein the androgenic alopecia is
male pattern baldness.
7. The method of claim 1 wherein the 5.alpha.-reductase 2 inhibitor
is administered orally.
8. The method of claim 1 wherein the 5.alpha.-reductase 2 inhibitor
is administered topically.
9. The method of claim 1 wherein the 5.alpha.-reductase 2 inhibitor
has the structural formula I 3or a pharmaceutically acceptable
salt thereof wherein: R.sup.1 is hydrogen, methyl or ethyl;
R.sup.2 is a hydrocarbon radical selected from straight and
branched chain alkyl of from 1-12 carbons or monocyclic aryl
optionally containing 1 or more lower alkyl substituents of
from 1-2 carbon atoms and/or 1 or more halogen (Cl, F or Br)
substitints.; R' is hydrogen or methyl; R" is hydrogen or .beta.-methyl;
and R'" is hydrogen, .alpha.-methyl or .beta.-methyl.
10. The method of claim 1 wherein the 5.alpha.-reductase 2 inhibitor
has the structural formula II 4or a pharmaceutically acceptable
salt thereof wherein R.sup.1 is hydrogen, or methyl; and R.sup.3
is branched chain alkyl of from 4-8 carbons.
11. A method of treating androgenic alopecia comprising administering
to a person in need of such treatment 17.beta.-(N-tert-butylcarbamoyl)-4-aza-5-
.alpha.-androst-1-ene-3-one in a dosage amount under 5.0 mgs/day.
12. The method of claim 11 wherein the androgenic alopecia is
male pattern baldness.
13. The method of claim 11 wherein the dosage amount is from
about 0.01 to 3.0 mgs/day.
14. The method of claim 11 wherein the dosage amount is from
about 0.05 to 1.0 mg/day.
15. The method of claim 11 wherein the dosage amount is about
0.05 mg/day.
16. The method of claim 11 wherein the dosage amount is about
0.2 mg/day.
17. The method of claim 11 wherein the 17p-(N-tert-butylcarbamoyl)-4-aza-5-
.alpha.-androst-1-ene-3-one is administered topically.
18. The method of claim 11 wherein the 17.beta.-(N-tert-butylcarbamoyl)-4--
aza-5.alpha.-androst-1-ene-3-one is administered orally.
19. The method of claim 18 wherein the dosage amount is about
0.2 mg/day.
20. The method of claim 18 wherein the dosage amount it about
0.05 mg/day.
21. A method of arresting and reversing androgenic alopecia
comprising administering to a person in need of such treatment
a 5.alpha.-reductase 2 inhibitor in a dosage amount under 5.0
mgs/day.
22. A method of lowering the level of 5.alpha.-dihydrotestosterone
in human scalp comprising administering to a person in need
of such treatment a 5.alpha.-reductase 2 inhibitor in a dosage
amount under 5.0 mgs/day.
23. The method of claim 22 wherein the dosage amount is from
about 0.01 to 3.0 mgs/day.
24. The method of claim 22 wherein the dosage amount is from
about 0.05 to 1.0 mg/day.
25. The method of claim 22 wherein the dosage amount is about
0.05 mg/day.
26. The method of claim 22 wherein the dosage amount is about
0.2 mg/day.
27. The method of claim 22 wherein the dosage amount is about
1.0 mg/day.
Description
[0001] This application is a continuation-in-part of Ser. No.
08/138,520 filed Oct. 15, 1993.
[0002] The present invention is concerned with the treatment
of androgenic alopecia, including male pattern baldness, with
compounds that are 5-alpha reductase isozyme 2 inhibitors.
BACKGROUND OF THE INVENTION
[0003] Certain undesirable physiological manifestations, such
as acne vulgaris, seborrhea, female hirsutism, androgenic alopecia
which includes female and male pattern bkdness, and benign prostatic
hyperplasia, are the result of hyperandrogenic stimulation caused
by an excessive accumulation of testosterone ("T") or similar
androgenic hormones in the metabolic system. Early attempts
to provide a chemotherapeutic agent to counter the undesirable
results of hyperandrogenicity resulted in the discovery of several
steroidal antiandrogens having undesirable hormonal activities
of their own. The estrogens, for example, not only counteract
the effect of the androgens but have a feminizing effect as
well. Non-steroidal antiandrogens have also been developed,
for example, 4'-nitro-3'-trifluoromethyl-isobutyranilide. See
Neri, et al., Endocrinol. 1972, 91 (2). However, these products,
though devoid of hormonal effects, compete with all natural
androgens for receptor sites, and hence have a tendency to feminize
a male host or the male fetus of a female host and/or initiate
feed-back effects which would cause hyperstimulation of the
testes.
[0004] The principal mediator of androgenic activity in some
target organs, e.g. the prostate, is 5.alpha.-dihydrotestosterone
("DHT"), formed locally in the target organ by the action of
testosterone-5.alpha.-reductase. Inhibitors of testosterone-5.alpha.-redu-
ctase will serve to prevent or lessen symptoms of hyperandrogenic
stimulation in these organs. See especially U.S. Pat. No. 4,377,584
assigned to Merck & Co., Inc., issued Mar. 22, 1983. It
is now known that a second 5.alpha.-reductase isozyme exists,
which interacts with skin tissues, especially in scalp tissues.
See, e.g., G. Harris, et al., Proc. Natl. Acad. Sci. USA, Vol.
89, pp. 10787-10791 (November 1992). The isozyme that principally
interacts in skin tissues is conventionally designated as 5.alpha.-reductase
1 (or 5.alpha.-reductase type 1), while the isozyme that principally
interacts within the prostatic tissues is designated as 5.alpha.-reductase
2 (or 5.alpha.-reductase type 2).
[0005] Finasteride (17.beta.-(N-tert-butylcarbamoyl)-4-aza-5.alpha.-andros-
t-1-ene-3-one), which is marketed by Merck & Co., Inc. under
the tradename PROSCAR.RTM., is an inhibitor of 5.alpha.-reductase
2 and is known to be useful for the treatment of hyperandrogenic
conditions. See e.g., U.S. Pat. No. 4,760,071. Finasteride is
currently marketed in the United States and worldwide for the
treatment of benign prostatic hyperplasia. Finasteride's utility
in the treatment of androgenic alopecia and prostatic carcinoma
is also disclosed in the following documents: EP 0 285,382,
published 5 October 1988; EP 0 285 383, published Oct 5, 1988;
Canadian Patent no. 1,302,277; and Canadian Patent no. 1,302,276.
The specific dosages exemplified in the above-noted disclosures
varied from 5 to 2000 mg per patient per day.
[0006] In the treatment of androgenic alopecia, which includes
both female and male pattern baldness, and other hyperandrogenic
conditions, it would be desirable to administer the lowest dosage
possible of a pharmaceutical compound to a patient and still
maintain therapeutic efficacy. Applicants have surprisingly
and unexpectedly discovered that a low daily dosage of a 5.alpha.-reductase
2 inhibitor is particularly useful in the treatment of androgenic
alopecia. Furthermore, a low daily dosage of a 5.alpha.-reductase
2 inhibitor may also be particularly useful in the treatment
of the hyperandrogenic conditions of acne vulgaris, seborrhea,
female hirsutism, and polycystic ovary syndrome.
DETAILED DESCRIPTION OF THE INVENTION
[0007] The instant invention involves a method of treating and/or
reversing androgenic alopecia and promoting hair growth, and
methods of treating acne vulgaris, seborrhea, and female hirsutism,
which comprises administering to a patient in need of such treatment
a 5.alpha.-reductase 2 inhibitor in a dosage amount under 5
mgs/day. In one embodiment of this invention, the 5.alpha.-reductase
2 inhibitor is administered in a dosage amount of from 0.01
to 3.0 mgs/day. In one class of this embodiment, the 5.alpha.-reductase
2 inhibitor is administered in a dosage amount of from 0.05
to 1.0 mg/day, and in a sub-class of this embodiment, the 5.alpha.-reductase
2 inhibitor is administered in dosage amounts of about 0.05
to 0.2 mg/day. Illustrating this subclass are dosage amounts
of about 0.05, 0.1, 0.15 and 0.2 mg/day. Exemplifying the sub-class
are dosages of 0.05 and 0.2 mg/day. Compounds which are inhibitors
of 5.alpha.-reductase 2 can be determined by employing the assay
described below in Example 3.
[0008] In a second embodiment of this invention, the method
of treating androgenic alopecia comprises administration of
5.alpha.-reductase 2 inhibitor compounds which have the structural
formula I 1
[0009] or a pharmaceutically acceptable salt thereof wherein:
[0010] R.sup.1 is hydrogen, methyl or ethyl;
[0011] R.sup.2 is a hydrocarbon radical selected from straight
and branched chain alkyl of from 1-12 carbons or monocyclic
aryl optionally containing 1 or more lower alkyl substituents
of from 1-2 carbon atoms and/or 1 or more halogen (Cl, F or
Br) substituents;
[0012] R' is hydrogen or methyl;
[0013] R" is hydrogen or .beta.-methyl; and
[0014] R'" is hydrogen, .alpha.-methyl or .beta.-methyl.
[0015] In one class of this second embodiment, the 5.alpha.-reductase
2 inhibitor compounds have the structural formula II 2
[0016] or a pharmaceutically acceptable salt thereof wherein
[0017] R.sup.1 is hydrogen, or methyl; and
[0018] R.sup.3 is branched chain alkyl of from 4-8 carbons.
[0019] Representative compounds that may be employed in the
present invention include the following:
[0020] 17.beta.-(N-tert-butylcarbamoyl)-4-aza-5-.alpha.-androst-
1-en-3-one,
[0021] 17.beta.-(N-isobutylcarbamoyl)-4-aza-5-.alpha.-androst-1-en-3-one,
[0022] 17.beta.-(N-tert-octylcarbamoyl)-4-aza-5.alpha.-androst-1-en-3-one,
[0023] 17.beta.-(N-octylcarbamoyl)-4-aza-5.alpha.-androst-1-en-3-one,
[0024] 17.beta.-(N- 1,1-diethylbutylcarbamoyl)-4-aza-5-.alpha.-androst-
1-en-3-one,
[0025] 17.beta.-(N-neopentylcarbamoyl)-4-aza-5.alpha.-androst-1-en-3-one,
[0026] 17.beta.-(N-tert-amylcarbamoyl-4-aza-5.alpha.-androst-1-en-3-one,
and
[0027] 17B-(N-tert-hexylcarbamoyl)-4-aza-5.alpha.-androst-1-en-3-one;
[0028] and the corresponding compounds wherein the 4-nitrogen
is substituted in each of the above named compounds by a methyl
or an ethyl radical.
[0029] Also included as representative compounds are any of
the above indicated compounds having the N-branched chain alkyl
substituent replaced by a methyl, ethyl, propyl, i-propyl, butyl,
phenyl; 2, 3 or 4 tolyl, xylyl, 2-bromo or 2-chlorophenyl, 2-6-dichloro,
or a 2,6-dibromophenyl substituent.
[0030] The compounds of formula I and II described above can
be synthesized according to procedures well known in the art,
and which are described, for example, in U.S. Pat. No. 4,760,071,
EP 0 285,382 and EP 0 285 383. The compound finasteride is currently
available as a prescription pharmaceutical from Merck &
Co. Inc. The synthesis of finasteride is described in U.S. Pat.
No. 4,760,071. A further synthesis of finasteride is described
in Synthetic Communications, 30 (17), p. 2683-2690 (1990).
[0031] The present invention has the objective of providing
methods of treating the hyperandrogenic conditions of androgenic
alopecia, including male pattern baldness and female pattern
baldness, acne vulgaris, seborrhea, female hirsutism, and polycystic
ovary syndrome by systemic, oral, parenteral or topical administration
of a 5.alpha.-reductase 2 inhibitor in a dosage amount under
5 mg/day, and particularly, from about 0.01 mg/day to 3.0 mg/day,
and more particularly 0.05 to 1 mg/day. The invention is further
illustrated by dosages of about 0.05 to 0.2 mg/day and specifically
dosages of about 0.05, 0.1, 0.15 and 0.2 mg/day. Exemplifying
the invention are dosages of 0.05 and 0.2 mg/day. The term "treating
androgenic alopecia" is intended to include the arresting and/or
reversing of androgenic alopecia, and the promotion of hair
growth. Also, a 5.alpha.-reductase 2 inhibitor, e.g. finasteride,
at a dosage under 5 mgs/day can be used in combination with
a potassium channel opener, such as minoxidil or a pharmaceutically
acceptable salt thereof, for the treatment of androgenic alopecia,
including male pattern baldness. The 5.alpha.-reductase 2 inhibitor
and the potassium channel opener may both be applied topically,
or each agent can be given via different administration routes;
for example, the 5.alpha.-reductase 2 inhibitor may be administered
orally while the potassium channel opener may be administered
topically.
[0032] The present invention also has the objective of providing
suitable systemic, oral, parenteral and topical pharmaceutical
formulations for use in the novel methods of treatment of the
present invention. The compositions containing 5.alpha.-reductase
2 inhibitor compounds as the active ingredient for use in the
treatment of the above-noted hyperandrogenic conditions can
be administered in a wide variety of therapeutic dosage forms
in conventional vehicles for systemic administration. For example,
the compounds can be administered in such oral dosage forms
as tablets, capsules (each including timed release and sustained
release formulations), pills, powders, granules, elixirs, tinctures,
solutions, suspensions, syrups and emulsions. Likewise, they
may also be administered in intravenous (both bolus and infusion),
intraperitoneal, subcutaneous, topical with or without occlusion,
or intramuscular form, all using forms well known to those of
ordinary skill in the pharmaceutical arts. For oral administration,
for example, the compositions can be provided in the form of
scored or unscored tablets containing 0.01, 0.05, 0.1, 0.2,
1.0, 2.0 and 3.0 milligrams of the activtingredient for the
symptomatic adjustment of the dosage to the patient to be treated.
[0033] For the treatment of androgenic alopecia including male
pattern baldness, acne vulgaris, seborrhea, and female hirsutism,
the 5.alpha.-reductase 2 inhibitor compounds may be administered
in a pharmaceutical composition comprising the active compound
in combination with a pharmaceutically acceptable carrier adapted
for topical administration. Topical pharmaceutical compositions
may be, e.g., in the form of a solution, cream, ointment, gel,
lotion, shampoo or aerosol formulation adapted for application
to the skin. Topical pharmaceutical compositions useful in the
method of treatment of the present invention may include about
0.001% to 0.1% of the active compound in admixture with a pharmaceutically
acceptable carrier.
[0034] Advantageously, compounds of the present invention may
be administered in a single daily dose, or the total daily dosage
may be administered in divided doses of two, three or four times
daily. The compounds for the present invention can be administered
in intranasal form via topical use of suitable intranasal vehicles,
or via transdermal routes, using those forms of transdermal
skin patches well known to those of ordinary skill in that art.
To be administered in the form of a transdermal delivery system,
the dosage administration will, of course, be continuous rather
than intermittent throughout the dosage regimen. Compounds of
the present invention may also be delivered as a suppository
employing bases such as cocoa butter, glycerinated gelatin,
hydrogenated vegetable oils, mixtures of polyethylene glycols
of various molecular weights and fatty acid esters of polyethylene
glycol.
[0035] The dosage regimen utilizing the compounds of the present
invention is selected in accordance with a variety of factors
including type, species, age, weight, sex and medical condition
of the patient; the severity of the condition to be treated;
the route of administration; the renal and hepatic function
of the patient; and the particular compound thereof employed.
A physician or veterinarian of ordinary skill can readily determine
and prescribe the effective amount of the drug required to prevent,
counter, arrest or reverse the progress of the condition. Optimal
precision in achieving concentration of drug within the range
that yields efficacy without toxicity requires a regimen based
on the kinetics of the drug's availability to target sites.
This involves a consideration of the distribution, equilibrium,
and elimination of a drug.
[0036] In the methods of the present invention, the 5.alpha.-reductase
2 inhibitor compounds herein described in detail can form the
active ingredient, and are typically administered in admixture
with suitable pharmaceutical diluents, excipients or carriers
(collectively referred to herein as "carrier" materials) suitably
selected with respect to the intended form of administration,
that is, oral tablets, capsules, elixirs, syrups and the like,
and consistent with conventional pharmaceutical practices.
[0037] For instance, for oral administration in the form of
a tablet or capsule, the active drug component can be combined
with an oral, non-toxic pharmaceutically acceptable inert carrier
such as ethanol, glycerol, water and the like. Capsules containing
the product of this invention can be prepared by mixing an active
compound of the present invention with lactose and magnesium
stearate, calcium stearate, starch, talc, or other carriers,
and placing the mixture in gelatin capsule. Tablets may be prepared
by mixing the active ingredient with conventional tableting
ingredients such as calcium phosphate, lactose, corn starch
or magnesium stearate. Moreover, when desired or necessary,
suitable binders, lubricants, disintegrating agents and coloring
agents can also be incorporated into the mixture. Suitable binders
include starch, gelatin, natural sugars such as glucose or beta-lactose,
corn sweeteners, natural and synthetic gums such as acacia,
tragacanth or sodium alginate, carboxymethylcellulose, polyethylene
glycol, waxes and the like. Lubricants used in these dosage
forms include sodium oleate, sodium stearate, magnesium stearate,
sodium benzoate, sodium acetate, sodium chloride and the like.
Disintegrators include, without limitation, starch, methyl cellulose,
agar, bentonite, xanthan gum and the like.
[0038] The liquid forms in suitably flavored suspending or dispersing
agents such as the synthetic and natural gums, for example,
tragacanth, acacia, methyl-cellulose and the like. Other dispersing
agents which may be employed include glycerin and the like.
For parenteral administration, sterile suspensions and solutions
are desired. Isotonic preparations which generally contain suitable
preservatives are employed when intravenous administration is
desired.
[0039] Topical preparations containing the active drug component
can be admixed with a variety of carrier materials well known
in the art, such as, e.g., alcohols, aloe vera gel, allantoin,
glycerine, vitamin A and E oils, mineral oil, propylene glycol,
PPG2 myristyl propionate, and the like, to form, e.g., alcoholic
solutions, topical cleansers, cleansing creams, skin gels, skin
lotions, and shampoos in cream or gel formulations. See, e.g.,
EP 0 285 382.
[0040] The compounds of the present invention can 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.
[0041] Compounds of the present invention may also be delivered
by the use of monoclonal antibodies as individual carriers to
which the compound molecules are coupled. The compounds of the
present invention may also be coupled with soluble polymers
as targetable drug carriers. Such polymers can include polyvinylpyrrolidone,
pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol,
or polyethyleneoxidepolylysine substituted with palmitoyl residues.
Furthermore, the compounds of the present invention may be coupled
to a class of biodegradable polymers useful in achieving controlled
release of a drug, for example, polylactic acid, polyepsilon
caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,
polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic
block copolymers of hydrogels.
[0042] The following example illustrates the present invention
and as such are not to be considered as limiting the invention
set forth in the claims appended hereto.
EXAMPLE 1
[0043] Finasteride is known to occur in two distinct polymorphic
crystal forms, termed "form 1" and "form II". Form I is the
marketed form of finasteride as a 5 mg tablet (PROSCAR.RTM.).
[0044] Finasteride Form I can be prepared by dissolving finasteride
in glacial acetic acid (ca. 100 mg/ml) and adding water with
stirring until the weight % of water equals or exceeds 84%.
The resulting solid phase is collected by filtration and dried
under vacuum and at about 50.degree. C. The resulting Form I
is characterized by a differential scanning calorimetry (DSC)
curve, at heating rate of 20.degree. C./min and in a closed
cup, exhibiting a minor endotherm with a peak temperature of
about 232.degree. C., an extrapolated onset temperature of about
223.degree. C. with an associated heat of about 11 joules/gm
and by a major melting endotherm with a peak temperature of
about of 261.degree. C., an extrapolated onset temperature of
about 258.degree. C. with an associated heat of about 89 J/gm.
The x-ray powder diffraction pattern is characterized by d-spacings
of 6.44, 5.69, 5.36, 4.89, 4.55, 4.31, 3.85, 3.59 and 3.14 .
The FT-IR spectrum shows bands at 3431, 3237, 1692, 1666, 1602
and 688 cm-1. The solubilities in water and cyclohexane at 25.degree.
C. are 0.05+0.02 and 0.27+0.05 mg/gm respectively. In addition,
Form I of finasteride can be prepared by recrystallization from
dry (H.sub.2O<1 mg/ml) ethyl acetate and isopropyl acetate.
The isolated solids are dried under vacuum at about 50.degree.
C. and have the same physical characterization data as given
above.
EXAMPLE 2
[0045] Form II of fmasteride can be prepared by dissolving finasteride
in glacial acetic acid (ca. 100 mg/ml) and adding water with
stirring until the weight % of water equals about 75% but not
in excess of 80%. The resulting solid phase is collected by
filtration and dried under vacuum and at about 100.degree. C.
The resulting Form II is characterized by a DSC curve, at heating
rate of 20.degree. C./min and in a closed cup, exhibiting a
single melting endotherm with a peak temperature of about of
261.degree. C., an extrapolated onset temperature of about 258.degree.
C. with an associated heat of about 89 J/gm. The x- ray powder
diffraction pattern is characterized by d-spacings of 14.09,
10.36, 7.92, 7.18, 6.40, 5.93, 5.66, 5.31, 4.68, 3.90, 3.60
and 3.25. The FT-IR spectrum shows bands at 3441, 3215, 1678,
1654, 1597, 1476 and 752 cm-1. The solubilities in water and
cyclohexane at 25.degree. C. are 0.16+0.02 and 0.42+0.05 mg/gm
respectively. In addition, Form II of finasteride can be prepared
by recrystallization from ethyl acetate containing between 2
to 30 mg/ml of water and isopropyl acetate containing between
2 to 15 mg/ml of water. The isolated solids are dried under
vacuum at about 80.degree. C. and have the same physical characterization
data as given above. Form II can also be prepared by heating
Form I up to about 150.degree. C., holding for about one hour
and cooling back to room temperature. The Form II prepared in
this manner has the same physical characterization data as given
above.
EXAMPLE 3
Preparation of Human Prostatic 5.alpha.-reductase.
[0046] Samples of human tissue were pulverized using a freezer
mill and homogenized in 40 mM potassium phosphate, pH 6.5, 5
mM magnesium sulfate, 25 mM potassium chloride, 1 mM phenylmethyl-sulfonyl
fluoride, 1 mM dithiothreitol (DTT) containing 0.25 M sucrose
using a Potter-Elvehjem homogenizer. A crude nuclear pellet
was prepared by centrifugation of the homogenate at 1,500.times.g
for 15 min. The crude nuclear pellet was washed two times and
resuspended in two volumes of buffer. Glycerol was added to
the resuspended pellet to a final concentration of 20%. The
enzyme suspension was frozen in aliquots at -80.degree. C. The
prostatic reductases were stable for at least 4 months when
stored under these conditions.
50.alpha.-reductase Assay
[0047] The reaction mixture for the type 2 5.alpha.-reductase
contained 40 mM sodium citrate, pH 5.5, 0.3 .mu.M [7-.sup.3H]-testosterone,
1 mM dithiothreitol and 500 .mu.M NADPH in a final volume of
100 .mu.l. Typically, the assay was initiated by the addition
of 50-100 .mu.g prostatic homogenate and incubated at 37.degree.
C. After 10-50 min the reaction was quenched by extraction with
250 .mu.l of a mixture of 70% cyclohexane: 30% ethyl acetate
containing 10 .mu.g each DHT and T. The aqueous and organic
layers were separated by centrifugation at 14,000 rpm in an
Eppendorf microfuge. The organic layer was subjected to normal
phase HPLC (10 cm Whatman partisil 5 silica column equilibrated
in 1 ml/min 70% cyclohexane: 30% ethyl acetate; retention times:
DHT, 6.8-7.2 min; androstanediol, 7.6-8.0 min; T, 9.1-9.7 min).
The HPLC system consisted of a Waters Model 680 Gradient System
equipped with a Hitachi Model 655A autosampler, Applied Biosystems
Model 757 variable UV detector, and a Radiomatic Model A120
radioactivity analyzer. The conversion of T to DHT was monitored
using the radioactivity flow detector by mixing the HPLC effluent
with one volume of Flo Scint 1 (Radiomatic). Under the conditions
described, the production of DHT was linear for at least 25
min. The only steroids observed with the human prostate preparation
were T, DHT and androstanediol.
Inhibition Studies
[0048] Compounds were dissolved in 100% ethanol. IC.sub.50 values
represent the concentration of inhibitor required to decrease
enzyme activity to 50% of the control. IC.sub.50 values were
determined using a 6 point titration where the concentration
of the inhibitor was varied from 0.1 to 1000 nM.
EXAMPLE 4
MACROPHOTOGRAPHY AND GLOBAL PHOTOGRAPHY PROCEDURE FOR DETECTION
OF HAIR GROWTH
[0049] A. Macrophotographic Procedure
[0050] Location: ID card
[0051] Haircount target area
[0052] Equipment: Film: Kodak-T-max 24 exposure each of same
emulsion lot number
[0053] Camera: Nikon N-6000
[0054] Lens: Nikkor 60 mm f2.8
[0055] Flashes: Nikon SB-21B Macroflash
[0056] Device: registration device
[0057] Photographic Procedure:
[0058] In these clinical photographs, the only variable allowed
is the haircount. Film emulsion, lighting, framing, exposure,
and reproduction ratios are held constant.
[0059] 1. The haircount area on the patient is prepared as follows:
[0060] A small (.about.1mm) dot tattoo is placed at the beginning
of the study at the leading edge of the bald area directly anterior
to the center of the vertex bald spot, using a commercial tattooing
machine or manually (needle and ink). An area approximately
one square inch in size, centered at the tattoo at the leading
edge of the balding area, is clipped short (.about.2mm). Cut
hairs are removed from the area to be photographed, using tape.
Compressed air and/or ethanol wipes may also be used to facilitate
removal of cut hairs.
[0061] 2. Magnification: Each lens supplied has a fixed reproduction
ratio of 1:1.2.
[0062] Aperture: Every photograph is taken at f/22.
[0063] Film: T-Max 100 (24 exposure) is used.
[0064] 3. Patient's haircount target area. Three exposures (-2/3,
0, and +2/3 f-stop).
[0065] A trained technician places a transparency over the photographic
print and, using a felt tip pen, places a black dot over each
visible hair. The dot map transparency is then counted using
image analysis with computer assistance.
[0066] Photographs are coded with a random number corresponding
to study site, visit number and patient allocation number to
insure blinding to time. At Month 6, baseline and Month 6 photographs
are counted and data analyzed for interim analysis. At Month
12, baseline, Month 6 and Month 12 photographs are counted and
data analyzed for the primary endpoint.
[0067] Methodology for detection of hair growth is also described
in Olsen, E.A. and DeLong, E., J. American Academy of Dermatology,
30 Vol. 23, p. 470 (1990).
[0068] B. Global Photographic Procedure
[0069] Locations: Color card/patient Id
[0070] Global photograph
[0071] Equipment: Film: Kodachrome KR-64 24 exposure each of
same emulsion lot number
[0072] Camera: Nikon N-6000
[0073] Lens: Nikkor 60 mm f2.8
[0074] Flashes: Nikon SB-23
[0075] Photographic Procedure
[0076] In these clinical photographs, the only variable allowed
is the global area's appearance. Anything extraneous to the
area (clothing, furniture, walls, etc.) is eliminated from the
fields to be photographed.
[0077] 1. Patients will have global photographs taken prior
to hair clipping with the head in a fixed position (determined
by the supplied stereotactic device). Hair on the patient's
head is positioned consistenty so as to not obscure the bald
area.
[0078] 2. Magnification: Each lens supplied has a fixed reproduction
ratio of 1:6.
[0079] Aperture: Every photograph will be taken at f/11.
[0080] Film: Kodachrome (24 exposure) is used.
[0081] 3. Patient's global photographs. Three exposures at zero
compensation.
[0082] Using the above-described methodology, it can be shown
that administration of 5.alpha.-reductase 2 inhibitors, including
finasteride, in dosages below 5 mg/day per patient, for example,
1 mg/day or 0.2 mg/day, are useful in the treatment of androgenic
alopecia, and promote hair growth in patients with this condition.
EXAMPLE 5
[0083] In another test, finasteride was orally administered
for 6 weeks to men with male pattern baldness at doses of 0.2
mg/day, 1.0 mg/day and 5.0 mgs/day. The results of this test
showed a significant reduction in DHT content in scalp tissue
of the test participants.
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