U.S. patent application number 13/576747 was filed with the patent office on 2013-03-14 for method of treatment or prevention of hair loss or for the enhancement of hair growth.
The applicant listed for this patent is Maria Halasz, Darren Jones, Sadatoshi Sakuma. Invention is credited to Maria Halasz, Darren Jones, Sadatoshi Sakuma.
Application Number | 20130067604 13/576747 |
Document ID | / |
Family ID | 44506072 |
Filed Date | 2013-03-14 |
United States Patent
Application |
20130067604 |
Kind Code |
A1 |
Sakuma; Sadatoshi ; et
al. |
March 14, 2013 |
Method of Treatment or Prevention of Hair Loss or for the
Enhancement of Hair Growth
Abstract
The present application relates to use of a Midkine family
protein for growing hair on a mammal, or in the manufacture of a
medicament for growing hair on a mammal, especially for treatment
or prevention of different forms of alopecia.
Inventors: |
Sakuma; Sadatoshi;
(Yokohama, JP) ; Halasz; Maria; (Sydney, AU)
; Jones; Darren; (Avalon, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sakuma; Sadatoshi
Halasz; Maria
Jones; Darren |
Yokohama
Sydney
Avalon |
|
JP
AU
AU |
|
|
Family ID: |
44506072 |
Appl. No.: |
13/576747 |
Filed: |
February 23, 2011 |
PCT Filed: |
February 23, 2011 |
PCT NO: |
PCT/AU2011/000194 |
371 Date: |
November 21, 2012 |
Current U.S.
Class: |
800/3 ;
514/7.6 |
Current CPC
Class: |
A61K 38/18 20130101;
A61K 2800/59 20130101; A61K 49/0004 20130101; A61P 17/14 20180101;
A61K 49/0008 20130101; A61Q 7/00 20130101; A61K 8/64 20130101 |
Class at
Publication: |
800/3 ;
514/7.6 |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61K 49/00 20060101 A61K049/00; A61P 17/14 20060101
A61P017/14 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 24, 2010 |
AU |
2010900771 |
Claims
1. A topical formulation comprising an amount of a midkine family
protein and a topical carrier, excipient or emollient effective to
treat or prevent hair loss and/or promote hair growth and/or
enhance hair growth.
2. The topical formulation of claim 1 wherein said formulation is
for administration to the dermis or skin of a subject who is
susceptible to hair loss or at risk of hair loss.
3. The topical formulation of claim 1 wherein said formulation is
for administration to the dermis or skin of a subject suffering
from hair loss.
4. The topical formulation according to claim 1 for treatment or
prevention of alopecia.
5. The topical formulation according to claim 4, wherein the
alopecia is an acute form of alopecia.
6. The topical formulation according to claim 4 wherein the
formulation is for treatment or prevention of alopecia in a subject
undergoing treatment with a cytotoxic agent or cytostatic agent or
to whom treatment with a cytotoxic agent or cytostatic agent has
been prescribed.
7. The topical formulation according to claim 4, wherein the
alopecia is androgenic alopecia.
8. The topical formulation according to claim 1, wherein the
midkine family protein is midkine, pleiotrophin, midkine-like
protein, or a truncated midkine protein.
9. The topical formulation according to claim 1, wherein the
midkine family protein is pleiotrophin.
10. The topical formulation according to claim 1, wherein the
midkine family protein is midkine.
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. A method of treatment or prevention of hair loss, said method
comprising administering to a subject in need thereof a formulation
comprising an amount of a midkine family protein and a carrier,
excipient or emollient, effective to prevent hair loss and/or
promote hair growth and/or enhance hair growth on the subject.
20. The method according to claim 19, wherein the formulation is a
topical formulation and wherein said method comprises administering
the topical formulation to an affected area of the skin of the
subject in which hair has been lost or to an area of skin from
which hair is likely to be lost.
21. The method according to claim 19, wherein the method comprises
administering the formulation to a subject suffering from alopecia
for a time and under conditions sufficient to reduce hair loss
and/or effect hair growth in the subject.
22. The method according to claim 19, wherein the method comprises
administering the formulation to a subject suffering from alopecia
for a time and under conditions sufficient to prevent or reduce
apoptosis of follicles of the subject.
23. The method according to claim 19, wherein the method comprises
administering the formulation to a subject suffering from alopecia
for a time and under conditions sufficient to extend an anagen
phase of follicles of the subject.
24. The method according to claim 19, wherein the method comprises
administering the formulation to a subject suffering from alopecia
for a time and under conditions sufficient to promote or advance
entry of normal telogen follicles of the subject into a following
hair cycle.
25. The method according to claim 19, wherein the method comprises
administering the formulation to a subject that is suffering from
alopecia.
26. The method according to claim 1, wherein the method comprises
administering the formulation to a subject that is susceptible to
developing alopecia.
27. The method according to claim 25, wherein the alopecia is an
acute form of alopecia.
28. The method according to claim 27, wherein the method comprises
administering the formulation to a subject undergoing treatment
with a cytotoxic agent or cytostatic agent or to whom treatment
with a cytotoxic agent or cytostatic agent has been prescribed.
29. The method according to claim 25, wherein the alopecia is
androgenic alopecia.
30. The method according to claim 19, wherein the midkine family
protein is midkine, pleiotrophin, midkine-like protein, or a
truncated midkine protein.
31. The method according to claim 19, wherein the midkine family
protein is pleiotrophin.
32. The method according to claim 19, wherein the midkine family
protein is midkine.
33. A method of promoting or enhancing hair growth or hair
initiation in a subject suffering from hair loss, said method
comprising administering to the subject a topical formulation
comprising an amount of a midkine family protein and a topical
carrier, excipient or emollient effective to prevent hair loss
and/or promote hair growth and/or enhance hair growth on the
subject.
34. The method according to claim 33, wherein the midkine family
protein is midkine or pleiotrophin.
35. A method of reducing hair loss in a subject undergoing
chemotherapy or to whom chemotherapy has been prescribed, said
method comprising administering to the subject a formulation
comprising an amount of a midkine family protein and a carrier,
excipient or emollient for a time and under conditions sufficient
to prevent or reduce hair loss due to the chemotherapy.
36. The method according to claim 35, wherein the method comprises
administering the formulation topically to a subject that has been
treated with the chemotherapy.
37. The method according to claim 35, wherein the method comprises
administering the formulation topically to a subject to whom
chemotherapy has been prescribed before commencement of the
chemotherapy.
38. The method according to claim 35, wherein the method comprises
administering the formulation topically to a subject to whom
chemotherapy has been prescribed before and after commencement of
the chemotherapy.
39. The method according to claim 35, wherein the midkine family
protein is midkine.
40. A method of identifying or isolating a compound that prevents
hair loss and/or promotes hair growth and/or enhances hair growth,
wherein said method comprises administering a test compound to a
mouse, said mouse being aged more than one year and suffering hair
loss and having a knockout of one allele of a gene encoding a
midkine protein and one other functional allele of the gene, and
then determining hair growth and/or reduced hair loss on the mouse,
and wherein reduced hair loss and/or increased hair growth and/or
an initiation of hair growth on the mouse in the presence of the
test compound relative to in the absence of the test compound is
indicative of the test compound preventing hair loss and/or
promoting hair growth and/or enhancing hair growth.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of priority from
Australian Application No. 2010900771 filed 24 Feb. 2010, the
contents of which are incorporated in their entirety by
reference.
FIELD OF THE INVENTION
[0002] The present application relates to the filed of therapy and
prevention of hair loss or hair thinning in mammals, such as in
humans suffering from or having a propensity to develop alopecia,
and to the promotion of hair growth in mammals, such as in humans
suffering from or having a propensity to develop alopecia.
BACKGROUND TO THE INVENTION
Hair and Hair Development
[0003] Hair is integral to our body image and can have a profound
influence on our self-esteem and self-confidence. The hair of
non-human mammal species is commonly referred to as "fur". Unless
specifically stated otherwise, or the context requires otherwise,
the term "hair" as used herein shall be taken to include "fur". The
term "hair" shall also be taken to include hair on any part of a
mammalian body, including the eyebrow, edge of the eyelid, armpit,
and inside of the nostril, unless the context requires otherwise.
Thus, hair may include head hair, eyebrow hair, eyelash, cilia, or
other body hair.
[0004] Each hair comprises two structures: the shaft and the
follicle. The primary component of the hair shaft is keratin. The
hair shaft contains three layers of keratin, however the inner
layer i.e., the medulla, may not be present. The middle layer i.e.,
the cortex, makes up the majority of the hair shaft. The outer
layer i.e., the cuticle, is formed by tightly-packed scales in an
overlapping structure. Pigment cells are distributed throughout the
cortex and medulla giving the hair its characteristic color. The
follicle contains several layers. At the base of the follicle is a
projection called a papilla, which contains capillaries, or tiny
blood vessels, that feed the cells. The living part of the hair,
the area surrounding the papilla called the bulb, is the only part
fed by the capillaries. The cells in the bulb divide every 23 to 72
hours, faster than any other cells in the body. The follicle is
surrounded by an inner root sheath and an outer root sheath. These
two sheaths protect and mould the growing hair shaft. The inner
root sheath follows the hair shaft and ends below the opening of a
sebaceous (oil) gland, which produces sebum, and sometimes an
apocrine (scent) gland. The outer root sheath continues all the way
up to the sebaceous gland. An erector pili muscle attaches below
the sebaceous gland to a fibrous layer around the outer sheath.
When this muscle contracts; it causes the hair to stand up.
[0005] Human skin comprises two types of hair: vellus hair and
terminal hair. Vellus hair is short, fine, "peach fuzz" body hair.
It is a very soft, generally pale, and short hair that grows in
most places on the human body in both sexes. Vellus hair is
generally less than two centimetres in length, and the follicles
from which vellus hair grows are not connected to sebaceous glands.
It is observed most easily in those having less terminal hair to
obscure it, such as women and children, It also is found in
pre-adolescents and in males exhibiting male-pattern baldness.
Terminal or "androgenic" hair is developed hair, which is generally
longer, coarser, thicker and darker than vellus hair. Phases of
growth in terminal hair are also more apparent than in vellus hair,
by virtue of a generally-longer anagen phase. Terminal hair has
associated sebaceous glands. In puberty, some vellus hair may
develop into terminal hair. Under other conditions, such as male
pattern baldness, terminal hair may revert to a vellus-like
state
[0006] There are three sequential stages of hair growth: catagen,
telogen, and anagen. Anagen is the active growth phase of the hair
during which the cells in the root of the hair are dividing
rapidly. Anagen hairs are anchored deeply into the subcutaneous fat
and cannot be pulled out easily. When a new hair is formed, it
pushes the club hair up the follicle, and eventually out. During
this phase, the hair grows about 1 cm every 28 days. Scalp hair
stays in this active phase of growth for 2-6 years. Human subjects
that have difficulty growing their hair beyond a certain length may
have a shortened anagen phase, whereas those having an ability to
grow longer hair quickly may have a longer anagen phase. In humans,
the hair on the arms, legs, eyelashes, and eyebrows generally has a
short anagen compared to head or scalp hair. The catagen phase is a
transitional stage that lasts for about 2-3 weeks in humans, during
which time growth stops, thereby forming "club" hair. Telogen is a
resting phase, lasting for about 100 days for scalp hair and much
longer for other body hair. During telogen, the hair follicle is at
rest, the club hair is formed, and compared to hair in anagen, the
hair in telogen is located higher in the skin and can be pulled out
readily. The root of telogen hair comprises a visible solid, hard,
dry, and white material. Shedding of telogen hair is normal, and up
to 75 hairs in telogen are shed from the human scalp daily. The
shed hairs are normally replaced as about the same number of
follicles enter anagen daily. At any time in normal scalp,
approximately 80% to 90% of follicles are in anagen, about 1% to 3%
are in catagen i.e., undergoing involution, and about 5% to 10% are
in telogen.
Conditions of Hair Loss and/or Reduced Hair Growth
[0007] Hair loss or hair thinning includes any condition that
results in a reduced ability to replace shed hairs or that results
in enhanced shedding without their concomitant or subsequent
replacement e.g., brittle hair growth, thin hair growth, short hair
growth, sparse hair growth, alopecia, or hair de-pigmentation. For
example, the hair cycle can become uncontrolled leading to
accelerated hair loss, which may be temporary or permanent. As used
herein, the term "alopecia" is used to refer to hair loss, unless
specifically stated otherwise or the context requires
otherwise.
[0008] Alopecia can have various causes. Hereditary androgenic
alopecia is the commonest form of alopecia: it is manifested by a
decrease in hair volume, or even baldness, and affects up to about
70% of men. Acute alopecia may be associated with treatment by
chemotherapy, stress, severe malnutrition, iron deficiency,
hormonal disorders, AIDS, or acute irradiation. Alopecia areata,
which seems to be of auto-immune origin (mechanism of cellular
mediation), is characterized by "patches" of varying size in one or
more body places. Alopecia totalis refers to a form of alopecia
areata that extends over the entire scalp, and alopecia universalis
refers to a form of alopecia areata that extending over the whole
body. Mechanistically, in all forms of alopecia, hair loss is
directly-related to a reduced ability, slowing or failure of the
follicle to enter the anagen phase, or a failure to maintain the
follicle in the anagen phase, such that formation of a hair shaft
reduces, is slowed or ceases altogether. Hair may move into the
catagen phase before sufficient growth is achieved in the anagen
phase, thus becoming in a sustained manner short and thin (i.e.
"hair thinning"). Chemotherapeutic agents, radiotherapeutic agents,
and other medicinal products may induce necrosis or apoptosis of
the follicle as a side-effect of the therapy, also preventing the
follicle to enter anagen. For example, alkylating agents e.g.,
temozolomide, busulfan, ifosamide, melphalan hydrochloride,
carmustine, lomustine or cyclophosphamide, and antimetabolites
e.g., 5-fluorouracil, capecitabine, gemcitabine, floxuridine,
decitabine, mercaptopurine, pemetrexed disodium, methotrexate or
dacarbazine, and natural products e.g., vincristine, vinblastine,
vinorelbine tartrate, paclitaxel, docetaxel, ixabepilone,
daunorubicin, epirubicin, doxorubicin, idarubicin, mitoxantrone,
mitomycin, dactinomycin, irinotecan, topotecan, etoposide,
teniposide, etoposide phosphate, or bleomycin sulfate, and
biologics e.g., filgrastim, pegfilgrastim, bevacizumab,
sargramostim or panitumumab, and hormones or hormone-related agents
e.g., megestrol acetate, fluoxymesterone, leuprolide, octreotide
acetate, tamoxifen citrate or fluxymesterone, and other therapeutic
agents e.g., sorafenib, erlotinib, oxaliplatin, dexrazoxane,
anagrelide, isotretinoin, bexarotene, vorinostat, adriamycin,
cytoxan, taxol, leucovorin, oxaliplatin, and combinations of the
foregoing agents are known to induce temporary or permanent
alopecia.
[0009] Alopecia caused by any of the foregoing factors is a source
of low self-esteem and anxiety for many patients. For those
undergoing chemotherapy or radiation therapy for cancer, alopecia
adds to discomfort from other adverse side-effects of the therapy
e.g., nausea, skin rash, etc. Many alopecia sufferers, including
patients receiving chemotherapy, choose to use wigs, hair pieces,
scarves, hats or turbans to cover their bald or thinning regions.
Those suffering from hair loss often experience embarrassment and
fear being ridiculed by others because they look different. Some
may take to wearing oversized eyeglasses in an attempt to hide the
absence of eyelashes and/or eyebrows. In some subjects, alopecia
may lead to depression.
Animal Models of Alopecia
[0010] There are several useful models of alopecia in humans, that
have been acknowledged in the art for use in testing efficacy of
alopecia remedies and other hair growth-promoting therapies.
[0011] For example, the stumptailed macaque possesses hereditary
balding characteristics similar in many respects to that of
androgenic alopecia in humans, is used to obtain a morphometric
assessment of the rate of cyclic change of the hair follicle,
including rates of cyclic progression (resting to regrowing phase,
and regrowing to late anagen phase) and overall changes in
follicular size. These primates are also reasonably good predictors
of compound efficacy, and for example, have been employed to test
efficacy of minoxidil on androgenic alopecia. Cessation of topical
minoxidil treatment resulted in a renewal of the balding process,
with folliculograms demonstrating increases in the proportion of
resting follicles. This withdrawal from treatment apparently had no
effect on hair regrowth during subsequent reapplications of
minoxidil. Such treatment resulted in regrowth similar to that in
the first treatment phase. Continuous treatment of topical
minoxidil for 4 years has not resulted in systemic or local side
effects in these animals. See e.g., Brigham et al., Clin. Dermatol.
6, 177-187,1998; Sundberg et al., Exp. Mol. Pathol. 67, 118-130
(1999), the contents of which are incorporated herein by reference
in their entirety).
[0012] Collectively, the findings obtained from studies on mouse
models support the concept of alopecia areata as an autoimmune
disease, and several rodent models with spontaneous and induced
alopecia areata have been identified. For example, the Dundee
Experimental Bald Rat (DEBR) was the first rodent model validated
that developed spontaneous alopecia areata and is utilized to
identify candidate alopecia areata susceptibility gene loci (Michie
et al., Br J Dermatol., 125, 94-100, 1991, incorporated herein by
reference). The most extensively-characterized and
readily-accessible alopecia areata model is the C3H/HeJ mouse model
(Sundberg et al., J Invest Dermatol., 102, 847-56, 1994,
incorporated herein by reference). Aging C3H/HeJ mice (females at
3-5 months of age or older and males at more than 6 months of age)
develop histopathological and immunohistochemical features of human
alopecia areata. Alopecia develops diffusely or in circular areas
on the dorsal surface of sufficiently-aged animals. Histologically,
the changes in this non-scarring alopecia appear limited to anagen
follicles surrounded by mononuclear cells composed primarily of
cytotoxic or cytostatic (CD8+) and helper (CD4+) T cells, this is
associated with follicular and hair shaft dystrophy. Pedigree
tracing of affected C3H/HeJ mice suggests that this non-scarring
alopecia may be an inherited and complex polygenic disease with a
female predominance at younger ages. C3H/HeJ mice with alopecia
areata can be used to study the efficacy of current treatments of
alopecia areata, to study the effectiveness and safety profile of
new treatment forms in established alopecia areata, and to assess
the influence of various factors on the development of alopecia
areata in order to prevent the onset of the disease.
[0013] Paus et al., Am. J. Pathol. 144, 719-734 (1994) have also
described a rodent model of acute alopecia, the entire content of
which is incorporated herein by reference. In this model, alopecia
is induced by a single intraperitoneal injection of
cyclophosphamide to C57 BL/6 mice. In depilated C57 BL/6 mice, the
hair follicles are synchronized to anagen. By day 9 after
depilation, all follicles are mature anagen VI follicles, and the
skin is characterized by grey-to-black coloured hair shafts.
Histologically, macroscopically, and functionally,
depilation-induced anagen VI follicles are indistinguishable from
spontaneously-developing anagen follicles. Around day 16 after
depilation, follicle regression occurs without loss of hair shafts
in the depilated animals, and skin colour converts from black to
pink, indicating both induction of catagen and cessation of
melanogenesis. The development of catagen follicles is indicated
macroscopically by a change in skin color from black to light grey,
and occurs in large waves appearing in the neck region first and
then the flanks and tail regions. At day 20 after depilation, all
follicles enter telogen again, characterized by change in skin
color from grey to pink. When cyclophosphamide is administered to
C57 BL/6 mice on day 9 after depilation, the animals show rapid and
reproducible visible signs of acute alopecia dose-dependent,
including significant loss of fur and premature termination of
anagen characterized by depigmentation leading to a grey skin
appearance by day 12-14. Thus, follicles of the neck region are
generally in catagen 3-5 days after cyclophosphamide treatment.
Hair shafts on the backs of animals are also removed easily by
rubbing at days 12-14, and by day 15, as much as 60% of the dorsal
surface may be exhibit alopecia. The color change and alopecia
induced by cyclophosphamide reflect the induction of dystrophic
forms of anagen and catagen in anagen VI follicles. In
cyclophosphamide-treated animals, follicles also progress to
telogen rapidly, as evidenced by pink skin, and rapid loss of fur
due to damage of the hair follicle. Telogen is shortened following
cyclophosphamide treatment, and normal telogen hair follicles enter
the next hair cycle, so that animals develop new hair shafts on
days 16-20 i.e., within about 7-10 days following treatment. These
new hair shafts are often de-pigmented due to the presence of
dystrophic anagen follicles that have not had time to produce new,
normally-pigmented hair shafts. Later, pigmented hair shafts
develop.
Therapy for Conditions of Hair Loss and/or Reduced Hair Growth
[0014] Existing therapies for alopecia include topical minoxidil
and derivatives thereof e.g., U.S. Pat. Nos. 4,139,619 and
4,596,812, and European Pat. Nos. EP-0353123, EP-20 0356271,
EP-0408442, EP-0522964, EP-0420707, EP-0459890 and EP-0519819,
spironolactone, cyproterone acetate, flutamide, finasteride,
progesterone or estrogen. Anti-androgen agents such as finasteride
and minoxidil are known for treating androgenic alopecia. None of
these treatments is broadly applicable. For example, such
treatments may not prevent hair loss during treatment with a
chemotherapeutic agent. On the other hand, such compounds may
produce undesirable side-effects. For example, minoxidil is a
potent vasodilator. Patients may also require frequent dosing with
such compounds to achieve an effective outcome. For example,
minoxidil provides very transient effects, because cessation of
topical minoxidil treatment results generally in a renewal of the
balding process, with folliculograms demonstrating increases in the
proportion of resting follicles. Minoxidil is also recommended for
administration twice-daily at 2% concentration. Notwithstanding
that finasteride provides an advance over minoxidil in being
deliverable orally, and is considered to be the best treatment
available, about 35% or more of balding male recipients show poor
or no response to that drug. Finasteride may also produce
significant side-effects for some users, as a number of male users
have reported erectile dysfunction, impotence, low libido, or
gynecomestica after using that drug. In those males suffering such
side-effects, the side effects may not disappear after ceasing
finasteride.
[0015] Various prostaglandin analogs have also been disclosed for
use in treatment of androgenic alopecia e.g., travoprost,
voprostol, and these may also require frequent administration e.g.,
at least daily, however single dosages of travoprost have been
described e.g., U.S. Pat. Publication 20100190853. Prostaglandin
analogs are also known for use in treatment of alopecia associated
with chemotherapy e.g., U.S. Pat. Publication 20110002286.
Prostaglandin analogs may have a variety of adverse effects e.g.,
muscular constriction mediating inflammation, calcium movement,
hormone regulation and cell growth control.
Midkine Family Proteins
[0016] Midkine (MK) is a growth/differentiation factor that was
first discovered as a gene product expressed transiently in the
process of differentiation induction of embryonic tumor cells (EC)
with retinoic acid. MK is known to produce a broad range of adverse
and beneficial biological effects. The expression of MK is
increased in human cancer cells, including esophageal cancer,
thyroid cancer, urinary bladder cancer, colorectal cancer, gastric
cancer, pancreatic cancer, chest cancer, liver cancer, lung cancer,
breast cancer, neuroblastoma, neuroblastoma, glioblastoma, uterine
cancer, ovarian cancer, and Wilms' tumor, and is believed to
promote the survival and migration of cancer cells and to
facilitate neovascularization. MK is also known to promote the
migration of inflammatory cells such as macrophages and neutrophil,
leading to inflammation. MK is also known to stimulate the
proliferation of cultured endometrial interstitial cells during
endometriosis. Thus, MK inhibitors e.g., antibodies, aptamers or
RNAi targeting MK protein or RNA, have been disclosed for use in
treatment of a broad range of inflammatory diseases such as
arthritis, autoimmune disease, rheumatic arthritis, osteoarthritis,
multiple sclerosis, postoperative adhesion, inflammatory colitis,
psoriasis, lupus, asthma, neutrophil functional abnormalities, and
endometriosis.
[0017] Beneficial effects of MK are also known, wherein MK is also
involved in promoting the formation of nascent intima following
blood vessel damage and the onset of nephritis in an ischemic
event, and in reducing postoperative adhesions in rheumatic
subjects. Thus, MK protein has been disclosed for use in treatment
of cerebral ischemia, cardiac ischemia, restenosis following
vascular reconstruction surgery, cardiac coronary arterial vascular
obstructive disease, cerebral vascular obstructive disease, renal
vascular obstructive disease, peripheral vascular obstructive
disease, and arteriosclerosis.
[0018] Pleiotrophin (PTN or HB-GAM) is a midkine family protein
having approximately 50% identity at the amino acid sequence level
to MK. Both MK and PTN comprise a high content of cysteine and
basic residues. All the 10 cysteine residues are conserved in MK
and PTN, and structurally, both can be divided into the N-domain
and the C-domain. As a result of NMR analysis, it is known that
these two molecules have very similar three-dimensional structures.
Each domain consists of three f3 sheets, connected via a flexible
linker region. K79, R81, and K102, considered to be important to
the binding of to chondroitin sulfate and heparin, are conserved
between the two proteins. MK and PTN also share three-dimensional
structures wherein these basic residues appear in the vicinity of
the protein surface. Accordingly, PTN has been disclosed previously
for the same medical indications as MK.
[0019] It is to be understood that, if any prior art publication is
referred to herein, such reference does not constitute an admission
that the publication forms a part of the common general knowledge
or background art in Australia or elsewhere.
SUMMARY OF THE INVENTION
[0020] In work leading up to the present invention, the inventor
sought to develop protein-based reagents, e.g., for topical
administration to a subject, that enhance hair growth or the
initiation of hair growth in subjects that are susceptible to hair
loss or suffer hair loss. The inventor also sought to develop an
effective topical protein-based formulation that can be applied
readily to the skin and exert an effect notwithstanding the barrier
that the dermis generally provides to external agents.
[0021] The inventor developed an animal model of hair loss
consisting essentially of an aged mouse that suffers hair loss
e.g., in the form of bald regions or alopecic sites. The inventor
found that topical application of midkine or pleotrophin protein to
the animal model induced hair growth or the recommencement of hair
growth in these bald regions or alopecic sites. Topical
formulations of midkine or pleiotrophin were found to be superior
to parenteral formulations in producing these hair growth promoting
effects in this animal model.
[0022] The data provided herein also show that, in contrast to
minoxidil, midkine and pleiotrophin failed to stimulate hair growth
visibly in healthy animals that have been shaved to synchronize
their follicle in anagen, and exhibited no clinical symptoms of
alopecia. This suggests that midkine and pleiotrophin may act by a
more specific mechanism than minoxidil that does not comprise a
vasodilatory effect of the dermal papillae. Thus, fur regrowth
stimulated by a midkine family protein may arise from an
anti-apoptotic effect of midkine on follicles to thereby extend
their anagen phase, or alternatively, or the re-entry of normal
telogen follicles into the next hair cycle.
[0023] In further work leading to the present invention, the
applicant utilized the accepted animal model of
chemotherapy-induced alopecia (CIA) described e.g., by Paus et al.,
Am. J. Pathol. 144, 719-734 (1994), the contents of which are
incorporated herein in their entirety. The applicant performed a
randomised trial in which all animals were shaved and waxed, and
five groups of animals were administered daily with a topical
formulation of midkine protein for 7 days before and 19 days
following i/p injection of cyclophosphamide, and another two groups
of animals were administered daily with a topical formulation of
midkine protein for 19 days following i/p injection of
cyclophosphamide. Control mice received a saline placebo. Data
presented herein indicate that topical administration of a midkine
family protein e.g., MK and/or PTN, before chemotherapy, or before
and after chemotherapy, significantly reduces the severity of fur
loss i.e., promotes fur growth at all time points following
administration of the chemotherapeutic agent. Treatment with a
midkine family protein also retains a greater proportion of
follicles in anagen following treatment with cyclophosphamide, as
evidenced by more animals having black or dark grey skin when
treated with midkine family protein than for the group receiving a
placebo. This suggests an anti-apoptotic effect on anagen
follicles. These beneficial effects are detectable in the period
immediate following chemotherapy when follicles have not yet
recovered, and during later follicle recovery after chemotherapy
has ceased. Data presented herein also indicate that topical
administration of a midkine family protein e.g., MK and/or PTN,
following chemotherapy at least improves recovery of follicles, as
demonstrated by a reduced incidence of alopecia compared to
subjects receiving a placebo. Thus, animals treated daily with
topical midkine protein following chemotherapy also lose less hair
in total than animals not receiving treatment, and the reduced hair
loss is nearly as effective as in animals treated with midkine
protein before and after chemotherapy. Subjects receiving midkine
therapy before or after chemotherapy also exhibit a higher rate of
hair growth during the follicle recovery period, suggesting an
anti-apoptotic effect of midkine on follicles to thereby extend
their anagen phase, or alternatively, enhanced re-entry of normal
telogen follicles into the next hair cycle following treatment with
midkine.
[0024] It is to be understood that the methods described herein for
the aged mouse model of hair loss are transferable to testing in
other animal models of alopecia of humans, including the
stump-tailed macaque 21-23 model of androgenic alopecia and/or the
hairless (hr/hr) model of papular atrichia, as described
herein.
[0025] The data provided herein therefore support the use of
compositions comprising a midkine family protein in promoting hair
growth and/or reducing hair loss and/or reducing hair thinning
and/or preventing hair loss and/or preventing hair thinning and/or
delaying hair loss and/or delaying hair thinning e.g., in aging
subjects, subjects suffering from alopecia such as androgenic
alopecia or acute alopecia. Typically, effective compositions f the
invention for treatment and/or prevention of alopecia are
formulated for topical administration, however parenteral
formulations may also be employed, such as for co-administration
with a cytotoxic or cytostatic agent of acute alopecia that is
gendrally administered parenterally e.g., a chemotherapeutic
agent.
[0026] Accordingly, one example of the present invention provides a
formulation e.g., a topical formulation, comprising an amount of a
midkine family protein, such as midkine, pleiotrophin or a variant
or homolog of midkine or pleiotrophin, and a topical carrier,
excipient or emollient, effective to prevent hair loss and/or
promote hair growth and/or enhance hair growth. By "topical
formulation" is meant that the formulation is capable of being
applied externally to the dermis of a mammal, or is applied to the
dermis. For example, the formulation may be useful for
administration to the dermis or skin of a male subject who is
susceptible to hair loss or at risk of hair loss, such as a subject
suffering from or at risk of developing male-pattern baldness. In
another example, the formulation of the invention is useful for
administration to the dermis or skin of a subject suffering from a
disease or condition associated with hair loss e.g., alopecia,
especially acute alopecia or androgenic alopecia.
[0027] In one example, the topical formulation comprises an amount
of a midkine family protein and a topical carrier, excipient or
emollient effective to treat or prevent hair loss and/or promote
hair growth and/or enhance hair growth. Preferably, the formulation
is for administration to the dermis or skin of a subject who is
susceptible to hair loss or at risk of hair loss, or for
administration to the dermis or skin of a subject suffering from
hair loss. It will be appreciated that the topical formulation is
suitable for treatment or prevention of alopecia e.g., an acute
form of alopecia or androgenic alopecia. For example, the
formulation may be used for treatment or prevention of alopecia in
a subject undergoing treatment with a cytotoxic agent or cytostatic
agent or to whom treatment with a cytotoxic agent or cytostatic
agent has been prescribed. The midkine family protein may be
midkine, pleiotrophin, midkine-like protein, or a truncated midkine
protein.
[0028] The present invention thus provides improved topical
formulations comprising a therapeutically-effective amount of a
midkine family protein e.g., an effective amount of a midkine
protein or pleiotrophin protein or biologically-active variant
thereof to enhance hair growth. The topical formulations of the
invention are particularly useful for the prevention or treatment
of alopecia, including androgenic alopecia, acute alopecia,
alopecia areata, alopecia totalis and alopecia universalis. The
topical formulations of the invention are preferably for treatment
of androgenic alopecia. The topical formulations of the invention
are even more preferably for the treatment of acute alopecia in
subjects undergoing therapy with a cytotoxic or cytostatic compound
that causes hair loss, especially a chemotherapeutic agent,
radiotherapeutic agent, or antiviral compound such as administered
to a subject infected with HIV-1. The topical formulations of the
invention are also preferably for the prevention of acute alopecia
in subjects about to undergo therapy with a cytotoxic or cytostatic
compound that causes hair loss, especially a chemotherapeutic
agent, radiotherapeutic agent, or antiviral compound such as
administered to a subject infected with HIV-1.
[0029] It is to be understood that the topical formulations of the
invention are useful for the stimulation of hair growth in subjects
suffering from or having a predisposition to develop androgenic
alopecia or male pattern baldness, and/or in subjects about to
undergo therapy with a cytotoxic or cytostatic compound that causes
hair loss, or undergoing such therapy.
[0030] It is also to be understood that the topical formulations of
the present invention are compatible with various types of
therapeutic agents or carriers with which they are combinable or
capable of being administered sequentially or simultaneously or
concomitantly. For example a topical formulation comprising a
midkine family protein may further comprise a second compound for
treatment of the same condition and/or be co-administered with such
second compound(s). In such circumstances, the efficacy of the
midkine family protein is supplemented by the action of the second
compound. For example, the midkine family protein is
co-administered with cestradiol and/or oxandrolone and/or minoxidil
and/or finasteride or an agent that blocks the conversion of
testosterone to dihydrotesterone. Alternatively, or in addition,
the topical formulation of the invention is co-administered with a
cytotoxic or cytostatic compound that causes hair loss e.g., in the
case of a subject undergoing chemotherapy or radiation therapy or
treatment for HIV-1 infection or AIDS. such circumstances, the
efficacy of the midkine family protein counteracts the hair-loss
effect of the cytotoxic or cytostatic compound.
[0031] Another example of the present invention provides for the
use of a midkine family protein in the manufacture of a medicament
e.g., a topical medicament for preventing and/or treating hair loss
in a subject suffering from alopecia or having a propensity to
develop alopecia. The medicament may be for administration to a
section of the population that is susceptible to hair loss or
suffers from hair loss, such as a subject suffering from or at risk
of developing male-pattern baldness. For example, the subject may
have no visible symptoms of alopecia, however suffers from a
genetic condition that predisposes the Subject to alopecia, or the
subject may be about to undergo therapy with a cytotoxic or
cytostatic agent or antiviral compound that induces alopecia. In
another example, the topical medicament is useful for
administration to the dermis or skin of a subject suffering from a
disease or condition associated with hair loss e.g., alopecia. For
example, the subject may have a pre-existing alopecia or be
undergoing therapy with a cytotoxic or cytostatic agent or
antiviral compound that induces alopecia whether or not visible
symptoms of alopecia have developed before midkine family protein
therapy is commenced. The midkine family protein is especially
useful in manufacture of a medicament for treatment or prevention
of acute alopecia in a subject undergoing chemotherapy, and such a
medicament may be formulated for administration by a topical or
parenteral route, however topical formulations are preferred.
[0032] By "topical medicament" is meant that the active agent i.e.,
midkine family protein, such as midkine, pleiotrophin or a variant
or homolog of midkine or pleiotrophin, is formulated with a topical
carrier, excipient or emollient for application to the dermis of a
mammal.
[0033] In one particularly preferred example, the use of a midkine
family protein is in the manufacture of a medicament for treatment
or prevention of alopecia, e.g., an acute form of alopecia or
androgenic alopecia, in a human or other mammalian subject. The
subject may be a human or mammalian subject a subject undergoing
treatment with a cytotoxic agent or cytostatic agent or to whom
treatment with a cytotoxic agent or cytostatic agent has been
prescribed. The medicament may be a formulation for topical
application. The midkine family protein may be midkine,
pleiotrophin, midkine-like protein, or a truncated midkine
protein.
[0034] Another example of the present invention provides a method
of treatment or prevention of hair loss, said method comprising
administering to a subject in need thereof e.g., a subject
suffering from alopecia or having a propensity to develop alopecia,
a formulation, e.g., a topical formulation, comprising an amount of
a midkine family protein, such as midkine, pleiotrophin or a
variant or homolog of midkine or pleiotrophin, and a topical
carrier, excipient or emollient, effective to prevent hair loss
and/or promote hair growth and/or enhance hair growth on the
subject. The subject will generally be a mammal such as a human.
For example, the method of the invention is useful for the
treatment of male subjects who are susceptible to or at risk of
hair loss such as in male-pattern baldness. Such subjects may have
no visible symptoms of alopecia, however suffers from a genetic
condition that predisposes the subject to alopecia, or the subject
may be about to undergo therapy or be undergoing therapy with a
cytotoxic or cytostatic agent or antiviral compound, that induces
alopecia. In another example, the method of the invention is useful
for the treatment of a subject suffering from a disease or
condition associated with hair loss e.g., alopecia. Such subjects
may have a pre-existing alopecia or be undergoing therapy with a
cytotoxic or cytostatic agent or antiviral compound that induces
alopecia wherein symptoms of alopecia have developed. The treatment
and prevention of subjects undergoing chemotherapy or to whom
chemotherapy has been prescribed is clearly encompassed by the
inventive method.
[0035] Another example of the present invention provides a method
of promoting or enhancing hair growth or hair initiation, such as
during or following therapy with a cytotoxic or cytostatic agent or
antiviral compound that induces alopecia, said method comprising
administering to a subject in need thereof a formulation e.g., a
topical formulation comprising an amount of a midkine family
protein, such as midkine, pleiotrophin or a variant or homolog of
midkine or pleiotrophin, and a topical carrier, excipient or
emollient, effective to prevent hair loss and/or promote hair
growth and/or enhance hair growth on the subject.
[0036] In another example, the present invention provides a method
of treatment or prevention of hair loss, said method comprising
administering to a subject in need thereof, e.g., a subject
suffering from alopecia such as an acute form of alopecia or
androgenic alopecia or susceptible to developing said alopecia, a
formulation comprising an amount of a midkine family protein and a
carrier, excipient or emollient, effective to prevent hair loss
and/or promote hair growth and/or enhance hair growth on the
subject. Preferably, the formulation is a topical formulation and
said method comprises administering the topical formulation to an
affected area of the skin of the subject in which hair has been
lost or to an area of skin from which hair is likely to be lost.
The formulation may be administered to a subject suffering from
alopecia for a time and under conditions sufficient to reduce hair
loss and/or effect hair growth in the subject. Alternatively, or in
addition, formulation may be administered to a subject suffering
from alopecia for a time and under conditions sufficient to prevent
or reduce apoptosis of follicles of the subject. Alternatively, or
in addition, formulation may be administered to a subject suffering
from alopecia for a time and under conditions sufficient to extend
an anagen phase of follicles of the subject. Alternatively, or in
addition, formulation may be administered to a subject suffering
from alopecia for a time and under conditions sufficient to promote
or advance entry of normal telogen follicles of the subject into a
following hair cycle. The method according to the invention is
particularly useful for subjects undergoing treatment with a
cytotoxic agent or cytostatic agent or to whom treatment with a
cytotoxic agent or cytostatic agent has been prescribed. In a
preferred example, the midkine family protein may be midkine,
pleiotrophin, midkine-like protein, or a truncated midkine
protein.
[0037] In another example, the invention provides a method of
promoting or enhancing hair growth or hair initiation in a subject
suffering from hair loss, said method comprising administering to
the subject a topical formulation comprising an amount of a midkine
family protein and a topical carrier, excipient or emollient
effective to prevent hair loss and/or promote hair growth and/or
enhance hair growth on the subject. Preferably, the midkine family
protein is midkine or pleiotrophin.
[0038] In another example, the invention provides a method of
reducing hair loss in a subject undergoing chemotherapy or to whom
chemotherapy has been prescribed, said method comprising
administering to the subject a formulation comprising an amount of
a midkine family protein and a carrier, excipient or emollient for
a time and under conditions sufficient to prevent or reduce hair
loss due to the chemotherapy. Preferably, the method comprises
administering the formulation topically to a subject that has been
treated with the chemotherapy or to whom chemotherapy has been
prescribed before commencement of the chemotherapy. In one example,
the method comprises administering the formulation topically to a
subject to whom chemotherapy has been prescribed before and after
commencement of the chemotherapy. As exemplified herein, the
midkine family protein may be midkine.
[0039] Another example of the present invention provides an animal
model of hair loss consisting essentially of an aged mouse that
suffers hair loss e.g., in the form of bald regions or alopecic
sites. The present invention clearly extends to the use of this
animal model to screen for novel therapeutic and prophylactic
compositions of matter that prevent hair loss and/or promote hair
growth and/or enhance hair growth, wherein said screen comprises
administering a test compound to the animal and determining hair
growth and/or reduced hair loss and wherein reduced hair loss
and/or increased hair growth and/or an initiation of hair growth on
the animal in the presence of the test compound relative to in the
absence of the test compound is indicative of the test compound
preventing hair loss and/or promoting hair growth and/or enhancing
hair growth. In one example of this screening method, the activity
of the test compound is compared to the activity of a midkine
family protein and a test compound having comparable or enhanced
activity relative to the midkine family protein is identified
and/or isolated and/or formulated for topical administration in a
method of the invention according to any example hereof.
[0040] In another example, the present invention provides a method
of identifying or isolating a compound that prevents hair loss
and/or promotes hair growth and/or enhances hair growth, wherein
said method comprises administering a test compound to a mouse,
said mouse being aged more than one year and suffering hair loss
and having a knockout of one allele of a gene encoding a midkine
protein and one other functional allele of the gene, and then
determining hair growth and/or reduced hair loss on the mouse, and
wherein reduced hair loss and/or increased hair growth and/or an
initiation of hair growth on the mouse in the presence of the test
compound relative to in the absence of the test compound is
indicative of the test compound preventing hair loss and/or
promoting hair growth and/or enhancing hair growth.
Definitions
[0041] This specification contains nucleotide and amino acid
sequence information prepared using PatentIn Version 3.6, presented
herein after the claims. Each nucleotide sequence is identified in
the sequence listing by the numeric indicator <210> followed
by the sequence identifier (e.g. <210>1, <210>2,
<210>3, etc). The length and type of sequence (DNA, protein
(PRT), etc), and source organism for each nucleotide sequence, are
indicated by information provided in the numeric indicator fields
<211>, <212> and <213>, respectively. Nucleotide
sequences referred to in the specification are defined by the term
"SEQ ID NO:" followed by the sequence identifier (e.g. SEQ ID NO: 1
refers to the sequence in the sequence listing designated as
<400>1).
[0042] As used herein the term "derived from" shall be taken to
indicate that a specified integer may be obtained from a particular
source albeit not necessarily directly from that source.
[0043] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising", is understood to imply the inclusion of a stated
step or element or integer or group of steps or elements or
integers but not the exclusion of any other step or element or
integer or group of elements or integers.
[0044] The term "hair" means any hair or fur on the body of a
mammal including a human, and includes, for example, head hair,
eyebrows, eyelashes, moustaches, beards, chest hair, back hair, arm
hair, leg hair, genital hair, nasal hair or ear hair.
[0045] As used herein, the term "treat" or "treating" or
"treatment" shall be taken to include therapeutic treatment of a
pre-existing condition, or prophylactic or preventative measures,
wherein the aim is to prevent, ameliorate, reduce or slow down
(lessen) hair thinning, hair loss or alopecia. It follows that hair
growth, or treatment of hair thinning, refers to normalization of
thinned hair, such as in alopecia, and/or increasing the length and
thickness of hair. A mammal in need of treatment may already have
the condition, or may be prone to have the condition or may be in
whom the condition is to be prevented. Such treatment preferably
involves an anti-apoptotic effect on follicles to thereby extend
their anagen phase, or alternatively, or the re-entry of normal
telogen follicles into the next hair cycle.
[0046] "Preventing", "prevention", "preventative" or "prophylactic"
refers to keeping from occurring, or to hinder, defend from, or
protect from the occurrence of a condition, disease, disorder, or
phenotype, including an abnormality or symptom. A mammal in need of
prevention may be prone to develop the condition.
[0047] The term "ameliorate" or "amelioration" refers to a
decrease, reduction or elimination of a condition, disease,
disorder, or phenotype, including an abnormality or symptom.
[0048] The term "therapeutically effective amount" refers to an
amount of the Midkine family protein capable of reducing hair
thinning, hair loss or alopecia in a mammal to a level which is
beneficial to treat or prevent hair thinning, hair loss or
alopecia. A therapeutically effective amount may be determined
empirically and in a routine manner in relation to treating hair
thinning, hair loss or alopecia.
[0049] Throughout this specification, unless specifically stated
otherwise or the context requires otherwise, reference to a single
step, composition of matter, group of steps or group of
compositions of matter shall be taken to encompass one and a
plurality (i.e. one or more) of those steps, compositions of
matter, groups of steps or group of compositions of matter.
[0050] Each example described herein is to be applied mutatis
mutandis to each and every other example unless specifically stated
otherwise.
[0051] Each definition or clarifying term described herein shall be
taken to apply mutatis mutandis to each and every example of the
invention unless the context requires otherwise.
[0052] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations or any two or more of said steps or features.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIG. 1 provides photographic representations showing a fur
growth promoting effect of thrice-weekly administration for 15 days
of topical formulations comprising midkine (MK) in 40% glycerol (1
.mu.g/ml) or 10 .mu.l of pleiotrophin (PTN) in 40% glycerol (1
.mu.g/ml) to different alopecic regions of alopecic Black 6 MK
heterozygous knockout (+/-) mice at 1.5 years of age. In the
drawing, the circles in each row indicate the positioning of dermal
patches of MK (circle nearer the neck region) and PTN (circles
nearer the tail) for each animal in the same row. Panel A shows an
animal on the day treatment was commenced. Panels B-L show mice at
day 1, 4, 6, 8, 11, 13, 15, 18, 20, 22, and 25 respectively from
commencement of treatment. Data indicate that animals treated with
a topical formulation of MK or PTN show significant fur regrowth in
this animal model, and that fur regrowth occurs more rapidly using
MK than PTN. After 15 days when treatment ceased (panel H), MK had
an ongoing beneficial effect to day 25. Fur regrowth is not
entirely localized to the treated region, since animals showed
improved fur condition over their entire dorsal region following
treatment.
[0054] FIG. 2 provides photographic representations showing the
effect of a parenteral, i.e., subcutaneous, formulations comprising
MK (1 .mu.g/ml) or 50 .mu.l of PTN (1 .mu.g/ml) to different
alopecic regions of aged Black 6 MK homozygous knockout (-/-) mice
at 1.5 years of age for 15 days, followed by treatment with topical
formulations described in the legend to FIG. 1 for a further 10
days. In the drawing, the circles in each row indicate the
positioning of subcutaneous injections and dermal patches of MK
(circle nearer the neck region) and PTN (circles nearer the tail)
for each animal in the same row. Panel A shows an animal on the day
treatment was commenced. Panels B-L show mice at day 1, 4, 6, 8,
11, 13, 15, 18, 20, 22, and 25 respectively from commencement of
treatment. Panel H shows an animal on the day that thrice-weekly
topical treatment commenced. Data indicate that animals treated
with a sub-cutaneous injection of MK or PTN showed no macroscopic
signs of fur regrowth 15 days later. However, 10 days following
commencement of topical therapy, administration of MK or PTN had
stimulated fur growth in this animal model (panel L). Fur regrowth
was not entirely localized to the treated region, since animals
showed improved fur condition over their entire dorsal region
following treatment.
[0055] FIG. 3 provides photographic representations showing a
fur-growth promoting effect of externally applying 100 .mu.l of MK
in 10% glycerol (2 .mu.g/ml) five times per week to alopecic
regions of Black 6 MK heterozygous knockout (+/-) mice at 1.5 years
of age. Panel A shows animals on the day treatment was commenced.
Panels B-F show mice at day 7, day 14, day 21, day 28, and day 35
respectively from commencement of treatment. Data show progressive
fur regrowth during the 35 day test period, with significant fur
regrowth evident after about 21-28 days (panels C and D).
[0056] FIG. 4 provides photographic representations showing a
fur-growth promoting effect of externally applying 100 .mu.l of PTN
in 10% glycerol (2 .mu.g/ml)five times per week to alopecic regions
of Black 6 MK heterozygous knockout (+/-) mice at 1.5 years of age.
Panel A shows animals on the day treatment was commenced. Panels
B-F show mice at day 7, day 14, day 21, day 28, and day 35
respectively from commencement of treatment. Data show progressive
fur regrowth during the 35 day test period, with significant fur
regrowth evident after about 21-28 days (panels C and D).
[0057] FIG. 5 provides photographic representations showing the
fur-growth promoting effect of externally applying 100 .mu.l of
minoxidil (RiUP.RTM., Taisho Pharmaceutical Co., Ltd.) to alopecic
regions of aged Black 6 MK heterozygous knockout (+/-) mice as a
positive control to the experiments of FIGS. 3 and 4. Panel A shows
animals on the day treatment was commenced. Panels B-F show mice at
day 7, day 14, day 21, day 28, and day 35 respectively from
commencement of treatment. Data show less effective treatment with
minoxidil than that observed following treatment with MK or PTN
because substantial areas of skin failed to regrow fur after 35
days treatment with minoxidil.
[0058] FIG. 6 provides photographic representations showing fur
regrowth in shaved regions of nine-week old male C3H/HeJ mice
following external application of 100 .mu.l of 10% (v/v)
glycerol/phosphate buffered saline (PBS) as a negative control for
the experiments of FIGS. 8 and 9. Panel A shows animals on the day
treatment was commenced. Panels B-D show mice at day 7, day 14, and
day 21 respectively from commencement of treatment. Data show slow
fur regrowth which becomes apparent macroscopically only on about
day 21.
[0059] FIG. 7 provides photographic representations showing fur
regrowth in shaved regions of nine-week old male C3H/HeJ mice
following external application of 100 .mu.l of minoxidil (e.g., 1%
w/v; RiUP.RTM., Taisho Pharmaceutical Co., Ltd.) as a positive
control for the experiments of FIGS. 8 and 9. Panel A shows animals
on the day treatment was commenced. Panels B-D show mice at day 7,
day 14, and day 21 respectively from commencement of treatment.
Data show slow fur re-growth which becomes apparent macroscopically
only on about day 21, consistent with the vasodilatory effect of
minoxidil on dermal papillae.
[0060] FIG. 8 provides photographic representations showing fur
regrowth in shaved regions of nine-week old male C3H/HeJ mice
following external application of 100 .mu.l of MK in 10%
glycerol/PBS (2 .mu.g/ml). Panel A shows animals on the day
treatment was commenced. Panels B-D show mice at day 7, day 14, and
day 21 respectively from commencement of treatment. Data show slow
fur regrowth which becomes apparent macroscopically only on about
day 21, suggesting a different mode of action for MK to minoxidil
in animals that have merely been synchronized in the anagen phase,
without exhibiting symptoms of alopecia.
[0061] FIG. 9 provides photographic representations showing fur
regrowth in shaved regions of nine-week old male C3H/HeJ mice
following external application of 100 .mu.l of PTN in 10%
glycerol/PBS (2.mu.g/ml). Panel A shows animals on the day
treatment was commenced. Panels B-D show mice at day 7, day 14, and
day 21 respectively from commencement of treatment. Data show slow
fur regrowth Which becomes apparent macroscopically only on about
day 21, suggesting a different mode of action for MK to minoxidil
in animals that have merely been synchronized in the anagen phase,
without exhibiting symptoms of alopecia
[0062] FIG. 10 provides a graphical representation showing the
percentage alopecia developed in a murine model of
cyclophosphamide-induced alopecia over time (days) following
synchronization in anagen phase by shaving and waxing on day 1, and
150 mg/kg cyclophosphamide (CYP) injection i/p on day 9. Percentage
alopecia was determined as the percentage of the area of skin to
which the dermal patches were applied showing fur loss on the
indicated day of measurement. Circles show alopecia in animals
receiving daily saline placebo i.e., without midkine protein, in
addition to CYP. Filled squares (MK 2-29) show percentage alopecia
in animals receiving 5 .mu.g midkine protein topically on a daily
basis from day 2 to day 29 when the experiment was terminated.
Filled triangles (MK-10-29) show percentage alopecia in animals
receiving 5 .mu.g midkine protein each on a daily basis from day 10
to day 29 when the experiment was terminated. Data demonstrate that
midkine protein reduces CYP-induced fur loss when administered
before and/or following CYP, and that midkine increases a rate of
follicle recovery as determined by fur growth following cessation
of CYP treatment. Under these conditions, maximum differentiation
between animals receiving the placebo control and animals receiving
midkine was detectable on days 12-14.
[0063] FIG. 11 provides a graphical representation of the
percentage alopecia on day 13 of the data presented in FIG. 10.
Percentage alopecia was determined as the percentage of the area of
skin to which the dermal patches were applied showing fur loss on
the indicated day of measurement. The open bar (PBS) shows 42%
alopecia in animals receiving daily saline placebo i.e., without
midkine protein, in addition to CYP. The hatched bar (MK 2-29) show
only 20% alopecia in animals receiving 5 .mu.g midkine protein
topically on a daily basis from day 2 to day 29 when the experiment
was terminated. The striped bar (MK-10-29) shows only 26% alopecia
in animals receiving 5 .mu.g midkine protein each on a daily basis
from day 10 to day 29 when the experiment was terminated. Data
demonstrate that midkine protein reduces CYP-induced fur loss when
administered before and/or following CYP, and that midkine
increases a rate of follicle recovery as determined by fur growth
following cessation of CYP treatment.
[0064] FIG. 12 provides photographic representations of the back
skin of 15 mice, each treated with phosphate buffered saline
placebo i.e., without midkine protein, administered daily as a
topical formulation (group of 5 mice labeled PBS), or 5 .mu.g
midkine protein administered daily as a topical formulation before
and/or after receiving a single cyclophosphamide (CYP) injection
i/p (group of 5 mice labeled MK 2-29), or treated topically with 5
.mu.g midkine protein on a daily basis after receiving a single 150
mg/kg cyclophosphamide (CYP) injection i/p (group of 5 mice labeled
MK 10-29). Animals were observed for signs of fur regrowth/alopecia
and skin pigmentation. Data indicate that mice treated with PBS
exhibit extensive fur loss and grey or pink skin colour, especially
in their neck regions, consistent with a predominance of follicles
in catagen (grey) or telogen (pink). Mice treated with midkine
protein before and after CYP treatment (MK2-29) showed reduced
areas of fur loss relative to the group receiving PBS, and skin was
either uniformly black or dark grey in color consistent with a
predominance of follicles in anagen (black) and a lower proportion
in catagen. Almost no pink skin was evident in this group,
suggesting a much reduced proportion of follicles in telogen.
Similarly, mice treated with midkine protein only following CYP
treatment (MK10-29) showed reduced areas of fur loss relative to
the group receiving PBS, and appeared macroscopically similar to
animals receiving midkine protein before and after chemotherapy.
The skin of mice receiving midkine protein only after chemotherapy
was uniformly dark grey in color, consistent with a predominance of
follicles in anagen or catagen.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0065] SEQ ID NO: 1 provides the amino acid sequence of human
midkine (Accession identifiers gi|4505135 and
ref|NP.sub.--002382.1).
[0066] SEQ ID NO: 2 provides the amino acid sequence of human
pleiotrophin (Accession identifiers gi|4506281 and
ref|NP.sub.--002816.1),
[0067] SEQ ID NO: 3 provides the amino acid sequence of a human
midkine-like protein corresponding with SEQ ID NO: 4 of WO
2004/052928).
[0068] SEQ ID NO: 4 provides the nucleotide sequence of Human
midkine mRNA (Accession identifiers gi|182650 and gb|M69148.1).
DETAILED DESCRIPTION OF THE INVENTION
Midkine Family Protein
[0069] Midkine is a 13-kDa protein, which was discovered as a
product from a gene whose expression is induced in an early stage
of lung tumour cell differentiation due to retinoic acid.
Pleiotrophin was discovered in the brain of a newborn rat as
heparin-binding protein with an ability of enhancing neurite
extension.
[0070] As used herein a "Midkine family protein" is a protein that
exhibits a similar amino acid sequence to midkine or a functional
region thereof and exhibits the same function as or a similar
function to that of midkine. A Midkine family protein includes a
functional variant of a Midkine family protein.
[0071] Even a protein with low identity with midkine may be a
Midkine family protein, provided that the protein has the same
function as or a similar function to that of midkine. An example of
such a Midkine family protein may be midkine (e.g. SEQ ID NO: 1),
midkine-like protein (e.g. SEQ ID NO: 3 (WO 2004/052928)),
truncated midkine protein, or pleiotrophin (e.g. SEQ ID NO: 2). The
Midkine family protein may be a functional variant of a Midkine
family protein, including a functional variant of midkine,
midkine-like protein, truncated midkine protein, or pleiotrophin.
The functional variant may be modified by substitution, deletion,
or addition of one or more amino acids relative to the non-modified
protein. The functional variant of the Midkine family protein will
exhibit a function of midkine.
[0072] In one embodiment, the Midkine family protein is midkine. In
another embodiment, the Midkine family protein is pleiotrophin.
[0073] The function of midkine may be a function of enhancing cell
proliferation, inhibiting apoptosis, binding to heparin, enhancing
cell migration, or inducing cell differentiation. In one
embodiment, the function of MK is inhibiting apoptosis.
[0074] "Functional variant" and "variant" as used herein includes
either natural protein variants or artificially modified protein
variants that exhibit a function of midkine.
[0075] As used herein, "modified" or "modification" includes
substitution, addition, and/or deletion of an amino acid
residue.
[0076] The Midkine family protein may have an amino acid sequence
that is at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% or is 100% identical over
its length or functional region to a segment (preferably a
continuous segment) of a wild-type Midkine family protein (e.g. any
one of SEQ ID NOs: 1 to 3).
[0077] A Midkine family protein may comprise 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10, or even 15, 20, 25, 30, 35, 40, 45, or 50
modifications e.g., at selected positions to maintain or enhance
midkine function.
[0078] Thus, the specifically stated Midkine family protein
sequences can vary, provided individual substitution, deletion
and/or addition of an amino acid does not strongly impair the
function of the Midkine family protein.
[0079] The Midkine family protein or their functional variants can
also be linked with other peptides or polypeptides or with further
chemical groups such as glycosyl groups, lipids, phosphates, acetyl
groups or the like, provided they do not strongly adversely
influence their midkine function. Thus, the modified Midkine family
protein may be a fusion construct. For example, the Midkine family
protei may be fused to a peptidyl moiety comprising an
art-recognized protein translocation domain to facilitate entry of
the Midkine family protein to the follicular cells.
[0080] In addition to naturally occurring amino acids,
non-naturally occurring amino acids, or modified amino acids, are
also contemplated and within the scope of the invention. In fact,
as used herein, "amino acid" refers to naturally occurring amino
acids, non-naturally occurring amino acids, and amino acid analogs,
and to the D or L stereoisomers of each.
[0081] Natural amino acids include alanine (A), arginine (R),
asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q),
glutamic acid (E), glycine (G), histidine (H), isoleucine (I),
leucine (L), lysine (K), methionine (M), phenylalanine (F), proline
(P), serine (S), threonine (T), tryptophan (W), tyrosine (Y),
valine (V), hydroxyproline (O and/or Hyp), isodityrosine (IDT), and
di-isodityrosine (di-IDT). Hydroxyproline, isodityrosine, and
di-isodityrosine are formed post-translationally: In some
embodiments, the natural amino acids, in particular the 20
genetically encoded amino acids, are used.
[0082] The substitutions may be conservative amino acid
substitutions, in which the substituted amino acid has similar
structural or chemical properties with the corresponding amino acid
in the reference sequence. Alternatively, the substitutions may be
non-conservative amino acid substitutions.
[0083] By way of example, conservative amino acid substitutions
involve substitution of one aliphatic or hydrophobic amino acids,
e.g., alanine, valine, leucine and isoleucine, with another;
substitution of one hydroxyl-containing amino acid, e.g., serine
and threonine, with another; substitution of one acidic residue,
e.g., glutamic acid or aspartic acid, with another; replacement of
one amide-containing residue, e.g., asparagine and glutamine, with
another; replacement of one aromatic residue, e.g., phenylalanine
and tyrosine, with another; replacement of one basic residue, e.g.,
lysine, arginine and histidine, with another; and replacement of
one small amino acid, e.g., alanine, serine, threonine, methionine,
and glycine, with another.
[0084] Functional variants may be obtained in which the Midkine
family protein has been chemically modified at the level of amino
acid side chains, of amino acid chirality, and/or of the peptide
backbone. These alterations are intended to provide proteins having
similar or improved therapeutic properties.
[0085] Modifications providing increased potency, prolonged
activity, ease of purification, and/or increased half-life will be
known to the person skilled in the art.
[0086] Functional variants of the Midkine family protein may be
identified by modifying the sequence of the protein and then
assaying the resulting protein for the ability to function
similarly to midkine, for example enhancing cell proliferation,
inhibiting apoptosis, binding to heparin, enhancing cell migration,
or inducing cell differentiation.
[0087] The Midkine family protein can be made by synthetic
chemistry of recombinant DNA mutagenesis techniques that are well
known to persons skilled in the art. For example, midkine may be
produced in accordance with a method disclosed in the Examples of
JP-A-H09-95454.
Formulations
[0088] The Midkine family protein may be formulated in any form
used in the pharmaceutical, quasi-drug, or cosmetic field,
preferably suitable for topical administration. For example, the
composition may be a hair-growing product, hair or scalp cosmetic
(e.g.
[0089] shampoo, hair conditioner, scalp lotion, scalp cream, hair
tonic, etc.), skincare product (e.g. lotion, cream, face cream,
face lotion, milk, pack, liquid facial wash, soap, etc.), body care
product (e.g. body cream, body lotion, soap, liquid wash, bath
additive, etc.), UV protective agent (e.g. sun block, sunscreen
lotion, tanning oil, etc.), or cosmetic (e.g. eyeliner, eyebrow
pencil, cream, lotion, etc).
[0090] Conveniently, the Midkine family protein may be formulated
for parenteral administration e.g., with one or more
chemotherapeutic drugs, such as by intravenous injection.
[0091] Excipients will typically be included in the dosage form
e.g., to improve solubility and/or bioadhesion. Suitable excipients
include solvents, co-solvents, emulsifiers, plasticizers,
surfactants, thickeners, pH modifiers, emollients, antioxidants,
and chelating agents, wetting agents, and water absorbing agents.
Formulations may also include one or more additives, for example,
dyes, colored pigments, pearlescent agents, deodorizers, and odor
maskers.
[0092] Diluents or fillers increase the bulk of a solid dosage form
so that a practical size is provided for compression of tablets or
formation of beads and granules. Suitable diluents include, but are
not limited to dicalcium phosphate dihydrate, calcium sulfate,
lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline
cellulose, kaolin, sodium chloride, dry starch, hydrolyzed
starches, pregelatinized starch, silicone dioxide, titanium oxide,
magnesium aluminum silicate and powdered sugar.
[0093] Formulations may also comprise one or more dispersants e.g.,
phosphate-buffered saline (PBS), saline, glucose, sodium lauryl
sulfate (SLS), polyvinylpyrrolidone (PVP), polyethylene glycol
(PEG), and hydroxypropylmethylcellulose (HPMC).
[0094] Formulations may also comprise one or more binders to impart
cohesive qualities to a solid dosage formulation, and thus ensure
that a tablet, bead or granule remains intact after the formation
of the dosage forms. Suitable binder materials include, but are not
limited to, starch, pregelatinized starch, gelatin, sugars
(including sucrose, glucose, dextrose, lactose and sorbitol),
polyethylene glycol, waxes, natural and synthetic gums such as
acacia, tragacanth, sodium alginate, cellulose, including
hydroxypropylmethylcellulose ("HPMC"), microcrystalline cellulose
("MCC"), hydroxypropylcellulose, ethylcellulose, and veegum, and
synthetic polymers such as acrylic acid and methacrylic acid
copolymers, methacrylic acid copolymers, methyl methacrylate
copolymers, aminoalkyl methacrylate copolymers, polyacrylic
acid/polymethacrylic acid and polyvinylpyrrolidone (PVP).
[0095] Formulations may also comprise one or more lubricants to
facilitate manufacture or ingestion of a solid dosage unit e.g., a
tablet. Examples of suitable lubricants include, but are not
limited to, magnesium stearate, calcium stearate, stearic acid,
glycerol behenate, polyethylene glycol, talc, and mineral oil.
[0096] Formulations may also comprise one or more disintegrants to
facilitate dosage form disintegration after administration, and
generally include, but are not limited to, starch, sodium starch
glycolate, sodium carboxymethyl starch, sodium
carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized
starch, clays, cellulose, alginine, gums or cross linked polymers,
such as cross-linked PVP.
[0097] Formulations may also comprise one or more stabilizers
and/or preservatives (e.g., E216, E218, and chlorobutanol
hemihydrate) to inhibit or retard drug decomposition reactions
e.g., by oxidation or bacterial action.
[0098] Formulations may also comprise one or more surfactants.
Surfactants may be anionic, cationic, amphoteric or nonionic
surface active agents. Suitable anionic surfactants include, but
are not limited to, those containing carboxylate, sulfonate and
sulfate ions. Examples of anionic surfactants include sodium,
potassium, ammonium of long chain alkyl sulfonates and alkyl aryl
sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium
sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl
sodium sulfosuccinates, such as sodium
bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as
sodium lauryl sulfate. Cationic surfactants include, but are not
limited to, quaternary ammonium compounds such as benzalkonium
chloride, benzethonium chloride, cetrimonium bromide, stearyl
dimethylbenzyl ammonium chloride, polyoxyethylene and coconut
amine. Examples of nonionic surfactants include ethylene glycol
monostearate, propylene glycol myristate, glyceryl monostearate,
glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose
acylate, PEG-150 laurate, PEG-00 monolaurate, polyoxyethylene
monolaurate, polysorbates, polyoxyethylene octylphenylether,
PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene
glycol butyl ether, steamyl monoisopropanolamide, and
polyoxyethylene hydrogenated tallow amide. Examples of amphoteric
surfactants include sodium N-dodecyl-.beta.-alanine, sodium
N-lauryl-.beta.-iminodipropionate, myristoamphoacetate, lauryl
betaine and lauryl sulfobetaine.
[0099] If desired, solid formulations e.g., tablets, beads,
granules, or particles may also contain an amount of a non-toxic
auxiliary substance such as a wetting or emulsifying agent, dye, or
pH buffering agent.
[0100] Pharmaceutical formulations may be presented in unit dose
forms containing a predetermined amount of active agent i.e.,
Midkine family protein, or a derivative or analog thereof, per unit
dose. The concentration of active agent may vary depending upon
whether or not the formulation is for prevention or therapy, the
route of administration, half-life of the compound following
administration by the selected route, and the age, weight and
condition of the patient including e.g., the severity of problem
drinking being treated. For example a unit dose may comprise about
1 .mu.g to 10 ug, or 0.01 mg to 1000 mg, or 0.1 mg to 250 mg, of
Midkine family protein, or a derivative or analog thereof. In
another example, Midkine family protein, or a derivative or analog
thereof may be formulated such that the concentration of active
agent is at least about 1% (w/w) or at least about 5% (w/w) or at
least about 10% (w/w) or at least about 25% (w/w) based on the
total weight of the pharmaceutical composition.
[0101] To prepare pharmaceutical formulations, one or more Midkine
family proteins is/are mixed with a pharmaceutically acceptable
carrier or excipient for example, by mixing with physiologically
acceptable carriers, excipients, or stabilizers in the form of,
e.g., lyophilized powders, slurries, aqueous solutions, or
suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's
The Pharmacological Basis of Therapeutics, McGraw-Hill, New York,
N.Y.; Gennaro (2000) Remington: The Science and Practice of
Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis,
et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral
Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)
Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman,
et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems,
Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity
and Safety, Marcel Dekker, Inc., New York, N.Y.).
[0102] The amount of the Midkine family protein in a formulation
for treatment of alopecia may vary depending on various parameters
such as age, sex, and condition of the mammal being treated. For
example, the Midkine family protein may be formulated to produce a
final concentration of 0.001 to 50% (w/w), 0.001 to 10% (w/w),
0.001 to 5% (w/w), or 0.001 to 1% of the total weight of the
medicament. The concentration of the Midkine family protein in a
liquid formulation may be, for example, 0.0001 .mu.g/ml to 2 mg/ml,
0.001 .mu.g/ml to 1 mg/ml, 0.01 .mu.g/ml to 500 .mu.g/ml, 0.1
.mu.g/ml to 100 .mu.g/ml, 0.1 .mu.g,/ml to 50 .mu.g/ml, 0.1
.mu.g/ml to 10 .mu.g/ml, or 1 .mu.g/ml to 2 .mu.g/ml.
[0103] A formulation comprising a midkine family protein and a
pharmaceutically acceptable carrier or diluent may further comprise
one or adjunctive therapeutic agents for treatment of the same
condition, wherein the adjunctive therapeutic agent is suitable for
administration by the same route as the midkine family protein(s).
For example, a midkine family protein may be formulated for topical
administration with cestradiol and/or oxandrolone and/or minoxidil.
The adjunctive therapeutic agent is generally present in the
formulation at a concentration in accordance with its known
prescribed level. The skilled artisan will appreciate that such
compositions may provide enhanced therapeutic benefit to the
patient, and may be more than additive in their effect.
[0104] Formulation of a pharmaceutical compound will vary according
to the route of administration selected (e.g., solution, emulsion,
capsule). For solutions or emulsions, suitable carriers include,
for example,, aqueous- or alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles can include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's or fixed oils, for
instance. Intravenous vehicles can include various additives,
preservatives, or fluid, nutrient or electrolyte replenishers and
the like (See, generally, Remington's Pharmaceutical Sciences, 17th
Edition, Mack Publishing Co., Pa., 1985). For inhalation, the agent
can be solubilized and loaded into a suitable dispenser for
administration (e.g., an atomizer, nebulizer or pressurized aerosol
dispenser).
[0105] Pharmaceutical formulations can be adapted for
administration by any appropriate route, for example by the oral
(including buccal or sublingual), topical (including buccal,
sublingual or transdermal), or parenteral (including subcutaneous,
intramuscular, intravenous or intradermal) route. Such formulations
can be prepared by any method known in the art of pharmacy, for
example by bringing into association the active ingredient with the
carrier(s), diluent(s) or excipient(s).
[0106] For example, the Midkine family protein is formulated for
topical administration. Topical compositions include those
pharmaceutical forms in which the Midkine family protein(s) is(are)
applied externally by direct contact with the skin surface to be
treated. Conventional pharmaceutical forms for this purpose include
ointments, liniments, creams, shampoos, lotions, pastes, jellies,
sprays, aerosols, and the like, and may be applied in patches or
impregnated dressings depending on the part of the body to be
treated. The term "ointment" embraces formulations (including
creams) having oleaginous, water-soluble and emulsion-type bases,
e.g., petrolatum, lanolin, polyethylene glycols, as well as
mixtures thereof.
[0107] For topical use on the eyelids or eyebrows, the active
Midkine family protein(s) can be formulated in aqueous alcohol
solutions, creams, ointments or oils exhibiting physiologically
acceptable osmolarity by addition of pharmacologically acceptable
buffers and salts. Such formulations may or may not, depending on
the dispenser, contain preservatives such as benzalkonium chloride,
chlorhexidine, chlorobutanol, parahydroxybenzoic acids and
phenylmercuric salts such as nitrate, chloride, acetate, and
borate, or antioxidants, as well as additives like EDTA, sorbitol,
boric acid etc. as additives. Furthermore, particularly aqueous
solutions may contain viscosity increasing agents such as
polysaccharides, e.g., methylcellulose, mucopolysaccharides, e.g.,
hyaluronic acid and chondroitin sulfate, or polyalcohol, e.g.,
polyvinylalcohol. Various slow releasing gels and matrices may also
be employed as well as soluble and insoluble ocular inserts, for
instance, based on substances forming in-situ gels. Depending on
the actual formulation and Midkine family protein(s) to be used,
various amounts of the drug and different dose regimens may be
employed.
[0108] For topical use on the skin and the scalp, the Midkine
family protein(s) can be advantageously 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 like. Various matrices for slow release delivery
may also be used.
[0109] In another example, a formulation comprising one or more
Midkine family proteins is adapted for parenteral administration
e.g., by subcutaneous or intravenous injection. Such formulations
include aqueous and non-aqueous sterile injection solutions which
may contain the antioxidants as well as buffers, bacteriostats and
solutes that render the formulation isotonic with the blood of the
intended recipient; and aqueous and non-aqueous sterile suspensions
which may include suspending agents and thickening agents. The
formulations may be presented in unit-dose or multi-dose
containers, for example sealed ampules and vials, and may be stored
in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example water for
injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets. In one example, one or more Midkine family
proteins is formulated as an intravenous lipid emulsion or a
surfactant micelle or polymeric micelle (see., e.g., Jones et al.,
Eur. J. Pharmaceutics Biopharmaceutics 48, 101-111, 1999; Torchilin
J. Clin, release 73, 137-172, 2001 for parenteral
administration.
[0110] Sustained release injectable formulations are produced e.g.,
by encapsulating one or more Midkine family proteins in porous
microparticles comprising a pharmaceutical agent and a matrix
material having a volume average diameter between about 1 .mu.m and
150 .mu.m, e.g., between about 5 .mu.m and 25 .mu.m diameter. In
one example; the porous microparticles have an average porosity
between about 5% and 90% by volume. In another example, the porous
microparticles further comprise one or more surfactants, such as a
phospholipid. The microparticles may be dispersed in a
pharmaceutically acceptable aqueous or non-aqueous vehicle for
injection. Suitable matrix materials for such formulations comprise
a biocompatible synthetic polymer, a lipid, a hydrophobic molecule,
or a combination thereof. For example, the synthetic polymer can
comprise, for example, a polymer selected from the group consisting
of poly(hydroxy acids) such as poly(lactic acid), poly(glycolic
acid), and poly(lactic acid-co-glycolic acid), poly(lactide),
poly(glycolide), poly(lactide-co-glycolide), polyanhydrides,
polyorthoesters, polyamides, polycarbonates, polyalkylenes such as
polyethylene and polypropylene, polyalkylene glycols such as
poly(ethylene glycol), polyalkylene oxides such as poly(ethylene
oxide), polyalkylene terepthalates such as poly(ethylene
terephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl
esters, polyvinyl halides such as poly(vinyl chloride),
polyvinylpyrrolidone, polysiloxanes, poly(vinyl alcohols),
poly(vinyl acetate), polystyrene, polyurethanes and co-polymers
thereof, derivativized celluloses such as alkyl cellulose,
hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro
celluloses, methyl cellulose, ethyl cellulose, hydroxypropyl
cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl
cellulose, cellulose acetate, cellulose propionate, cellulose
acetate butyrate, cellulose acetate phthalate, carboxylethyl
cellulose, cellulose triacetate, and cellulose sulphate sodium salt
(jointly referred to herein as "synthetic celluloses"), polymers of
acrylic acid, methacrylic acid or copolymers or derivatives thereof
including esters, poly(methyl methacrylate), poly(ethyl
methacrylate), poly(butylmethacrylate), poly(isobutyl
methacrylate), poly(hexylmethacrylate), poly(isodecyl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), and poly(octadecyl acrylate) (jointly
referred to herein as "polyacrylic acids"), poly(butyric acid),
poly(valeric acid), and poly(lactide-co-caprolactone), copolymers,
derivatives and blends thereof. In a preferred example, the
synthetic polymer comprises a poly(lactic acid), a poly(glycolic
acid), a poly(lactic-co-glycolic acid), or a
poly(lactide-co-glycolide).
[0111] In another example, pharmaceutical formulations are adapted
for oral administration e.g., as capsules, soft gels, or tablets;
powders or granules, solutions or suspensions in aqueous or
non-aqueous liquids, edible foams or whips, or oil-in-water liquid
emulsions or water-in-oil liquid emulsions. An oral formulation may
comprise an intragranular phase comprising an effective amount of a
Midkine family protein and at least one carbohydrate alcohol and an
aqueous binder. The pharmaceutical formulation may be substantially
lactose-free. Preferred carbohydrate alcohols for such formulations
are selected from the group consisting of mannitol, maltitol,
sorbitol, lactitol, erythritol and xylitol. Preferably, the
carbohydrate alcohol is present at a concentration of about 15% to
about 90%. A preferred aqueous binder is selected from the group
consisting of hydroxypropyl cellulose, hydroxypropyl
methylcellulose, carboxymethyl cellulose sodium, polyvinyl
pyrrolidones, starches, gelatins and the like. A binder is
generally present in the range of from about 1% to about 15% by
weight. The intragranular phase can also comprise one or more
diluents, such as, for example, a diluent selected from the group
consisting of microcrystalline cellulose, powdered cellulose,
calcium phosphate-dibasic, calcium sulfate, dextrates, dextrins,
alginates and dextrose excipients. Such diluents are also present
in the range of about 15% to about 90% by weight. The intragranular
phase can also comprise one or more disintegrants, such as, for
example, a disintegrant selected from the group consisting of a low
substituted hydroxypropyl cellulose, carboxymethyl cellulose,
calcium carboxymethylcellulose, sodium carboxymethyl cellulose,
sodium starch glycollate, crospovidone, croscarmellose sodium,
starch, crystalline cellulose, hydroxypropyl starch, and partially
pregelatinized starch. A disintegrant is generally present in the
range of from about 5% to about 20% by weight. Such a formulation
can also comprise one or more lubricants such as, for example, a
lubricant selected from the group consisting of talc, magnesium
stearate, stearic acid, hydrogenated vegetable oils, glyceryl
behenate, polyethylene glycols and derivatives thereof, sodium
lauryl sulphate and sodium stearyl fumarate. A lubricant is
generally present in the range of from about 0.5% to about 5% by
weight. Such formulations are made into a tablet, capsule, or soft
gel e.g., by a process comprising mixing a Midkine family protein
and at least one carbohydrate alcohol to form a dry blend, wet
granulating the dry blend with an aqueous binder so as to obtain an
intragranular phase, and further formulating the resulting
intragranular phase so as to provide the formulation. Typically,
tablet or capsules is prepared to contain an appropriate unit
dosage e.g., from 0.001 mg to 1000 mg.
[0112] Alternatively, a liquid or semi-solid pharmaceutical
formulation for oral administration e.g., a hard gel or soft gel
capsule comprising one or more Midkine family proteins may be
prepared. The formulation may comprise a first carrier component
and optional second carrier component, which carriers comprise,
independently, one or more of lauroyl macrogel glycerides,
caprylocaproyl macrogel glycerides, stearoyl macrogel glycerides,
linoleoyl macrogel glycerides, oleoyl macrogel glycerides,
polyalkylene glycol, polyethylene glycol, polypropylene glycol,
polyoxyethylene-polyoxypropylene copolymer, fatty alcohol,
polyoxyethylene fatty alcohol ether, fatty acid, polyethoxylated
fatty acid ester, propylene glycol fatty acid ester, fatty ester,
glycerides of fatty acid, polyoxyethylene-glycerol fatty ester,
polyoxypropylene-glycerol fatty ester, polyglycolized glycerides,
polyglycerol fatty acid ester, sorbitan ester, polyethoxylated
sorbitan ester, polyethoxylated cholesterol, polyethoxylated castor
oil, polyethoxylated sterol, lecithin, glycerol, sorbic acid,
sorbitol, or polyethoxylated vegetable oil.
[0113] The formulation may also comprise an
emulsifying/solubilizing component comprising one or more of
metallic alkyl sulfate, quaternary ammonium compounds, salts of
fatty acids, sulfosuccinates, taurates, amino acids, lauroyl
macrogol glycerides, caprylocaproyl macrogolglycerides, stearoyl
macrogol glycerides, linoleoyl macrogol glycerides, oleoyl macrogol
glycerides, polyalkylene glycol, polyethylene glycol, polypropylene
glycol, polyoxyethylene-polyoxypropylene copolymer, polyoxyethylene
fatty alcohol ether, fatty acid, polyethoxylated fatty acid ester,
propylene glycol fatty acid ester, polyoxyethylene-glycerol fatty
ester, polyglycolized glycerides, polyglycerol fatty acid ester,
sorbitan ester, polyethoxylated sorbitan ester, polyethoxylated
cholesterol, polyethoxylated castor oil, polyethoxylated sterol,
lecithin, or polyethoxylated vegetable oil.
[0114] The formulation may also comprise an
anti-crystallization/solubilizing component which, when present,
generally comprises one or more of metallic alkyl sulfate,
polyvinylpyrrolidone, lauroyl macrogol glycerides, caprylocaproyl
macrogolglycerides, stearoyl macrogol glycerides, linoleoyl
macrogol glycerides, oleoyl macrogol glycerides, polyalkylene
glycol, polyethylene glycol, polypropylene glycol,
polyoxyethylene-polyoxypropylene copolymer, fatty alcohol,
polyoxyethylene fatty alcohol ether, fatty acid, polyethoxylated
fatty acid ester, propylene glycol fatty acid ester, fatty ester,
glycerides of fatty acid, polyoxyethylene-glycerol fatty ester,
polyglycolized glycerides, polyglycerol fatty acid ester, sorbitan
ester, polyethoxylated sorbitan ester, polyethoxylated cholesterol,
polyethoxylated castor oil, polyethoxylated sterol, lecithin, or
polyethoxylated vegetable oil.
[0115] A Midkine family protein may be formulated with a
hydrophobic polymer; preferably a bioadhesive polymer and
optionally encapsulated in or dispersed throughout a microparticle
or nanoparticle. The bioadhesive polymer improves gastrointestinal
retention via adherence of the formulation to the walls of the
gastrointestinal tract. Suitable bioadhesive polymers include
polylactic acid, polystyrene, poly(bis carboxy phenoxy
propand-co-sebacic anhydride) (20:80) (poly (CCP:SA)), alginate
(freshly prepared); and poly(fumaric anhydride-co-sebacic anhydride
(20:80) (poly (FA:SA)), types A (containing sudan red dye) and B
(undyed). Other high-adhesion polymers include p(FA:SA) (50:50) and
non-water-soluble polyacrylates and polyacrylamides. Preferred
bioadhesive polymers are typically hydrophobic enough to be
non-water-soluble, but contain a sufficient amount of exposed
surface carboxyl groups to promote adhesion e.g., non-water-soluble
polyacrylates and polymethacrylates; polymers of hydroxy acids,
such as polylactide and polyglycolide; polyanhydrides;
polyorthoesters; blends comprising these polymers; and copolymers
comprising the monomers of these polymers. Preferred biopolymers
are bioerodable, with preferred molecular weights ranging from 1000
to 15,000 kDa, and most preferably 2000 to 5000 Da. Polyanhydrides
e.g., polyadipic anhydride ("p(AA)"), polyfumaric anhydride,
polysebacic anhydride, polymaleic anhydride, polymalic anhydride,
polyphthalic anhydride, polyisophthalic anhydride, polyaspartic
anhydride, polyterephthalic anhydride, polyisophthalic anhydride,
poly carboxyphenoxypropane anhydride and copolymers with other
polyanhydrides at different mole ratios, are particularly
preferred. Blends of hydrophilic polymers and bioadhesive
hydrophobic polymers can also be employed. Suitable hydrophilic
polymers include e.g., hydroxypropylmethylcellulose,
hydroxypropylcellulose, carboxymethylcellulose, polyvinylalcohols,
polyvinylpyrollidones, and polyethylene glycols. Other mucoadhesive
polymers include DOPA-maleic anhydride co polymer, isopthalic
anhydride polymer, DOPA-methacrylate polymers, DOPA-cellulosic
based polymers, and DOPA-acrylic acid polymers.
[0116] Alternatively, the Midkine family protein may be
encapsulated or molecularly dispersed for oral administration in a
polymer to reduce particle size and increase dissolution. The
polymers may include polyesters such as poly(lactic acid) or P(LA),
polycaprylactone, polylactide-coglycolide or P(LGA), poly
hydroxybutyrate poly .beta.-malic acid); polyanhydrides such as
poly(adipic)anhydride or P(AA), poly(fumaric-co-sebacic)anhydride
or P(FA:SA), poly(sebacic)anhydride or P(SA); cellulosic polymers
such as ethylcellulose, cellulose acetate, cellulose acetate
phthalate, etc; acrylate and methacrylate polymers such as Eudragit
RS 100, RL 100, E100 PO, L100-55, L100, S100 (distributed by Rohm
America) or other polymers commonly used for encapsulation for
pharmaceutical purposes and known to those skilled in the art. Also
suitable are hydrophobic polymers such as polyimides. Blending or
copolymerization sufficient to provide a certain amount of
hydrophilic character can be useful to improve wetability of the
materials. For example, about 5% to about 20% of monomers may be
hydrophilic monomers. Hydrophilic polymers such as
hydroxylpropylcellulose (HPC), hydroxpropylmethylcellulose (HPMC),
carboxymethylcellulose (CMC) are commonly used for this
purpose.
[0117] Oral formulations may be "immediate release" formulations
e.g., that release at least 85% (wt/wt) of the Midkine family
protein within 60 minutes in vitro. Alternatively, the formulation
may be a "controlled release" formulation that releases drug more
slowly than an immediate release formulation i.e., it takes longer
than 60 minutes to release at least 85% (wt/wt) of the drug in
vitro. To extend the time period for release, the ratio of active
agent to polymer can be increased. Increased relative drug
concentration is believed to have the effect of increasing the
effective compound domain size within the polymer matrix thereby
slowing dissolution. In the case of a polymer matrix containing
certain types of hydrophobic polymers, the polymer will act as a
mucoadhesive material and increase the retention time of the active
compound in the gastrointestinal tract. Increased drug dissolution
rates combined with the mucoadhesive properties of the polymer
matrix increase uptake of the active compound and reduce
differences found in the fed and fasted states for the
compounds.
[0118] The compositions described herein may further comprise
components which are generally used in cosmetics, for example,
oils, detergents, UV absorbers, alcohols, chelating agents, pH
modifiers, preservatives, thickeners, pigments, fragrances, and
skin nutritional supplements. Specifically, the composition may
comprise active ingredients used for skin cosmetics, such as zinc
oxide microparticles, titanium oxide, UV absorbers such as Parsol
MCX and Parsol 1789, vitamins such as ascorbic acid, moisturising
agents such as hyaluronate sodium, petrolatum, glycerin, and urea,
hormonal agents, skin-lightening agents such as kojic acid,
arbutin, placenta extract, and rucinol, steroid drugs, inhibitors
of production or release of a chemical mediator such as
arachidonate metabolite and histamine (e.g. indometacin and
ibuprofen), anti-inflammatory drugs such as receptor antagonist,
anti-androgenic agents, sebum secretion suppressing agents such as
vitamin A acid, royal jelly extract, and royal jelly acid,
peripheral blood-vessel dilators such as tocopherol nicotinate,
alprostadil, isoxsuprine hydrochloride, and tolazoline
hydrochloride, carbon dioxide with peripheral blood-vessel dilating
activity, blood circulation promoting agents such as minoxidil,
carpronium chloride, capsicum tincture, vitamin E variants, ginkgo
extract, and Swertia japonica extract, cellular stimulants such as
pentadecanoic acid glyceride and nicotinic-aid amide,
antimicrobials such as hinokitiol, L-menthol, and
isopropylmethylphenol, glycyrrhizipic acid and variants or salts
thereof, ceramide and ceramide analogs.
Dosage Units and Frequency of Administration
[0119] The dose and frequency of the Midkine family protein or the
medicament may be appropriately modified depending on the
situation.
[0120] In general, the Midkine family protein, or the medicament
comprising the Midkine family protein(s), may be used with any
frequency. Typically, the Midkine family protein(s) are applied
repeatedly for a sustained period of time topically on the part of
the body to be treated, for example, the eyelids, eyebrows, skin or
scalp. The preferred dosage regimen will generally involve regular,
such as daily, weekly, twice-weekly, or thrice-weekly,
administration for a period of treatment of at least one about one
month, more preferably at least three months, and most preferably
at least six months. For example, the Midkine family protein or the
medicament may be used 1, 2, 3, 4, 5, 6 or 7 times per week,
corresponding with one use per day that the Midkine family protein
or medicament is applied. On any day, use of the Midkine family
protein or medicament may correspond with application 1, 2, 3, 4 or
5 times per day. In one embodiment, the Midkine family protein or
medicament is applied to the mammal once per day and three or five
times per week.
[0121] Typically, a topical formulation comprising a Midkine family
protein may be applied in the amount of 0.001 to 1,000
.mu.g/cm.sup.2/day, 0.005 to 500 .mu.g/cm.sup.2/day, 0.01 to 100
.mu.g/cm.sup.2/day, 0.05 to 50 .mu.g/cm.sup.2/day, or 0.1 to 10
.mu.g/cm.sup.2/day. Typically, the dose to be applied topically 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 depending on the
Midkine family protein(s) and the formulation.
[0122] Typically, the daily amount of Midkine family protein(s) for
treatment of the eyelid may be about 0.1 ng to about 100 mg per
eyelid.
[0123] The Midkine family protein may be applied alone as an active
ingredient, or may be applied with other active ingredients.
Similarly, the medicament may comprise further active ingredients
in addition to the Midkine family protein. Such active ingredients
other than the Midkine family protein may be, but are not limited
to, cellular stimulants, blood circulation promoting agents,
anti-androgen drugs, sebum secretion suppressing agents,
immunosuppressants, antihistamine agents, antimicrobials, focal
stimulants, emollients, antiphlogistics, or low-molecular
anti-apoptotic agents. Specifically, said other active ingredients
may be at least one of pantothenic acid or variants thereof,
placenta extract, photosensitizers, ginseng extract, biotin,
mononitro guaiacol, carpronium chloride or hydrates thereof,
vitamin E or variants thereof, Swertia japonica extract, capsicum
tincture, cepharanthine, nicotinic acid or variants thereof,
estradiol, ethynylestradiol, randic acid, minoxidil or
analogs/variants thereof, 5.alpha.-reductase inhibitor,
12-tetradecanoylphorbol-13-acetate, herbal medicine such as
Polygonatum rhizome, Uncaria, Silybum marianum, henna, and
Glycyrrhiza, estradiol benzoate, diphenhydramine, resorcin,
hinokitiol, 1-menthol, salicylic acid, Polygonum root extract,
Panax japonicus rhizome extract, panthenol, selenium disulfide,
pyridoxine hydrochloride, dipyrithione zinc, pyrithione zinc,
sulfur, piroctone olamine, pyrithione zinc, sulfur, glycyrrhetinic
acid stearyl, glycyrrhizinate dipotassium, allantoin,
dialkylmonoamine variants, Perilla frutescens extract, Poria
sclerotium extract, .beta.-glycyrrhetinic acid, miconazole nitrate,
benzoic acid, sodium salicylate, phytosterol, wine yeast extract,
takanal, ethinyl estradiol, isopropylmethylphenol, cepharanthine
biotin, D-pantothenyl alcohol, Paeonia extract, Tilia extract,
Sophora extract, Sophora flavescens extract, Zingiber Officinale
(Ginger) root extract, 6-benzylaminoprine, pentadecanoic glyceride,
t-flavanone, sweet Hydrangea leaf extract, adenosine, and
pantothenylethylether.
[0124] In one example, a formulation comprising a midkine family
protein is administered sequentially or simultaneously with an
adjunctive therapeutic agent for treatment of the same condition
e.g., cestradiol and/or oxandrolone and/or minoxidil and/or
finasteride or an agent that blocks the conversion of testosterone
to dihydrotesterone. The adjunctive therapeutic agent is
co-administered under conditions and in accordance to a standard
treatment regime for that agent. The skilled artisan will
appreciate that such treatment regimens provide enhanced
therapeutic benefit to the patient, and may be more than additive
in their effect.
[0125] Alternatively, or in addition, a formulation comprising a
midkine family protein is administered sequentially or
simultaneously with a cytotoxic or cytostatic compound that causes
hair loss e.g., in the case of a subject undergoing chemotherapy or
radiation therapy or treatment for HIV-1 infection or AIDS. In such
circumstances, the efficacy of the midkine family protein
counteracts the hair-loss effect of the cytotoxic or cytostatic
compound. The cytotoxic or cytostatic compound will generally be
administered in accordance to a standard treatment regime for that
agent. Conveniently, the Midkine family protein and the
cytotxin/cytostatin are administered via the same route e.g.,
parenterally.
Subjects
[0126] The compositions of the present invention are suitable for
medical treatment of humans and other mammals, including treatment
companion animals such as dogs and cats, and domestic animals such
as horses, zoo animals such as felids, canids, bovids, ungulates
and primates, or laboratory animals such as rodents, lagomorphs and
primates. The compositions are particularly suitable for treatment
of any mammal that suffers alopecia of any form, especially humans,
primates dogs, cats, or horses.
[0127] The subject to be treated may be afflicted with hair
thinning, hair loss or alopecia, or may not be afflicted with hair
thinning, hair loss or alopecia (i.e., free of detectable disease),
but is prone to develop hair thinning, hair loss or alopecia.
[0128] A Midkine family protein, or a medicament comprising a
Midkine family protein, may be used to treat hair thinning, hair
loss or alopecia caused by cytotoxic or cytostatic agents. The
cytotoxic or cytostatic agents may be endogenous, e.g. as generated
in response to stress, or may be exogenous, e.g. as administered
during chemotherapy for treatment of cancer.
[0129] The present invention is particularly suited to treatment
and prevention of alopecia in subjects that are either undergoing
treatment with a cytotoxic or cytostatic compound described herein,
or to whom such therapy has been prescribed. As exemplified herein,
the subject may be treated before therapy with a cytotoxic or
cytostatic compound commences, or before and after such therapy has
commenced. The present invention also provides for commences of
midkine family protein therapy after treatment with a cytotoxic or
cytostatic compound has commenced.
[0130] For example, a subject may apply a composition described
herein as a fine line at the skin-eyelash border of each eyelid,
and as a cream to the scalp, once a day several weeks e.g., two
weeks or three weeks, prior to the initiation of a chemotherapy
regimen (e.g., doxorubicin, cyclophosphamide, and paclitaxel, or
5-fluoruracil, leucovorin and oxaliplatin). The patient may
continue applying the composition throughout and after cessation of
the chemotherapy regimen. The patient would not generally
experience the total hair loss normally associated with
chemotherapy, and may recover more rapidly when chemotherapy
ceases. A few weeks after completion of the chemotherapy, the
patient may stop applying the composition. If hair is lost at this
stage, treatment is resumed.
[0131] The present invention is illustrated in detail below with
references to examples, but is not to be construed as being limited
thereto.
Example 1
Method for Producing Recombinant Midkine
[0132] A cDNA fragment comprising human MK open reading frame
(nucleotide positions 1-432; SEQ ID NO: 4) was inserted into the
yeast expression vector pPIC9 (Invitrogen). This recombinant
plasmid was transfected into yeast (Pichia pastoris GS115; Research
Corporation Technologies), and the desired clones were selected
with histidine and G418.
[0133] Human MK protein secreted by yeast into the culture medium
was purified by employing the following column chromatography in
the order below: [0134] 1. SP Steamlines (Pharmacia; adsorption and
wash with 20 mM pH 5.5 acetate buffer, elution with 20 mM pH 3.5
acetate suffer containing NaCl); [0135] 2. Sulfated Cellulofine
(Seikagaku Kogyo, Japan; adsorption with 10 mM pH 7.2 phosphate
buffer, wash with buffer containing 0.7 M NaCl, elution with buffer
containing 2.0 M NaCl); [0136] 3. Superdex 75 pg (Pharmacia; gel
filtration with saline); [0137] 4. Poly. Sulfoethyl A (Poly LC Co.;
adsorption with 20 mM buffer containing 0.6 M NaCl, wash with
buffer containing 0.88 M NaCl, elution with buffer containing 2M
NaCl); and [0138] 5. Superdex 75 pg (Pharmacia; gel filtration with
saline).
[0139] The Midkine preparation was dialyzed against saline. The
activity of purified MK protein was detected using as an index the
activity of MK for promoting the survival of embryonic neurons.
Example 2
[0140] Hair-growing effects of external applications of midkine
(MK) and pleiotrophin (PTN). 1 .mu.g/ml of MK or PTN in 40%
glycerol/phosphate buffered saline (PBS) (v/v) was prepared. 10
.mu.l of the solution of MK or PTN was directly applied three times
per week to alopecic regions of aged Black 6 mice (1.5 years old,
MK heterozygous knockout (+/-)). It was observed for 25 days after
starting the application whether each of MK and PTN had a
hair-growing effect. FIG. 1 shows the results of the observation.
As clearly understood from FIG. 1 and the brief description
thereof, application of MK or PTN promoted hair growth on the
alopecic regions of the aged mice. In this experiment, the
applications of MK and PTN stopped on Day 15, because the
hair-growing effect of MK and PTN was already confirmed on Day 15.
From Day 15 to Day 25, the amount of hair increased even though the
applications of MK and PTN were stopped (FIG. 1). This shows that
the hair-growing effects of MK and PTN last for some time even
after their applications are stopped.
Example 3
Hair-Growing Effects of Subcutaneous Injection of MK and PTN
[0141] 1 .mu.g/ml of MK or PTN in PBS was prepared. 50 .mu.l of the
solution of MK or PTN was injected subcutaneously into aged Black 6
mice (1.5 years old, MK homozygous knockout (-/-)). The
subcutaneous injections of MK and PTN showed no hair-growing effect
on Day 15 (FIG. 2) under these conditions in this animal model.
After changing on Day 15 from the subcutaneous injection to
external applications of MK or PTN in accordance with Example 2,
the hair-growing effects of MK or PTN were observed on Day 25 (FIG.
2).
Example 4
Hair-Growing Effect of Intravenous Injection of MK
[0142] 1 .mu.g/ml of MK in PBS was prepared. 100 .mu.l of the
solution of MK was injected intravenously into aged Black 6 mice
(1.5 years old, MK homozygous knockout (-/-)). The intravenous
injection of MK showed no hair-growing effect under these
conditions and in this animal model (data not shown).
Example 5
Hair-Growing Effects of External Applications of MK and PTN
[0143] 2 .mu.g/ml of MK or PTN in 10% glycerol/PBS (v/v) was
prepared. 100 .mu.l of the solution of MK or PTN was applied
directly five times per week to alopecic regions of aged Black 6
mice (1.5 years old, MK heterozygous knockout (+/-)). It was
observed for 35 days after starting the application whether each of
MK and PTN had a hair-growing effect. FIGS. 3 and 4 show the
results of the observation. Minoxidil (e.g., 1% w/v; RiUP.RTM.,
Taisho Pharmaceutical Co., Ltd.) was used as a positive control
(FIG. 5). As clearly understood from FIGS. 3 and 4 and the brief
descriptions thereof, both applications of MK and PTN under these
conditions showed hair-growing effects on the alopecic regions of
the aged mice.
Example 6
Hair-Growing Effects of External Applications of MK and PTN are
Different to the Effect of Minoxidil
[0144] 2 .mu.g/ml of MK or PTN in 10% glycerol/PBS (v/v) was
prepared. 100 .mu.l of the solution of MK or PTN was applied
directly to shaved regions of C3H/HeJ mice (male, nine weeks old).
10% glycerol/PBS (v/v) and minoxidil (e.g., 1% w/v; RiUP.RTM.,
Taisho Pharmaceutical Co., Ltd.) were used as negative and positive
controls, respectively (FIGS. 6 and 7). It was observed for 21 days
after starting the application whether each of MK and PTN showed
enhancement of hair growth in the anagen phase. FIGS. 8 and 9 show
the results of the observation. The application of minoxidil
(RiUP.RTM.) showed enhancement of hair growth (FIG. 7). In
contrast, MK and PTN showed no such enhancement (FIGS. 8 and 9).
The available data suggest a different mode of action for MK to
minoxidil in animals that have merely been synchronized in the
anagen phase, without exhibiting symptoms of alopecia.
Example 7
Effect of External Application of MK on Acute Alopecia
Animal Ethics
[0145] All procedures were approved by the Royal Melbourne
Institute of Technology (RMIT) Animal Experimentation and Ethics
Committee (AEEC Project #1042), Melbourne, Australia.
Animals
[0146] A total of thirty-five mice were used in the study.
[0147] Pathogen-free female C57B1/6J mice were obtained from Animal
Resource Centre, Canning Vale, Western Australia, Australia.
Animals were housed under standard clean conventional animal house
conditions in a single room within the RDDT Animal Facility
(201.01), maintained at target valueS of 22.+-.2.degree. C. and
30-70% relative humidity, with a 12 h light:dark cycle. Animals
were fed irradiated Rat and Mouse Chow (Specialty Feeds, Glen
Forrest, Western Australia, Australia) and supplied municipal town
water ad libitum. Fresh water was supplied at least twice weekly.
Periodic testing of feed, bedding and water was undertaken to
identify any contaminants that might interfere with the study
results.
[0148] Animals were randomised into study groups, housed in groups
of two to three per cage and subjected to at least a two-day
quarantine period on receipt. The animals were identified by
implantation of microchip transponders by sub-cutaneous injection
between the shoulder blades. Secondary identification of animals
within each cage was made by use of tail markings with a permanent
marker. Mice were housed individually prior to the commencement of
treatment if there appeared to be excessive grooming of depilated
mouse skin by cage-mates.
[0149] As the commencement of treatment, mice were aged between
five weeks and seven weeks, and varied in weight by no more than
20% of their mean weight.
Induction of Acute Alopecia
[0150] Acute alopecia was induced according to the procedures
described by Paus et al., Am. J. Pathol. 144, 719-734 (1994), i.e.,
by a single intraperitoneal injection of cyclophosphamide to C57
BL/6 mice. The study includes 35 female C57B1/6J mice divided into
seven experimental groups of five mice each as outlined in the text
table below:
TABLE-US-00001 Cyclophosphamide Midkine Treatment No. of Group
mg/kg Study Day .mu.g/mL Study Day animals 1 0 9 0 2-29 5 2 50 9 0
2-29 5 3 50 9 10 2-29 5 4 50 9 10 10-29 5 5 150 9 0 2-29 5 6 150 9
10 2-29 5 7 150 9 10 10-29 5
[0151] The following protocol was employed:
[0152] Study Day 1: Mice were anaesthetized by intraperitoneal
injection with a combination of ketamine (50 mg/kg), xylazine (5
mg/kg) and acepromazine (0.8 mg/kg). Fur was removed from a test
area of approximately 2.times.4 cm on the backs of the mice, by
shaving with an electric clipper. The skin was rinsed with warm
water to clean the area and patted dry. The remaining fur was
removed by use of a commercial hair remover wax strip.
[0153] Study Days,2-9: Mice in groups 1, 2, 3, 5 and 6 were
administered a final dose concentration of 5.mu.g
midkine/mouse/day, or a control phosphate buffered saline (PBS)
solution, by dermal application to the test area in a volume of 0.5
mL. Mice in groups 4 and 7 were administered a control phosphate
buffered saline (PBS) solution, by dermal application to the test
area in a volume of 0.5 mL.
[0154] Study Day 9: Mice in all groups were treated with either
vehicle (water) or cyclophosphamide by intraperitoneal injection in
a dose volume of 10 mL/kg.
[0155] Study Days 10-29: Mice in groups 4 and 7 were administered a
final dose concentration of 5 .mu.g midkine/mouse/day, by dermal
application to the test area in a volume of 0.5 mL. Mice in groups
1, 2, 3, 5 and 6 were administered a final dose concentration of
5.mu.g midkine/mouse/day, or a control phosphate buffered saline
(PBS) solution, by dermal application to the test area in a volume
of 0.5 mL. Animals in groups 1, 2, 3, 5, and 6 that received the
control solution on days 2-9 also received the control solution on
days 10-29. Animals in groups 1, 2, 3, 5, and 6 that received the
midkine solution on days 2-9 also received the midkine solution on
days 10-29.
[0156] Study Day 30: Mice were sacrificed by exposure to a rising
concentration of carbon dioxide. A portion of skin in the target
area was collected and preserved in 10% neutral buffered formalin
for macroscopic and microscopic examination.
[0157] Mice were monitored daily from Study Day 1-29 for clinical
signs of toxicity. These observations will include assessment of
any changes in the following: [0158] Skin and fur (roughness,
piloerection, lack of grooming, fur loss); [0159] Eyes and mucous
membranes (discharge, cloudiness, sores around eyes); [0160]
Respiratory, circulatory, autonomic or central nervous system
functions; [0161] Somatomotor activity and behaviour patterns
(check for abnormal posture, gait and any abnormal activity);
[0162] Any tremors, convulsions, salivation, diarrhoea, lethargy,
excessive sleeping or coma.
Skin/Fur Observations
[0163] From Study Day 9-29 onwards, mice were observed daily for
signs of alopecia (fur loss) and changes in skin colour
pigmentation. The degree of fur loss was determined as a percentage
of target area with alopecia (fur loss), and skin pigmentation was
scored as black, grey or pink. Group incidence findings for
alopecia and skin pigmentation changes were determined for control
and test groups by One-Way Analysis of Variance tests using
GraphPad Prism 5.0 for Windows, GraphPad Software, San Diego Calif.
USA.
[0164] The extent of fur loss/re-growth was also documented by
twice-weekly photographs. To facilitate the photography process,
the mice were sedated using ketamine (50 mg/kg), xylazine (5
mg/kg), acepromazine (0.8 mg/kg) anaesthetic.
Terminal Measurements
[0165] Skin samples were collected from the target area of each
animal and preserved in 10% neutral buffered formalin for
histopathological assessment.
[0166] Data in FIG. 11 demonstrate that midkine protein reduces
cyclophosphamide-induced fur loss when administered before and/or
following cyclophosphamide, and that midkine increases a rate of
follicle recovery as determined by fur growth following cessation
of cyclophosphamide treatment. Data in FIG. 12 indicate that mice
treated with PBS exhibit extensive fur loss and grey or pink skin
colour, especially in their neck regions, consistent with a
predominance of follicles in catagen (grey) or telogen (pink). Mice
treated with midkine protein before and after CYP treatment
(MK2-29) showed reduced areas of fur loss relative to the group
receiving PBS, and skin was either uniformly black or dark grey in
color consistent with a predominance of follicles in anagen (black)
and a lower proportion in catagen. Almost no pink skin was evident
in this group, suggesting a much reduced proportion of follicles in
telogen. Similarly, mice treated with midkine protein only
following CYP treatment (MK10-29) showed reduced areas of fur loss
relative to the group receiving PBS, and appeared macroscopically
similar to. animals receiving midkine protein before and after
chemotherapy. The skin of mice receiving midkine protein only after
chemotherapy was uniformly dark grey in color, consistent with a
predominance of follicles in anagen or catagen.
Sequence CWU 1
1
41143PRTHomo sapiens 1Met Gln His Arg Gly Phe Leu Leu Leu Thr Leu
Leu Ala Leu Leu Ala1 5 10 15Leu Thr Ser Ala Val Ala Lys Lys Lys Asp
Lys Val Lys Lys Gly Gly 20 25 30Pro Gly Ser Glu Cys Ala Glu Trp Ala
Trp Gly Pro Cys Thr Pro Ser 35 40 45Ser Lys Asp Cys Gly Val Gly Phe
Arg Glu Gly Thr Cys Gly Ala Gln 50 55 60Thr Gln Arg Ile Arg Cys Arg
Val Pro Cys Asn Trp Lys Lys Glu Phe65 70 75 80Gly Ala Asp Cys Lys
Tyr Lys Phe Glu Asn Trp Gly Ala Cys Asp Gly 85 90 95Gly Thr Gly Thr
Lys Val Arg Gln Gly Thr Leu Lys Lys Ala Arg Tyr 100 105 110Asn Ala
Gln Cys Gln Glu Thr Ile Arg Val Thr Lys Pro Cys Thr Pro 115 120
125Lys Thr Lys Ala Lys Ala Lys Ala Lys Lys Gly Lys Gly Lys Asp 130
135 140 2168PRTHomo sapiens 2Met Gln Ala Gln Gln Tyr Gln Gln Gln
Arg Arg Lys Phe Ala Ala Ala1 5 10 15Phe Leu Ala Phe Ile Phe Ile Leu
Ala Ala Val Asp Thr Ala Glu Ala 20 25 30Gly Lys Lys Glu Lys Pro Glu
Lys Lys Val Lys Lys Ser Asp Cys Gly 35 40 45Glu Trp Gln Trp Ser Val
Cys Val Pro Thr Ser Gly Asp Cys Gly Leu 50 55 60Gly Thr Arg Glu Gly
Thr Arg Thr Gly Ala Glu Cys Lys Gln Thr Met65 70 75 80Lys Thr Gln
Arg Cys Lys Ile Pro Cys Asn Trp Lys Lys Gln Phe Gly 85 90 95Ala Glu
Cys Lys Tyr Gln Phe Gln Ala Trp Gly Glu Cys Asp Leu Asn 100 105
110Thr Ala Leu Lys Thr Arg Thr Gly Ser Leu Lys Arg Ala Leu His Asn
115 120 125Ala Glu Cys Gln Lys Thr Val Thr Ile Ser Lys Pro Cys Gly
Lys Leu 130 135 140Thr Lys Pro Lys Pro Gln Ala Glu Ser Lys Lys Lys
Lys Lys Glu Gly145 150 155 160Lys Lys Gln Glu Lys Met Leu Asp
1653156PRTHomo sapiens 3Met Gln His Arg Gly Phe Leu Leu Leu Thr Leu
Leu Ala Leu Leu Ala1 5 10 15Leu Thr Ser Ala Val Ala Lys Lys Lys Asp
Lys Val Lys Lys Gly Gly 20 25 30Pro Gly Ser Glu Cys Ala Glu Trp Ala
Trp Gly Pro Cys Thr Pro Ser 35 40 45Ser Lys Asp Cys Gly Val Gly Phe
Arg Glu Gly Thr Cys Gly Ala Gln 50 55 60Thr Gln Arg Ile Arg Cys Arg
Val Pro Cys Asn Trp Lys Lys Glu Phe65 70 75 80Gly Ala Asp Cys Lys
Tyr Lys Phe Glu Asn Trp Gly Ala Cys Asp Gly 85 90 95Gly Thr Gly Thr
Lys Val Arg Gln Gly Thr Leu Lys Lys Ala Arg Tyr 100 105 110Asn Ala
Gln Cys Gln Glu Thr Ile Arg Val Thr Lys Pro Cys Thr Pro 115 120
125Lys Thr Lys Ala Lys Ala Lys Gly Gln Arg Lys Glu Lys Gly Val Gly
130 135 140Leu Ser Arg Gly Ala Ala Pro Pro Pro Pro Arg Leu145 150
1554562DNAHomo sapiens 4atgcagcacc gaggcttcct cctcctcacc ctcctcgccc
tgctggcgct cacctccgcg 60gtcgccaaaa agaaagataa ggtgaagaag ggcggcccgg
ggagcgagtg cgctgagtgg 120gcctgggggc cctgcacccc cagcagcaag
gattgcggcg tgggtttccg cgagggcacc 180tgcggggccc agacccagcg
catccggtgc agggtgccct gcaactggaa gaaggagttt 240ggagccgact
gcaagtacaa gtttgagaac tggggtgcgt gtgatggggg cacaggcacc
300aaagtccgcc aaggcaccct gaagaaggcg cgctacaatg ctcagtgcca
ggagaccatc 360cgcgtcacca agccctgcac ccccaagacc aaagcaaagg
ccaaagccaa gaaagggaag 420ggaaaggact agacgccaag cctggatgcc
aaggagcccc tggtgtcaca tggggcctgg 480cccacgccct ccctctccca
ggcccgagat gtgacccacc agtgccttct gtctgctcgt 540tagctttaat
caatcatgcc cc 562
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