U.S. patent application number 09/955241 was filed with the patent office on 2002-03-21 for method of preventing and treating chemotherapy-induced alopecia.
Invention is credited to Jimenez, Joaquin J., Yunis, Adel A..
Application Number | 20020035097 09/955241 |
Document ID | / |
Family ID | 27505559 |
Filed Date | 2002-03-21 |
United States Patent
Application |
20020035097 |
Kind Code |
A1 |
Jimenez, Joaquin J. ; et
al. |
March 21, 2002 |
Method of preventing and treating chemotherapy-induced alopecia
Abstract
The present invention relates, in general, to a method of
inhibiting alopecia, or hair loss, and, in particular, to a method
of inhibiting alopecia induced by chemotherapeutic agents. The
invention further relates to a composition suitable for use in such
a method. Active agents for use in the above method include
proteinaceous growth factors, such as EGF, and/or vitamin D.sub.3
or metabolites thereof.
Inventors: |
Jimenez, Joaquin J.; (Miami,
FL) ; Yunis, Adel A.; (Miami, FL) |
Correspondence
Address: |
Greenberg Traurig, LLP
885 Third Avenue
New York
NY
10022
US
|
Family ID: |
27505559 |
Appl. No.: |
09/955241 |
Filed: |
September 17, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09955241 |
Sep 17, 2001 |
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08477103 |
Jun 7, 1995 |
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6291443 |
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08477103 |
Jun 7, 1995 |
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08250646 |
May 27, 1994 |
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5486509 |
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08250646 |
May 27, 1994 |
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07903829 |
Jun 24, 1992 |
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07903829 |
Jun 24, 1992 |
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07810412 |
Dec 20, 1991 |
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07810412 |
Dec 20, 1991 |
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07786788 |
Nov 1, 1991 |
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07786788 |
Nov 1, 1991 |
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07722500 |
Jun 28, 1991 |
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Current U.S.
Class: |
514/167 ;
514/49 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 38/1825 20130101; A61K 31/675 20130101; B82Y 5/00
20130101; Y10S 514/922 20130101; A61K 31/70 20130101; A61K 38/1808
20130101; A61K 31/675 20130101; A61Q 7/00 20130101; A61K 38/1825
20130101; A61K 31/70 20130101; A61K 8/64 20130101; A61K 8/67
20130101; A61K 38/1808 20130101; Y10S 514/88 20130101 |
Class at
Publication: |
514/167 ;
514/49 |
International
Class: |
A61K 031/7105; A61K
031/59 |
Claims
What is claimed is:
1. A method of preventing or reducing chemotherapy-induced alopecia
comprising administering to a patient undergoing chemotherapy
vitamin D.sub.3, or derivative, analog or structural analog
thereof, in an amount sufficient to effect said prevention or
reduction.
2. The method according to claim 1 wherein said derivative is
1,25-dihydroxyvitamin D.sub.3 or
1,25-dihydroxy-16-ene-23-yne-cholecalcif- erol.
3. The method according to claim 1 wherein said vitamin D.sub.3 or
derivative, analog or structural analog thereof is administered
topically.
4. The method according to claim 1 wherein said chemotherapeutic
agent is cell cycle specific.
5. The method according to claim 4 wherein said chemotherapeutic
agent is cytosine arabinoside.
6. The method according to claim 1 wherein said chemotherapeutic
agent is non cell cycle specific.
7. The method according to claim 6 wherein said chemotherapeutic
agent is Cytoxan.
8. The method according to claim 1 wherein the chemotherapeutic
agent is a cell cycle specific agent in combination with a non cell
cycle specific agent.
9. The method according to claim 1 wherein vitamin D.sub.3, or
derivative, analog or structural variant thereof, is administered
prior to the initiation of said chemotherapy.
10. The method according to claim 1 further comprising
administering to said patient at least one proteinaceous growth
factor in an amount sufficient to effect said prevention or
reduction.
11. The method according to claim 10, wherein said growth factor is
epidermal growth factor.
12. The method according to claim 10, wherein said growth factor is
fibroblast growth factor.
13. A method of preventing or reducing chemotherapy-induced
alopecia comprising administering to a patient undergoing
chemotherapy a proteinaceous growth factor in an amount sufficient
to effect said prevention or reduction.
14. The method according to claim 13, wherein said growth factor is
epidermal growth factor.
15. A method of stimulating hair growth comprising administering to
a warm-blooded animal in need of such stimulation Vitamin D.sub.3
or metabolite, analog or derivative thereof, in an amount
sufficient to effect said stimulation.
16. The method according to claim 15 further comprising
administering to said animal a proteinaceous growth factor.
17. The method according to claim 16 wherein said growth factor is
epidermal growth factor.
18. The method according to claim 15 wherein said warm-blooded
animal is a human suffering from sale pattern baldness.
19. A composition suitable for use in stimulating hair growth
comprising an effective amount of a combination of vitamin D.sub.3,
or metabolite, analog or derivative thereof, and a proteinaceous
growth factor, together with a pharmaceutically acceptable
carrier.
21. The composition according to claim 20 wherein said composition
is in the form a gel, cream, lotion or ointment.
Description
[0001] This is a continuation-in-part of application Ser. No.
07/810,412, filed Dec. 20, 1991, which is a continuation-in-part of
application Ser. No. 07/786,788, filed Nov. 1, 1991, which itself
is a continuation-in-part of application Ser. No. 07/722,500, filed
on Jun. 28, 1991, the entire contents of each of these applications
being hereby incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates, in general, to a method of
preventing or treating alopecia, and, in a specific embodiment, to
a method of preventing or treating alopecia induced by
chemotherapeutic agents.
BACKGROUND
[0003] Alopecia is a common and distressing side effect of many
chemotherapeutic agents and for which there is currently no
effective preventive measure. In a recent study, thirty-five of
forty-six patients receiving chemotherapy ranked alopecia as more
important than vomiting (Tierney et al, B. J. Cancer, 62:527-528,
1990).
[0004] Recently, using the young rat model, Applicants demonstrated
that ImuVert, a biologic response modifier prepared from the
bacterium Serratia marcescens, protected the animals from alopecia
induced by cytosine arabinoside or adriamycin (Hussein et al,
Science 249: 1564-1566, 1990). In subsequent studies, similar
protection from ARA-C-induced alopecia was observed from
recombinant interleukin-1 (IL-1) beta (Jimenez et al FASEB J.
1991).
[0005] The present invention provides an independent method of
preventing and treating chemotherapy-induced alopecia. This method
involves the use of a growth factor, such as epidermal growth
factor (EGF) or fibroblast growth factor (FGF). It should be noted
that, as far as Applicants are aware, ImuVert has not been shown to
stimulate the production of EGF or FGF, nor has it been proposed to
stimulate such production.
[0006] The present invention also relates to the use of Vitamin
D.sub.3, or a metabolite thereof, alone or in combination with EGF
to prevent or treat alopecia. Vitamin D.sub.3 is absorbed after
ingestion of fish liver oils or irradiated yeast. Plants and animal
sources contain only the inactive vitamin D precursors,
7-dehydrocholesterol or ergosterol. 7-Dehydrocholesterol is stored
in the skin and can be converted by sunlight into vitamin D.sub.3.
However, whether ingested or formed by ultraviolet irradiation in
the skin, Vitamin D has to be transformed into active metabolites.
Vitamin D.sub.3, is converted to 25-hydroxycholecalciferol by liver
enzymes. Then in the kidneys two compounds
1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalcifero- l
are formed. The vitamin D active metabolites play an important role
in the absorption of calcium from the intestinal tract, bone
deposition and bone reabsorption.
[0007] 1, 25-Dihydroxyvitamin D.sub.3, an active metabolite of
Vitamin D.sub.3, has been shown to increase EGF receptors on breast
cancer cells (Falette et al, Molec. and Cell. Endocrinol., 63
(1-2):189-198, 1989) and on a cell line established from rat
calvaria (Petkovich et al, J. Biol. Chem. 262 (28):13424-13428,
1987). However, as far as Applicants are aware, the effect of
Vitamin D.sub.3, or a metabolite thereof, on alopecia has not been
shown or proposed.
SUMMARY OF THE INVENTION
[0008] It is a general object of the invention to provide a method
of treating or preventing alopecia. It is a specific object of the
invention to prevent or treat alopecia in patients undergoing
treatment with chemotherapeutic agents, including cycle specific
agents (such as cytosine arabinoside (ARA-C)) and non cycle
specific agents (such as Cytoxan), individually or in
combination.
[0009] Further objects and advantages of the invention will be
clear from the description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a photograph of 10 rats from Experiment I, Table I
(see below). All rats received ARA-C 50 mg/kg.times.7 days. Five
rats on top received buffer solution s.c. Five rats on bottom
received murine EGF 2 .mu.g s.c. daily.times.7 days.
[0011] FIG. 2 is a photograph of 6 rats from Experiment III, Table
I (see below). All rats received ARA-C 50 mg/kg.times.7 days. Three
rats on top received buffer solution S.C. Three rats on bottom
received rHu-EGF 2 .mu.g s.c. daily.times.7 days.
[0012] FIG. 3 is a photograph of 4 rats from topical murine-EGF
experiment (see below). All rats received ARA-C 50 mg/kg.times.7
days. Two rats on the left received murine EGF 10 .mu.g in DMSO
daily.times.7 days rubbed topically between the shoulder blades
over an area of 1 cm.sup.2. Two rats on the right received buffer
solution topically.
[0013] FIG. 4 is a photograph of 12 rats from ARA-C-aFGF experiment
(see below). All rats received ARA-C 50 mg/kg.times.7 days. Six
rats on top received buffer solution s.c. Six rats on bottom
received aFGF 2 .mu.g s.c. daily.times.7 days.
[0014] FIG. 5 is a photograph of 8 rats treated with combination
chemotherapy Cytoxan and Adriamycin. Four rats on top treated in
addition with murine EGF. Four rats on bottom treated with buffer
solution.
[0015] FIG. 6 is a photograph of 10 rats treated with VP-16. All
rats received 1.5 mg/kg.times.3 days i.p. of VP-16. Five rats on
top received buffer solution for four days prior to treatment. Five
rats on bottom received Vitamin D.sub.3 50 .mu.g/day for 4 days for
four days prior to treatment.
[0016] FIG. 7 is a photograph of 6 rats treated with combination
chemotherapy Cytoxan and Adriamycin (25 mg/kg i.p..times.1 day and
2.5 mg/kg i.p..times.3 days, respectively). Three rats on top
received buffer solution for four days prior to treatment. Three
rats on bottom received Vitamin D.sub.3 , 50 .mu.g/day for four
days prior to treatment.
[0017] FIGS. 8(A-C). For each experiment, five day old rats were
randomly divided into equal numbers. The experimental group of rats
(top group) received 0.2 .mu.g of 1,25-dihydroxyvitamin D.sub.3, in
0.15 ml of absolute ethanol daily over the head and neck for 5
days. Control rats (bottom group) were similarly treated with 0.15
ml of absolute ethanol. One day after the last topical treatment,
the rats from FIG. 8A were treated with Cytoxan (CTX), rats from
FIG. 8B with the Etoposide (VP-16) regimen and rats from FIG. 8C
with CTX+Adriamycin (ADM) regimen.
[0018] FIG. 9. Twenty 5-day old rats were randomly divided into two
groups of 10 rats each. The experimental group of rats (top group)
received 0.1 .mu.g of 1,25 (OH)2D.sub.3 in 0.1 ml of absolute
ethanol daily over the head only for 5 days. Control rats (bottom
group) were similarly treated with 0.1 ml of absolute ethanol. one
day after the last topical treatment, all rats were treated with
the VP-16 regimen.
[0019] FIG. 10. Thirteen 9-day old rats were randomized into two
groups. Experimental, 7 rats (top group), received 1 .mu.g of RO
23-7553 in 0.2 ml absolute ethanol daily topically over the neck
and back for 6 days. Control, 6 rats (bottom group), were similarly
treated with 0.2 ml of absolute ethanol. One day after the last
treatment all rats were treated with the VP-16 regimen.
[0020] FIG. 11, shows the effect of 1,25-dihydroxyvitamin D.sub.3
on hair growth.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention relates generally to a method of
preventing or reducing alopecia, particularly in patients
undergoing chemotherapy. Applicants have shown that a growth
factor, such as EGF, and Vitamin D.sub.3 appear to render the hair
follicle resistant to the toxic effect of chemotherapeutic agents
thus preventing hair lose.
[0022] In one embodiment of the present method, a growth factor is
administered to a patient undergoing chemotherapy in an amount
sufficient to prevent or reduce the hair loss that normally
accompanies this treatment regimen.
[0023] Growth factors suitable for use in the present method
include EGF, FGF, transforming growth factors (TGF), and
platelet-derived growth factor (PDGF). The growth factors can be
derived from natural sources (for example, human tissue or rodent
tissue); however, recombinant production is preferred as large
quantities can be produced at relatively low cost. Chemically
synthesized factors can also be used. The use of portions or
derivatives of growth factors, such as EGF and FGF, is also
contemplated as long as those portions or derivatives can effect
the same result observed with the factor itself.
[0024] In another embodiment of the present invention, Vitamin
D.sub.3 or metabolite, analog, derivative or structural variant
thereof (for example 1,25-dihydroxy-16-ene-23-yne-cholecalciferol;
1 60 -hydroxyvitamin D.sub.3+B; 1 .alpha.-24-dihydroxyvitamin
D.sub.3, MC 903, etc.) is administered to a warm blooded animal,
for example, a human, in an amount sufficient to prevent or reduce
the hair loss or stimulate hair growth. Hair loss treatable or
preventable using vitamin D.sub.3 can be due to chemotherapy or
other cause, including, but not limited to, male pattern baldness.
Examples of Vitamin D.sub.3 metabolites suitable for use in the
present method include, but are not limited to,
1,25-dihydroxyvitamin D.sub.3 and 1,25-dihydroxy-16-ene-23-yne
cholecalciferol.
[0025] Compositions suitable for use in the claimed method include
as an active agent a growth factor, Vitamin D.sub.3 (or a
metabolite or analog thereof) or a combination of both. Such
compositions can be formulated by combining an active agent
together with a pharmaceutically acceptable vehicle (carrier,
diluent or excipient), in an amount sufficient to effect the
preventative effect when administered in accordance with an
appropriately designed treatment protocol. The composition can be
in dosage unit form.
[0026] Though not limiting the present method to a particular mode
of action, it is suggested that Vitamin D.sub.3 protects against
alopecia by increasing the receptors for EGF at the hair follicle
level. Accordingly, administering a combination of a growth factor
and Vitamin D.sub.3 can be expected to provide for greater
protection.
[0027] Compositions suitable for use in the method to which the
invention relates can be in a form suitable for topical
administration. In that event, the composition can take the form of
a solution, lotion, cream, gel or ointment. When the composition is
to be administered by injection, it advantageously takes the form
of a solution. The vehicle used, regardless of the form taken by
the composition, can be inert or can itself possess a
physiologically or pharmaceutically beneficial effect.
[0028] Various additives can be included in the composition. In
this regard, inclusion in the composition of an agent that
stimulates production of the patients'own growth factor is
contemplated. Inclusion in compositions suitable for topical
administration of penetration enhancing agents, such as DMSO or
ethanol, is preferred. Stabilizers that extend shelf life can also
be included in the composition, regardless of the manner in which
it is formulated.
[0029] One skilled in the art will appreciate that various
concentrations of growth factor and/or Vitamin D.sub.3 can be used
in the above-described composition. Optimum concentrations can be
readily determined by one skilled in the art.
[0030] As noted above, the method to which the invention relates
can involve either topical application of the active agent (a
growth factor and/or Vitamin D.sub.3 or metabolite thereof) or
administration by injection. The amount of the active agent and the
frequency of administration can vary depending on the individual
and can readily be optimized by one skilled in the art. As an
example, however, a solution of 2-100 .mu.g/ml of
1,25-dihydroxyvitamin D.sub.3 in absolute ethanol can be prepared
and 3-5 ml of that solution applied directly to the scalp at
various points with a dropper followed by scalp message for 3-5 min
to ensure even distribution. When chemotherapy is involved, this
treatment is, advantageously, administered once or twice daily
beginning 5-8 days prior to initiation of chemotherapy and
continued through the course of chemotherapy. However, it is also
contemplated that the active agent can be administered
substantially simultaneously with, or subsequent to, the
administration of the chemotherapeutic agent.
[0031] The method to which the invention relates is shown in the
Examples that follow to be effective when the cell cycle specific
drug, ARA-C, is the chemotherapeutic agent used and when the
combination of Adriamycin (cell cycle specific) and Cytoxan (non
cell cycle specific) is used. It is contemplated, however, that,
using the present method, hair loss resulting from treatment with
other chemotherapeutic agents can be prevented. In addition, it is
contemplated that a growth factor and/or vitamin D.sub.3 can be
used to prevent or retard hair loss in male pattern baldness if it
is used on a regular basis and, advantageously, at the first sign
of baldness, for example, once daily or every other day to the
predisposed area of the scalp.
[0032] The alopecia preventative effect observed by Applicants was
wholly unexpected. Growth factors, such as EGF, are presumed
stimulants of skin cell growth. Accordingly, these agents would be
expected to induce the hair follicle to enter the cell cycle thus
rendering the follicle more susceptible to chemotherapeutic agents,
particularly cell cycle specific drugs, such as ARA-C. Thus,
administration of growth factors to patients receiving chemotherapy
would have been expected to aggravate hair loss. The reverse
effect, however, was achieved. It should be noted therefore that
the observations recorded herein with EGF and FGF are novel and
have not been proposed or described in the literature. Similarly,
nothing about the role of Vitamin D.sub.3 in the body suggested
that the vitamin would provide such excellent protection against
alopecia, chemotherapeutically induced or otherwise.
[0033] The following non-limiting Examples describe certain aspects
of the invention in greater detail.
EXAMPLES
[0034] The following experimental details relate to Examples I-IV
set forth below.
[0035] Sprague Dawley rats were purchased from Charles River
Laboratories, Wilmington, Mass. Cytosar-U (ARA-C) was from the
Upjohn Company, Kalamazoo, Mich. Receptor grade EGF from mouse
submaxillary glands, human recombinant EGF, dimethyl sulfoxide
(DMSO) and Vitamin D.sub.3 were purchased from Sigma Chemical Co.,
St. Louis, Mo. aFGF was purchased from AMGEN Corp., Thousand Oaks,
Calif.
[0036] All rats from each experiment were treated with ARA-C 50
mg/kg intraperitoneally (i.p.) daily for 7 days. For subcutaneous
(s.c.) injections, EGF and FGF were prepared in PBS 1% BSA.
Alopecia was always recorded on day 12 of experiment, and scored as
previously described (Hussein et al, Science, 249:1564-1566, 1990;
Jimenez et al, FASEB J., 1991).
[0037] For topical treatment, murine EGF was prepared as follows:
One vial of EGF (100 .mu.g) was dissolved in 0.2 ml of PBS 1% BSA
and 0.12 ml of this solution was added to 0.48 of DMSO. Three hours
prior to ARA-C injection, 0.1 ml of the EGF-DMSO mixture was
applied to each rat over a 1 cm.sup.2 area between the shoulders
using a rubber tip applicator. Rats were then kept individually
separated for a period of three hours, following which the treated
area was carefully washed with soap and water and dried. Treatment
was continued for 7 days. Control animals were similarly treated
using DMSO without EGF.
Example I
Protective Effect of EGF
[0038] Two separate experiments were conducted to test the ability
of murine EGF to protect from ARA-C-induced alopecia. In Experiment
I, twenty-two 7-day old rats were randomized in two groups of
eleven rats each. In addition to ARA-C, Group I received 2 .mu.g of
mouse EGF s.c. in the back between the two hind legs 3 hours prior
to ARA-C injections daily for 7 days. Group II, received buffer
solution similarly and served as control. Ten of eleven rats in
Group II developed virtually total body alopecia and one rat
developed more than 50% hair loss. In contrast, in Group I, 5 rats
had no detectable hair loss and 6 rats had mild hair loss (Table I,
Experiment I (FIG. 1)). In Experiment II, twelve 7-day old rats
were randomized in two groups of 6 rats each. In addition to ARA-C,
Group I received mouse EGF 1 .mu.g s.c. daily for 7 days. Group II
received buffer s.c. All 6 rats in Group II developed moderately
severe to severe alopecia, whereas in Group I, one rat had no
detectable hair loss and 5 rats developed only minimal hair loss
(Table I, Experiment II).
[0039] For the next experiment, rHu-EGF was used. Twelve 7-day old
rats were randomized in two groups of six rats each. In addition to
ARA-C, Group I received rHu-EGF 2 .mu.g s.c. in the flank area
daily for 7 days. Group II received buffer s.c. All 6 rats in Group
II developed total body alopecia, whereas in Group I none of the
rats had total body alopecia, one rat had no detectable hair loss,
four rats had mild alopecia and one rat had moderate alopecia
(Table I, Experiment III, (FIG. 2)).
1TABLE I OCCURRENCE OF ALOPECIA IN RATS TREATED WITH ARA-C. EFFECT
OF MURINE EGF AND rHU-EGF. Alopecia* 0 1+ 2+ 3+ Experiment I ARA-C
0 0 1 10 ARA-C + Murine EGF 2 .mu.g 5 6 0 0 Experiment II ARA-C 0 0
3 3 ARA-C + Murine EGF 1 .mu.g 1 5 0 0 Experiment III ARA-C 0 0 0 6
ARA-C + rHu-ECF 2 .mu.g 1 4 1 0 Seven day old rats were used for
all experiments. All rats received ARA-C 50 mg/kg .times. 7 days
I.P. in 0.1 ml. Murine EGF and rHu-EGF in PBS 1% BSA were given 3
hours prior to ARA-C once daily in 0.1 ml s.c. .times. 7 days.
Controls received PBS 1% BSA 0.1 ml s.c. .times. 7 days. Data
recorded on day 12. *No detectable alopecia, 0; mild alopecia
defined as less than 50% hair Loss, 1+; moderately sever alopecia,
with more than 50% hair Loss, 2+; and total or virtually total
(>90%) hair Loss, 3+.
[0040] In the next experiment, twelve 7-day old rats were
randomized in two groups of six rats each. Group I, in addition to
ARA-C, received murine-EGF 10 .mu.g in DMSO daily.times.7 days
rubbed topically with a cotton tip applicator between the shoulder
blades over an area of 1 cm.sup.2. Group II received control
solution topically. In Group II, all six rats developed complete
body alopecia. In Group I, all rats developed complete body
alopecia except where the EGF was applied topically (FIG. 3).
Example II
Protective Effect of aFGF
[0041] Fourteen 7-day old rats were randomized in two groups. All
rats received ARA-C 50 mg/kg/day for seven days. In addition, Group
I received aFGF 2 .mu.g s.c. on back of head daily for seven days.
Group II received buffer injections and served as controls.
Alopecia was recorded on day 12 of experiment. All rats in Group II
developed complete body alopecia. In contrast, all rats in Group I
were protected locally at the site of injection (FIG. 4).
Example III
Protection from Cytoxan/Adriamycin-Induced Alopecia
[0042] Eight 4-day old Sprague Dawley rats were randomized in two
groups of 4 rats each. Group I, received EGF 2.mu. s.c. on the head
daily for 7 days. Group II received buffer injections and served as
controls. One day after stopping EGF or buffer injections all rats
received Cytoxan 25 mg/kg i.p..times.1 day and Adriamycin 2.5 mg/kg
i.p..times.3 days. In Group II, all rats had 3+ alopecia over head
and neck area. In contrast, in Group I one rat had mild alopecia,
one rat minimal alopecia, and two rats no detectable alopecia over
head and neck. (FIG. 5). ImuVert when used under similar conditions
did not protect from alopecia caused by the Cytoxan/Adriamycin
combination.
Example IV
Protective Effect of Vitamin D.sub.3
[0043] Twelve 7 day old rats were randomized in two groups. Group I
was treated daily with buffer 0.1 ml s.c. for four days. The second
Group was treated daily with Vitamin D.sub.3 50 .mu.g s.c. over
head for four days. After stopping the buffer or Vitamin D.sub.3
treatment, all rats received 1.5 mg/kg i.p. of VP-16 (Etoposide)
daily for three days. All rats in Group I developed complete body
alopecia while the rats in Group II were protected (FIG. 6 shows 4
rats from each group).
[0044] In other experiments, rats pretreated with Vitamin D.sub.3
demonstrated excellent protection against alopecia produced by
Etoposide, Cytoxan, Cytarabine and the combination of Cytoxan and
Adriamycin (FIG. 7). The results are set forth below in Table
II.
2TABLE II PROTECTION FROM CHEMOTHERAPY-INDUCED ALOPECIA BY
PRETREATMENT WITH VITAMIN D3. Alopecic Total No. of Total No.
Protection drug tested experiments of animals from alopecia
Etoposide (VP-16) 2 22 Yes* Cytoxan (CTX) 7 89 Yes* Cytarabine
(ARA-C) 1 8 Yes* Adriamycin + CTX 6 77 Yes* Combination
Chemotherapeutic agents were given as follows: VP-16 1.5 mg/kg i.p.
daily for3 days; Cytoxan 32.5 mg/kg as a single injection; ARA-C 50
mg/kg i.p. daily for 7 days; for combination (Adriamycin 2.5 mg/kg
i.p. daily for 3 days plus Cytoxan 25 mg/kg as a single injection).
Vitamin D.sub.3 was given in 50 .mu.g daily doses i.p. or s.c. for
4 days prior to chemotherapy *In these experiments, protection from
chemotherapy-induced alopecia was uniformly observed in all animals
treated with Vitamin D.sub.3
[0045] In other experiments, 1,25-dihydroxyvitamin D.sub.3 applied
topically (0.5 .mu.g daily) in 50% ethanol or DMSO also protected
rats from VP-16-induced alopecia.
EXAMPLE V
Protective Effect of Vitamin D.sub.3 Pretreatment
[0046] Topical Application of 1,25-dihydroxyvitamin D.sub.3
[0047] 1,25-Dihydroxyvitamin D.sub.3 was dissolved in absolute
ethanol and applied topically with an applicator. Control animals
were similarly treated with the same amount of ethanol. Animals
were then kept individually separated for a period of three hours
following which the treated area was carefully washed with soap and
water and dried. Treatment was given daily beginning on day 5 after
birth and ending on day 10.
[0048] Chemotherapy
[0049] All chemotherapies were given I.P. and started at 11 days of
age. CTX, 35 mg/kg, was given for one day only. VP-16, 1.5 mg/kg,
was given for three days. For CTX and ADM combination, CTX, 25
mg/kg was given for one day and ADM, 2.5 mg/kg, for three days. At
these doses neither CTX nor ADM alone will produce alopecia.
Alopecia was recorded on the tenth day from beginning
chemotherapy.
[0050] A total of 4 experiments were carried out. In the first
experiment, protection from Cytoxan-induced alopecia was examined.
The experimental group was pretreated with 0.2 .mu.g of
1,25-dihydroxyvitamin D.sub.3 in 0.15 ml of absolute ethanol
applied topically over the head and neck and the control group
received 0.15 ml of alcohol. All 10 rats in the control group
became totally alopecic. In contrast, all animals in the
experimental group were protected (FIG. 8A). The second experiment
was carried out under similar conditions to examine protection from
VP-16-induced alopecia. All 10 rats in the control group developed
total body alopecia. In contrast, all rats in the experimental
group were protected (FIG. 8B). The third experiment was designed
to examine protection from alopecia induced by Cytoxan-Adriamycin
combination. There were 11 rats in each group. Six rats in the
control group developed alopecia over the head and neck and 5 rats
developed total body alopecia. In contrast, all rats in the
experimental group were protected (FIG. 8C). In the fourth
experiment, protection from VP-16-induced alopecia was similarly
examined except that the dose of 1,25-dihydroxyvitamin D.sub.3 was
reduced to 0.1 .mu.g in 0.1 ml absolute ethanol applied topically
over the head area only. All 10 rats in the control group became
completely alopecic. In contrast, all rats in the experimental
group were protected primarily at the site of 1,25-dihydroxyvitamin
D.sub.3 application (FIG. 9).
[0051] It is noteworthy that protection from 0.2 .mu.g
1,25-dihydroxyvitamin D.sub.3 was not limited to the site of
application but involved the entire body, suggesting systemic
absorption. When the dose was reduced to 0.1 .mu.g applied to the
head area only, protection from VP-16-induced alopecia was less
generalized and was more limited to the site of application.
EXAMPLE VI
Protection from VP-16-Induced Alopecia by Topical Application of RO
23-7553
[0052] RO 23-7553 (1,25 dihydroxy-16-ene-23-yne-cholecalciferol)
was dissolved in absolute ethanol and applied topically with an
applicator. Control animals were similarly treated with the same
amount of ethanol. Animals were then kept individually separated
for a period of three hours following which the treated area was
carefully washed with soap and water and dried. Treatment was given
daily beginning on day 9 after birth and ending on day 14.
[0053] On day 15 all animals received VP-16, 1.5 mg/kg i.p., for
three days. Alopecia was recorded on day 25.
[0054] Thirteen rats were randomized in two groups. Experimental
group, 7 rats; control group, 6 rats. The experimental group was
pretreated with 1 .mu.g of RO 23-7553 in 0.2 ml absolute ethanol
applied topically over the neck and back and the control group
received 0.2 ml of absolute ethanol. All six rats in the control
group became totally alopecic over the neck and back. In contrast,
all animals in the experimental group were protected (FIG. 10). It
should be noted that when the chemotherapy is started at the age of
14 days, the head area does not become alopecic.
Example VII
Stimulation of Hair Growth by Vitamin D.sub.3
[0055] During the course of the above-described studies on the
protection from chemotherapy-induced alopecia by Vitamin D.sub.3
and its active analog, 1,25-dihydroxyvitamin D.sub.3 it was noted
that rats treated with 1,25-dihydroxyvitamin D.sub.3 not only were
protected from chemotherapy-induced alopecia, but these rats had a
better coat of hair and longer hair in the treated area. These
observations prompted the following further experiments on the
stimulation of hair growth by 1,25-dihydroxyvitamin D.sub.3.
[0056] The backs of nineteen 25 day old Sprague Dawley rats were
shaven and randomized in two groups.
[0057] Group I (control 10 rats) received 0.1 ml of ethanol applied
topically once daily to the shaven area for 14 days.
[0058] Group II (Calcitriol 9 rats) received 50 ng of
1,25-dihydroxyvitamin D.sub.3 in 0.1 ml of ethanol applied
topically once daily to the shaven area for 14 days.
[0059] On day 15 stimulation of hair regrowth was assessed by
reshaving an area 6 cm.times.6 cm in diameter. The hair was
collected and weighed. The difference between two groups was highly
statistically significant. P. value 0.003 (see Table III and FIG.
11).
3TABLE III STIMULATION OF HAIR GROWTH BY CALCITRIOL IN RATS Hair
Weight in Mg. Control Calcitriol 98 202 131 143 72 150 84 253 102
130 144 177 115 140 129 147 125 135 130 Mean S.E.M. Mean S.E.M. 113
.+-. 8 164 .+-. 13
[0060] Based on these data showing stimulation of hair growth by
1,23-dihydroxyvitamin D.sub.3 administered topically in the rat, it
is expected that 1,25-dihydroxyvitamin D.sub.3 can be used as a
stimulant of hair growth in cases of alopecia of any cause.
Additionally, the data suggest that vitamin D.sub.3 and its
metabolites is/are necessary for optimal hair growth and therefore
can be used to prevent hair loss from any cause, including male
pattern baldness.
Example VIII
Formulations
[0061] The following are four formulations that include 1,25
-dihydroxyvitamin D.sub.3 as active ingredient, and the methods of
their manufacture.
4 1. TOPICAL SOLUTION Ingredients % (W/W) 1,25-Dihydroxyvitamin
D.sub.3 0.0002-0.10 Propylene Glycol 10.00 Propylene Glycol 30.00
Dicarprylate/Dicaprate.sup.a Butylated Hydroxytoluene (BHT) 0.05
Butylated Hydroxyanisole (BHA) 0.05 Ethyl Alcohol, Absolute q.s. to
100.00 .sup.aCan be substituted by the following materials (1)
medium chain triglycerides; 2) dimethyl isosorbide; (3)
polyethylene glycols; (4) ethoxydiglycol
[0062] Manufacturing Procedure
[0063] i. Weigh the appropriate amount of propylene glycol
dicaprylate/dicaprate, ethyl alcohol, propylene glycol in a
stainless steel container.
[0064] ii.Dissolve BHT and BHA into the solution from step (i).
[0065] iii. Add the 1,25-dihydroxyvitamin D.sub.3 into the mixture
from step (ii) and stir until dissolved.
5 2. BUFFERED TOPICAL SOLUTION Ingredients % (W/W)
1,25-Dihydroxyvitamin D.sub.3 0.0002-0.10 Propylene Glycol 50.00
Hydroxypropyl cellulose (Klucel MF) 0.50 Methylparaben 0.20
Butylated Hydroxytoluene (BHT) 0.05 Butylated Hydroxyanisole (BHA)
0.05 Sodium Phosphate, Monobasic 0.43 Sodium Phosphate Dibasic 0.70
Sodium Hydroxide (q.s. to pH = 7) 0.04 Ethyl Alcohol, 95% Proof
30.00 Water q.s. to 100.00
[0066] Manufacturing Procedure
[0067] i. Dissolve the sodium phosphate, monobasic, sodium
phosphate, dibasic, and sodium hydroxide in the water in a
stainless steel container. Measure the pH of the solution. The pH
of the solution should be 7.0; if not adjust the pH.
[0068] ii. Add the propylene glycol and ethyl alcohol to the
solution from step (i).
[0069] iii. Dissolve the 1,25-dihydroxyvitamin D.sub.3,
methylparaben, BHT and BHA to the solution from step (ii).
[0070] iv. Dissolve Klucel MF to the solution from step (iii).
6 3. OIL-IN WATER BUFFERED TOPICAL LOTION Ingredients % (W/W)
1,25-dihydroxyvitamin D.sub.3 0.0002-0.10 Cetyl Alcohol 0.25
Stearyl Alcohol 0.50 Sorbitan Monosterate 2.00 Glyceryl
Monostearate and 4.00 Polyoxyethylene Stearate Blend (Arlacel 165)
Polysorbate 60 1.00 Mineral Oil 4.00 Propylene Glycol 5.00
Butylated Hydroxyanisole 0.05 Propylparaben 0.05 Buffering Agent
q.s. to pH 7.00 Sorbitol Solution 2.00 Edetate Disodium 0.10
Methylparaben 0.18 Water q.s. to 100.00
[0071] Manufacturing Procedure
[0072] i. Prepare the buffer solution (pH 7.0) in a stainless steel
container.
[0073] ii. In a stainless steel vessel, at 70.degree. C. , melt the
cetyl alcohol, stearyl alcohol, sorbitan monostearate, Arlacel 165,
Polysorbate 60, mineral oil, butylated hydroxyanisole,
propylparaben, and 50% propylene glycol together.
[0074] iii. Add the sorbitol solution to step (i) and heat the
solution to 70.degree. C.
[0075] iv. Add the edetate disodium and methylparaben to the
solution from step (iii).
[0076] v. Dissolve the 1,25-dihydroxyvitamin D.sub.3 in
approximately 40% propylene glycol in a beaker and add this to the
material from step (ii) while mixing. Rinse the container from 10%
propylene glycol and add this to the mixture from step (ii).
[0077] vi. Add step (v) to step (iv) when both phases are at
70.degree. C. and homogenize. Cool the emulsion to 200 m
temperature.
7 4. TOPICAL GEL Ingredients % (W/W) 1,25-dihydroxyvitamin D.sub.3
0.0002-0.10 Butylated Hydroxytoluene (BHT) 0.05 Butylated
Hydroxyanisole (BHA) 0.05 Hydroxypropyl Cellulose 3.00 Ethyl
Alcohol, 95% Proof 50.00 Water q.s. to 100.00
[0078] Manufacturing Procedure
[0079] i. Weigh the ethyl alcohol and water in a stainless steel
container.
[0080] ii. Dissolve the 1,25-dihydroxyvitamin D.sub.3, BHT and BHA
to the solution from step (i).
[0081] iii. Dissolve the hydroxypropyl cellulose to the solution
from step (ii).
[0082] The entire contents of all references cited above are
incorporated herein by reference.
[0083] While the present invention has been described in some
detail for purposes of clarity and understanding, one skilled in
the art will appreciate that various changes in form and detail can
be made without departing from the true scope of the invention.
* * * * *