Hair loss abstract
The present invention discloses a topical scalp and transdermal
preparation with excellent penetration to the skin and follicle,
containing a [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cylosporin
derivative which is a non-immunosuppressive component with hair
growth stimulating ability. The topical scalp and transdermal preparation
is prepared by incorporating the cyclosporin derivative into a liposome,
microcapsule, micro-sphere, composite particle or emulsion, capable
of being employed as a hair growth stimulating agent and applied
for the prevention of hair loss.
Hair loss claims
We claim:
1. A topical scalp and transdermal preparation with good penetration
to the skin and follicle, prepared by encapsulating a [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
cylosporin derivative, represented by Chemical Formula 1 below,
having an excellent hair restoring effect without immunosuppresive
activity, in a carrier selected from the group consisting of a liposome,
microcapsule, microsphere, composite particle and emulsion: 6in
which A represents N-methyl-(4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine,
(2S,3R,4R,6E)-3-sulfhydryl-4-methyl-2-(methylamino)-6-octenoic acid
or (2S,4R,6E)-3-oxo-4-methyl-2-(methylamino)-6-octenoic acid; B
represents L-.alpha.-aminobutyric acid (Abu), L-alanine (Ala), L-threonine
(Thr), L-valine (Val) or L-norvaline (Nva); C represents a D-amino
acid represented by the general formula ("1"), CH.sub.3NH--CH(R)--COOH
in which, R is one selected from the group consisting of hydrogen,
C.sub.1-C.sub.6 straight or branched alkyl, alkenyl or alkynyl moieties,
substituted or unsubstituted, with one or more moieties selected
from the group consisting of amino, hydroxy, halo, haloalkyl, ester,
alkoxy, cyano, nitro, alkylamino, and dialkylamino, and general
formula ("2") X--R' in which, X is oxygen or sulfur, and
R' is one selected from the group consisting of hydrogen, and C.sub.1-C.sub.6
straight or branched alkyl, alkenyl or alkynyl moieties, substituted
or unsubstituted, with one or more moieties selected from the group
consisting of amino, hydroxy, halo, haloalkyl, ester, alkoxy, cyano,
nitro, alkylamino, and dialkylamino; HMeLeu represents .gamma.-hydroxy
N-methyl-L-leucine; D represents L-valine, L-norvaline or L-leucine;
E represents N-methyl-L-leucine, .gamma.-hydroxy N-methyl-L-leucine,
or L-leucine; F represents L-alanine or L-alanine thioamide ([.sup.7.psi..sup.8
CS--NH], NH--CHCH.sub.3--CS--); G represents D-hydroxyisovaleric
acid or a D-amino acid represented by the general formula ("3),
--NH--CH(CH.sub.2R)--COOH in which, R is hydrogen or general formula
("4") X--R' in which, X is oxygen or sulfur, and R' is
one selected from the group consisting of hydrogen, and C.sub.1-C.sub.6
straight or branched alkyl, alkenyl or alkynyl moieties, substituted
or unsubstituted, with one or more moieties selected from the group
consisting of amino, hydroxy, halo, haloalkyl, ester, alkoxy, cyano,
nitro, alkylamino, and dialkylamino; H represents N-methyl-L-leucine,
y-hydroxy-N-methyl-L-leucine or L-leucine; I represents N-methyl-L-leucine,
y-hydroxy-N-methyl-L-leucine or L-leucine; and J represents N-methyl-L-valine
or L-valine.
2. The topical scalp and transdermal preparation as set forth in
claim 1, wherein the cyclosporin derivative is represented by Chemical
Formula 2: 7in which MeBmt represents N-methyl-(4R)-4-[(E)-2-butenyl]-4-methyl-L-thr-
eonine; A' represents L-.alpha.-aminobutyric acid, L-alanine, L-threonine,
L-valine or L-norvaline; B' represents N-methyl-D-alanine, D-2-(methylamino)pent-4-enoyl,
N-methyl-D-aminobutyric acid, N-methyl-D-norvaline, D-2-(methylamino)hexa-4-ynoyl,
D-2-(methylamino)pent-4-ynoyl, D-2-methylthio-sarcosine, N-methyl-O-propenyl-D-serine
or N-methyl-D-serine; HMeLeu represents .gamma.-hydroxy-N-methyl-L-leucine;
Val represents L-valine; MeLeu represents N-methyl-L-leucine; C'
represents L-alanine or L-alanine thioamide ([.sup.7.psi..sup.8
CS--NH], NH--CHCH.sub.3--CS--); DAla represents D-alanine; D' represents
N-methyl-L-leucine, .gamma.-hydroxy-N-methyl-L-leucine or L-leucine;
and MeVal represents N-methyl-L-valine.
3. The topical scalp and transdermal preparation as set forth in
claim 1, wherein the cyclosporin derivative is represented by Chemical
Formula 3: 8in which MeBmt represents N-methyl-(4R)-4-[(E)-2-butenyl]-4-methyl-L-thr-
eonine; A" represents L-.alpha.-aminobutyric acid, L-alanine,
L-threonine, L-valine or L-norvaline; B" represents N-methyl-D-alanine,
D-2-(methylamino)pent-4-enoyl, N-methyl-D-aminobutyric acid, N-methyl-D-norvaline,
D-2-(methylamino)hexa-4-ynoyl, D-2-(methylamino)pent-4-ynoyl, D-2-methylthio-sarcosine,
N-methyl-O-propenyl-D-serine or N-methyl-D-serine; HMeLeu represents
.gamma.-hydroxy-N-methyl-L-leucine; Val represents L-valine; Ala
represents L-alanine; MeLeu represents N-methyl-L-leucine; DAla
represents D-alanine; and MeVal represents N-methyl-L-valine.
4. The topical scalp and transdermal preparation as set forth in
claim 1, wherein the cyclosporin derivative is [.gamma.-hydroxy-N-methyl-L-leucine-
.sup.4] cylosporin A.
5. The topical scalp and transdermal preparation as set forth in
claim 1, wherein the cyclosporin derivative is [.gamma.-hydroxy-N-methyl-L-leucine-
.sup.4] cylosporin C.
6. The topical scalp and transdermal preparation as set forth in
claim 1, wherein the cyclosporin derivative is [N-methyl-D-alanine.sup.3]
[.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cyclosporin A.
7. The topical scalp and transdermal preparation as set forth in
claim 1, wherein the cyclosporin derivative is [.gamma.-hydroxy-N-methyl-L-leucine-
.sup.4] [.gamma.-hydroxy-N-methyl-L-leucine.sup.9] cyclosporin A.
8. The topical scalp and transdermal preparation as set forth in
claim 1, wherein the cyclosporin derivative is [.gamma.-hydroxy-N-methyl-L-leucine-
.sup.4] [alanine thiomide.sup.7] cyclosporin A (or [.sup.7.psi..sup.8
CS--NH] cyclosporin A).
9. The topical scalp and transdermal preparation as set forth in
claim 1, wherein the cyclosporin derivative is [L-threonine].sup.2[L-leucine].sup.-
5[.gamma.-hydroxy-N-methyl-L-leucine.sup.4] [D-hydroxyisovaleric
acid].sup.8[L-leucine].sup.10 cyclosporin A.
10. The topical scalp and transdermal preparation as set forth
in claim 1, wherein the cyclosporin derivative is [.gamma.-hydroxy-N-methyl-L-leucine-
.sup.4] [D-serine] cyclosporin A.
11. The topical scalp and transdermal preparation as set forth
in claim 1, wherein the liposome encapsulating the cyclosporin derivative
is prepared by the steps of: dissolving amphiphilic molecules and
the cyclosporin derivative in organic solvent; evaporating the organic
solvent at ambient temperature, thereby giving a mixture of dry
lipid film consisting of the amphiphilic molecules and the cyclosporin
derivative; hydrating the dry phospholipid film by adding a certain
amount of an aqueous solution; and homogenizing the resultant film
using a mechanical dispersion instrument.
12. The topical scalp and transdermal preparation as set forth
in claim 11, wherein the organic solvent is selected from the group
consisting of acetone, methanol, ethanol, isopropanol, chloroform
and dichloromethane.
13. The topical scalp and transdermal preparation as set forth
in claim 11, wherein a weight ratio of the cyclosporin derivative/the
amphiphilic molecules is 1/100 to 1/1.
14. The topical scalp and transdermal preparation as set forth
in claim 11, wherein the mechanical dispersion instrument is selected
from the group consisting of a colloid mill, a roll mill, a sonicator,
a high-pressure dispersion instrument (microfluidizer, Microfluidics
Corp., USA), Ultra Turrax (Janke and Kunkel, Germany), Nanoget (Nanoget
Engineering, Germany) and Brogli (Italy).
15. The topical scalp and transdermal preparation as set forth
in claim 11, wherein the amphiphilic molecules are selected from
the group consisting of phospholipids such as phosphatidylcholine
(PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI),
cholesterol, cationic surfactants such as stearamidopropyldimethylamine
(SAPDA), distearyldimethylammoniumchloride (DSDAC) and stearyltrimethylammoniumchl-
oride (STAC), and stearic acid (SA).
16. The topical scalp and transdermal preparation as set forth
in claim 11, wherein the liposome has a diameter of several nanometers
to several micrometers.
17. The topical scalp and transdermal preparation as set forth
in claim 1, wherein the microcapsules encapsulating the cyclosporin
derivative is prepared by the steps of: dissolving the cyclosporin
derivative in an oil phase; emulsifying the oil phase in an aqueous
solution; creating a chemical reaction of capsule wall materials
in the aqueous phase of the emulsion.
18. The topical scalp and transdermal preparation as set forth
in claim 17, wherein the capsule wall materials are selected from
the group consisting of gelatin, carboxymethyl cellulose, arabic
gum, acacia gum, methylol melamine, and methylol urea.
19. The topical scalp and transdermal preparation as set forth
in claim 17, wherein the capsule wall materials in the aqueous phase
of the emulsion are subjected to condensation reaction.
20. The topical scalp and transdermal preparation as set forth
in any one of claim 1, wherein the microcapsule encapsulating the
cyclosporin derivative is prepared by the steps of: dissolving the
cyclosporin derivative and a polymer in an oil phase; dispersing
the oil phase in a second immiscible phase; and evaporating the
oil phase.
21. The topical scalp and transdermal preparation as set forth
in claim 20, wherein the polymer is selected from the group consisting
of poly(lactic acid) (PLA), poly(lactic acid-co-glycolic acid) (PLGA),
poly(.epsilon.-caprolactone) (PECL), and cellulose-acetate phthalate.
22. The topical scalp and transdermal preparation as set forth
in claim 20, wherein the oil phase is selected from the group consisting
of chloroform, dichloromethane, a mixture of dichloromethane/acetone,
and a mixture of dichloromethane/acetone.
23. The topical scalp and transdermal preparation as set forth
in claim 20, wherein the second phase immiscible with the oil phase
is an aqueous phase or gas phase.
24. The topical scalp and transdermal preparation as set forth
in claim 1, wherein the composite particle encapsulating the cyclosporin
derivative is prepared by the steps of: mixing the cyclosporin derivative
and surfactant in an aqueous phase; and forcibly dispersing the
solution using a mechanical dispersion instrument.
25. The topical scalp and transdermal preparation as set forth
in claim 24, wherein the surfactant is selected from the group consisting
of cationic surfactants such as distearyl dimethyl ammonium chloride
(DSDAC), anionic surfactants such as sodium lauryl sulfate (SLS),
amphiphlic surfactants such as cocodimethyl sulphopropyl betaine
(CDSPB), and nonionic surfactants such as Tween 60.
26. The topical scalp and transdermal preparation as set forth
in claim 24, wherein the cyclosporin derivative is contained at
0.01 to 35 weight % in a mixture of the cyclosporin derivative/surfactant/water.
27. The topical scalp and transdermal preparation as set forth
in claim 24, wherein a weight ratio of the surfactant to the cyclosporin
derivative is 1/100 to 100/1 in a mixture of the cyclosporin derivative/surfactant/water.
28. The topical scalp and transdermal preparation as set forth
in claim 24, wherein the mechanical dispersion instrument is selected
from the group consisting of a colloid mill, a roll mill, a sonicator,
a high-pressure dispersion instrument (microfluidizer, Microfluidics
Corp., USA), Ultra Turrax (Janke and Kunkel, Germany), Nanoget (Nanoget
Engineering, Germany) and Brogli (Italy) and mechanical dispersion
instruments equivalent thereto.
29. The topical scalp and transdermal preparation as set forth
in claim 1, wherein the emulsion encapsulating the cyclosporin derivative
is prepared by emulsifying the cyclosporin derivative in an oil
phase in an aqueous phase containing an emulsifying agent.
30. The topical scalp and transdermal preparation as set forth
in claim 29, wherein the oil phase is selected from the group consisting
of plant or animal oil such as sweet almond oil, avocado oil, castor
oil, olive oil, jojoba oil, sunflower oil, wheat germ oil, sesame
oil, ground nut oil, raisin seed oil, sova oil, rape seed oil, safflower
oil, coconut oil, corn oil, hazelnut oil, palm oil and apricot-kernel
oil, mineral oil such as fluid paraffin, synthetic oil such as caprylic/capric
triglycerides and triglycerides (C.sub.10-C.sub.18), and fatty acid
triglyceride.
31. The topical scalp and transdermal preparation as set forth
in claim 29, wherein the emulsifying agent is selected from the
group consisting of substances which are relatively hydrophilic
and have a surface activating ability, including polyvinyl alcohol,
gelatin, polysorbate 80, sodium alginate, sodium oleate, methyl
cellulose, albumin, sodium dodecyl sulfate, sodium lauryl sulfate,
polysorbate 20 and fluroric (F68).
32. The topical scalp and transdermal preparation as set forth
in claim 29, wherein a volume ratio of the oil phase to the aqueous
phase is 0.01/1 to 1.2/1.
33. A hair growth-promoting composition for use on hair, formulated
by applying the topical scalp and transdermal preparation as set
forth in claim 1, in which a cyclosporin derivative is encapsulated
in a carrier selected from the group consisting of a liposome, microcapsule,
microsphere, composite particle and emulsion.
Hair loss description
TECHNICAL FIELD
[0001] The present invention discloses a topical scalp and transdermal
preparation with excellent penetration to the skin and follicle,
containing a [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cylosporin
derivative which has a hair restoring ability with no immunosuppressive
activity. The preparation is characterized in that the cyclosporin
derivative is encapsulated in a liposome, microcapsule, microsphere,
composite particle or emulsion, capable of being employed as a hair
restoring agent and applied for the prevention of hair loss.
BACKGROUND ART
[0002] On average, the human scalp contains about 100,000 to 150,000
hairs. Each hair has three main stages of growth: anagen, catagen
and telogen, after which the hair falls out. This hair growth cycle
is repetitive and the duration of one cycle is different from other
cycles, ranging from approximately 3 to 6 years. Thus, the average
adult normally loses about 50 to 100 hairs every day. In general,
alopecia refers to a phenomenon wherein duration of the anagen growth
phase is shortened and the percentage of hairs in the catagen and
telogen phases increases, whereby the number of lost hairs is abnormally
increased.
[0003] There are many theories to explain loss of hair, including
for example, poor blood circulation, excessive functioning of male
sex hormone, excessive production and secretion of sebum, deterioration
of scalp by peroxides, bacteria, etc., hereditary factors, aging,
stress, etc. However, explicit mechanisms have not been revealed.
Recently, the population suffering from hair loss is tending to
increase, since changing dietary habits and stress imposed on individuals
due to modern social environments, etc. have increased. Also, the
age of the individuals affected by alopecia is dropping and furthermore,
the population of female alopecia sufferers is rising.
[0004] One of preparations which are most commonly used for treatment
and prevention of alopecia is one that contains minoxidil. There
are two hair-regrowth agents which have received approval from the
U.S. Food and Drug Administration, and minoxidil is one of those
approved hair-regrowth agents. Minoxidil was originally developed
as a hypertension drug for the purpose of reducing blood pressure.
However, when using this drug, as a side effect, a trichogenous
effect was observed and thereafter, this drug became famous as a
hair-regrowth agent. Although mechanisms by which minoxidil works
as a hair-regrowth agent is not clearly understood, it is inferred
that minoxidil increases blood flow by expansion of blood vessels,
whereby roots of hairs are supplied with more nutrition and eventually,
growth of hairs are promoted.
[0005] Such a model of blood flow increase has been indirectly
supported by a recent report that minoxidil enhances the expression
of vascular endothelial growth factor (VEGF), a growth factor associated
with vasodilatation in the dermal papilla which is a main cell making
up the hair roots (Br. J. of Dermatol., 1998, 138:407-411). Also,
other than the vasodilative effect of the minoxidil in the hair-restoring
mechanism, it has been reported that minoxidil promotes activation
of dermal papilla cells in the roots of hair incubated in vitro,
and growth of hair follicles in a tissue culture of follicles in
vitro (Skin Pharmacol., 1996, 9:3-8 and J. Invest. Dermatol., 1989,
92:315-320). These facts indicate that minoxidil may work directly
on the roots of hair as a growth factor.
[0006] In addition, finasteride, a main component of Propecia which
has started to be sold by Merck, is used for treatment of alopecia.
It inhibits conversion of the male hormone testosterone into dihydrotestosterone,
which is a more potent male hormone than testosterone. On December
of 1997, the 1 mg finasteride tablet was approved by the US FDA
as a hair-regrowth agent for treatment of male pattern hair loss
in men only, and is now commercially available. In clinical studies,
it has been demonstrated to have a significant trichogenous effect.
However, there has been a report that finasteride may inhibit male
sexual function as a side effect (J. Am. Acad Dermatol., 1998, 39:578-589).
Since neither finasteride nor minoxidil show superior effect in
clinical tests, and there is concern about side effects, many researches
are conducted to develop a new and improved hair-regrowth agents.
[0007] The cyclosporin family of drugs has immunosuppressive activity.
It is also effective to inhibit growth of virus, fungus, protozoan,
etc. and has various physiological effects such as nephrotoxicity,
hepatotoxicity, hypertension, enlargement of periodontium, trichogenous
effect, and so on, as side effects (Advances in Pharmacol., 1996,
35:114-246 and Drug Safety, 1994, 10:310-317). Cyclosporin A, a
representative cyclosporin, is a cyclic peptide having the following
Chemical Formula, which comprises 11 amino acids, including several
N-methyl amino acids and D-alanine at No. 8 residue. 1
[0008] where MeBrnt is N-methyl-(4R)-4-[(E)-2-butenyl]-4-methyl-L-threonin-
e, Abu is L-.alpha.-aminobutyric acid, Sar is sarcosine, MeLeu is
N-methyl-L-leucine, Val is L-valine, Ala is L-alanine, DAla is D-alanine,
and MeVal is N-methyl-L-valine.
[0009] The amino acid form of cyclosporin A of the above Chemical
Formula 1 is L-configuration, unless otherwise specified. The residue
numbering of amino acids starts from MeBmt and proceeds clockwise,
i.e. 1 for MeBmt and 11 for the last MeVal (N-methyl-L-valine) as
shown in the Structure Formula 1. Nomenclature of various derivatives
including cyclosporin derivatives A to Z, follows methods commonly
used (Helv. Chim. Acta, 1987, 70:13-36). For example, if Abu, the
No. 2 residue of cyclosporin A is substituted with L-alanine, L-threonine,
L-valine or L-norvaline, the derivatives thus prepared are named
cyclosporin B, cyclosporin C, cyclosporin D or cyclosporin G, respectively.
Further, when the amino acid residues of the cyclosporin derivatives
differ from those of cyclosporin A, the derivatives are named by
describing the substituent.
[0010] For example, if sarcosine, the No. 3 residue of cyclosporin
A, is substituted with N-methyl-D-Abu.sup.3 and N-methyl-D-Nva.sup.3,
the derivatives thus prepared are named [N-methyl-D-Abu.sup.3] cyclosporin
A and [N-methyl-D-Nva.sup.3] cyclosporin A, respectively.
[0011] Similarly, when a hydroxyl group is added to the gamma carbon
in N-methyl-L-leucine, the residue No. 4 of cyclosporin, by microbial
metabolism, being substituted to [.gamma.-hydroxy-N-methyl-L-leucine.sup.-
4], the derivatives are named by describing the substituted residue.
For example, if cyclosporin A is modified, the derivative is named
[.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cyclosporin A, and if
cyclosporin B and cyclosporin C are modified, the derivatives are
named [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cyclosporin B and
[.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cyclosporin C, respectively.
Also, in the case that two or more residues are substituted, the
derivatives are named in a similar manner. For example, if both
of residues No. 3 and No. 4 are substituted, the derivative is named
[N-methyl-D-alanine.sup.3] [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
cyclosporin A. If both of residues No. 4 and No. 9 are substituted,
the derivative is named [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
[.gamma.-hydroxy-N-methyl-L-leucine.sup.9] cyclosporin A. Also,
if substitution at the residues No. 4 and No. 7 concurs, the derivative
is described as [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] [alanine
thiomide.sup.7, [.sup.7.psi..sup.8 CS--NH] cyclosporin A.
[0012] Regarding a peptolide in which the residue No. 8, D-alanine,
is substituted with D-hydroxyisovaleric acid, forming an ester bond
in its amino sequence, if the residue No. 4, N-methyl-L-leucine
is converted to [.gamma.-hydroxy-N-methyl-L-leucine.sup.4], the
cyclosporin derivative is described as [L-threonine.sup.2] [L-leucine.sup.5]
[.gamma.-hydroxy-N-methyl-L-leucine.sup.4] [D-hydroxyisovaleric
acid.sup.8] [L-leucine.sup.10] cyclosporin A. Further, as for a
derivative of cyclosporin which is substituted with sulfur instead
of a carbonyl oxygen at the residue No. 7, the name of the derivative
may be cyclosporin 7-thioamide or [.sup.7.psi..sup.8 CS--NH] cyclosporin,
according to different references (Helv. Chim. Acta. 74: 1953-1990,
1991; J. Org. Chem. 58: 673-677, 1993; J. Org. Chem. 59: 7249-7258,
1994).
[0013] Meanwhile, a common method for abbreviating amino acids
is employed, that is, N-methyl-L-leucine is abbreviated by MeLeu,
N-methyl-L-isoleucine by MeIle, N-methyl-L-Valine by MeVal, N-methyl-L-alanine
by MeAla, N-methyl-L-norvaline by MeNva, L-leucine by Leu, L-isoleucine
by Ile, sarcosine by Sar, L-serine by Ser, L-valine, Val, L-alanine
by Ala, D-alanine by DAla, L-.alpha.-aminobutyric acid by Abu, L-threonine
by Thr, and L-norvaline by Nva. In the invention, the term `cyclosporin
derivatives` generally refers to [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
cylosporin derivatives.
[0014] So far, possible development of cyclosporin as a hair-regrowth
agent has been studied by many research groups. Particularly, research
involving animal hair regrowth tests (Arch, Dermatol. Res., 1996,
288:408-410), human alopecia greata (J. Am. Acad. Dermatol., 1990,
22:242-250), human male pattern alopecia (J. Am. Acad. Dermatol.,
1990, 22:251-253 and Skin Pharmacol., 1994, 7:101-104), and inhibition
effect of hair loss by chemotherapy in animal models (Clin. Lab.
Invest., 1995, 190:192-196 and Am. J. Pathol., 1997, 150:1433-1441)
have been widely conducted. In comparative experiments on mouse's
back, it is shown that cyclosporin has a hair regrowth effect about
100 times superior to minoxidil. Based on such findings, there have
been attempts to utilize cyclosporin as a treatment for male pattern
alopecia, and many applications for patents have been filed.
[0015] For example, Japanese Patent Publication Kokai Nos. Sho
60-243008, Sho 62-19512 and Sho 62-19513 disclose use of cyclosporin
derivatives as a hair regrowth agent. Also, European Patent Publication
No. 0414632 B1 discloses cyclosporin derivatives modified at residue
No. 8, and PCT Patent Publication No. WO 93/17039 discloses isocyclosporin
provided as a hair regrowth agent. In U.S. Pat. No. 5,807,820 and
U.K. Patent No. 2,218,334 A, preparations containing cyclosporin
with excellent transdermal absorption are suggested for new application
of a hair regrowth agent. Furthermore, PCT Patent Publication No.
WO 00/51558 discloses hair regrowth using immunosuppressive cyclosporin
derivatives. These references do not include a structure of [.gamma.-hydroxy-N-methyl--
L-leucine.sup.4] cyclosporin A derivative claimed herein.
[0016] Although cyclosporin derivatives disclosed in the above
references have good hair restoring effects versus common hair loss,
they all show a strong immunosuppressive activity, limiting their
application as hair regrowth agents. In addition to their severe
side effect of immunosuppression, those cyclosporin derivatives
disclosed in the prior art have a problem with their delivery to
the skin and follicle.
DISCLOSURE OF THE INVENTION
[0017] Therefore, the present invention has been made in view of
the above problems, and it is an object of the present invention
to provide compositions of carrier particles for use on the scalp
and the skin, and methods of preparing them, by which penetration
of a novel hair growth promoting agent to the follicle and the skin
can be maximized, thereby exerting an excellent hair restoring effect.
The present inventors found that as the hair growth promoting agent,
[.gamma.-hydroxy-N-methyl-L-leuc- ine.sup.4] cyclosporin derivatives,
which are modified at residue No. 4 in their amino acid sequence,
uniquely exhibit an excellent hair restoring effect without immunosuppressive
activity. Further, the inventors have studied to develop various
formulations so that [.gamma.-hydroxy-N-methyl- -L-leucine.sup.4]
cyclosporin derivatives may be well penetrated to the follicle and
the skin. As a result, carriers having an excellent drug delivery
effect, such as liposomes, microcapsules, microspheres, composite
particles and emulsions, were manafactured. Those formulations can
be used in manufacturing a hair restoring agent and an agent for
the prevention of hair loss, although the cyclosporin derivatives
are difficult agents to penetrate to the follicle and the skin,
due to their high molecular weight.
[0018] More specifically, the invention is directed to compositions
of carrier particles with a high degree of skin penetration and
improved in vivo hair restoring effects. The compositions of carrier
particles of the invention were prepared by encapsulating a non-immunosuppresive
cyclosporin derivative, which is an active ingredient with hair
restoration property, in carriers such as liposomes, microcapsules,
microspheres, composite particles and emulsions. Since the compositions
exhibit excellent penetration to the follicle and the skin, they
are useful for hair restoring and for the prevention of hair loss.
[0019] In accordance with one aspect of the invention, the above
and other objects can be accomplished by the provision of a composition
of a carrier particle with a high degree of penetration to the follicle
and the skin, and thus excellent hair restoring effects, and a method
of preparing the same. The composition of the carrier particle of
the invention is prepared by encapsulating one of [.gamma.-hydroxy-N-methyl-L-
-leucine.sup.4] cyclosporin derivatives, represented by Chemical
Formula 1 below, having an excellent hair restoring effect without
immunosuppresive activity, in the carrier such as a liposome, microcapsule,
microsphere, composite particle or emulsion. 2
[0020] wherein:
[0021] A represents N-methyl-(4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine,
(2S,3R,4R,6E)-3-sulfhydryl-4-methyl-2-(methylamino)-6-octenoic acid
or (2S,4R,6E)-3-oxo-4-methyl-2-(methylamino)-6-octenoic acid; B
represents L-.alpha.-aminobutyric acid (Abu), L-alanine (Ala), L-threonine
(Thr), L-valine (Val) or L-norvaline (Nva); C represents a D-amino
acid represented by the general formula 1,
CH.sub.3NH--CH(R)--COOH [General formula 1]
[0022] in which R is one selected from the group consisting of
hydrogen, C.sub.1-C.sub.6 straight or branched alkyl, alkenyl or
alkynyl moieties, substituted or unsubstituted with one or more
selected from the group consisting of amino, hydroxy, halo, haloalkyl,
ester, alkoxy, cyano, nitro, alkylamino, and dialkylamino, and X--R'
represented by the general formula 2 below,
--X--R' [General formula 2]
[0023] in which X is oxygen or sulfur, and R' is one selected from
the group consisting of hydrogen, and C.sub.1-C.sub.6 straight or
branched alkyl, alkenyl or alkynyl moieties, substituted or unsubstituted
with one or more selected from the group consisting of amino, hydroxy,
halo, haloalkyl, ester, alkoxy, cyano, nitro, alkylamino, and dialkylamino;
HMeLeu represents .gamma.-hydroxy N-methyl-L-leucine; D represents
L-valine, L-norvaline or L-leucine; E represents N-methyl-L-leucine,
y-hydroxy N-methyl-L-leucine, or L-leucine; F represents L-alanine
or L-alanine thioamide ([.sup.7.psi..sup.8 CS--NH], NH--CHCH.sub.3--CS--);
G represents D-hydroxyisovaleric acid or a D-amino acid represented
by the general formula 3,
--NH--CH(CH.sub.2R)--COOH [General formula 3]
[0024] in which R is hydrogen or X--R' represented by the general
formula 4,
--X--R' [General formula 4]
[0025] in which X is oxygen or sulfur, and R' is one selected from
the group consisting of hydrogen, and C.sub.1-C.sub.6 straight or
branched alkyl, alkenyl or alkynyl moieties, substituted or unsubstituted
with one or more selected from the group consisting of amino, hydroxy,
halo, haloalkyl, ester, alkoxy, cyano, nitro, alkylamino, and dialkylamino;
H represents N-methyl-L-leucine, y-hydroxy-N-methyl-L-leucine or
L-leucine; I represents N-methyl-L-leucine, .gamma.-hydroxy-N-methyl-L-leucine
or L-leucine; and J represents N-methyl-L-valine or L-valine.
[0026] In accordance with another aspect of the invention, there
is provided a composition of a carrier particle with a high degree
of penetration to the follicle and the skin, and thus excellent
hair restoring effects, and a method of preparing the same. The
composition of the carrier particle of the invention is prepared
by encapsulating one of [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
cyclosporin derivatives, represented by Chemical Formula 2 below,
having an excellent hair restoring effect without immunosuppresive
activity, in the carrier such as a liposome, microcapsule, microsphere,
composite particle or emulsion. 3
[0027] wherein:
[0028] MeBmt represents N-methyl-(4R)-4-[(E)-2-butenyl]-4-methyl-L-threoni-
ne; A' represents L-.alpha.-aminobutyric acid, L-alanine, L-threonine,
L-valine or L-norvaline; B' represents N-methyl-D-alanine, D-2-(methylamino)pent-4-enoyl,
N-methyl-D-aminobutyric acid, N-methyl-D-norvaline, D-2-(methylamino)hexa-4-ynoyl,
D-2-(methylamino)pent-4-ynoyl, D-2-methylthio-sarcosine, N-methyl-O-propenyl-D-serine
or N-methyl-D-serine; HMeLeu represents y-hydroxy-N-methyl-L-leucine;
Val represents L-valine; MeLeu represents N-methyl-L-leucine; C'
represents L-alanine or L-alanine thioamide ([.sup.7.psi..sup.8
CS--NH], NH--CHCH.sub.3--CS--); DAla represents D-alanine; D' represents
N-methyl-L-leucine, -hydroxy-N-methyl-L-leucine or L-leucine; and
MeVal represents N-methyl-L-valine.
[0029] In accordance with another aspect of the invention, there
is provided a composition of a carrier particle with a high degree
of penetration to the follicle and the skin, and thus excellent
hair restoring effects, and a method of preparing the same. The
composition of the carrier particle of the invention is prepared
by encapsulating one of [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
cyclosporin derivatives, represented by Chemical Formula 3 below,
having an excellent hair restoring effect without immunosuppresive
activity, in the carrier such as a liposome, microcapsule, microsphere,
composite particle or emulsion. 4
[0030] wherein:
[0031] MeBmt represents N-methyl-(4R)-4-[(E)-2-butenyl]-4-methyl-L-threoni-
ne; A" represents L-.alpha.-aminobutyric acid, L-alanine, L-threonine,
L-valine or L-norvaline; B" represents N-methyl-D-alanine,
D-2-(methylamino)pent-4-enoyl, N-methyl-D-aminobutyric acid, N-methyl-D-norvaline,
D-2-(methylamino)hexa-4-ynoyl, D-2-(methylamino)pent-4-ynoyl, D-2-methylthio-sarcosine,
N-methyl-O-propenyl-D-serine or N-methyl-D-serine; HMeLeu represents
.gamma.-hydroxy-N-methyl-L-leucine; Val represents L-valine; Ala
represents L-alanine; MeLeu represents N-methyl-L-leucine; DAla
represents D-alanine; and MeVal represents N-methyl-L-valine.
[0032] In accordance with another aspect of the invention, there
is provided a composition of a carrier particle with a high degree
of penetration to the follicle and the skin, and thus excellent
hair restoring effects, and a method of preparing the same. The
composition of the carrier particle of the invention is prepared
by encapsulating [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cylosporin
A (Compound 1), having an excellent hair restoring effect without
immunosuppresive activity, in the carrier such as a liposome, microcapsule,
microsphere, composite particle or emulsion.
[0033] In accordance with another aspect of the invention, there
is provided a composition of a carrier particle with a high degree
of penetration to the follicle and the skin, and thus excellent
hair restoring effects, and a method of preparing the same. The
composition of the carrier particle of the invention is prepared
by encapsulating [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cylosporin
C (Compound 2), having an excellent hair restoring effect without
immunosuppresive activity, in the carrier such as a liposome, microcapsule,
microsphere, composite particle or emulsion.
[0034] In accordance with another aspect of the invention, there
is provided a composition of a carrier particle with a high degree
of penetration to the follicle and the skin, and thus excellent
hair restoring effects, and a method of preparing the same. The
composition of the carrier particle of the invention is prepared
by encapsulating [N-methyl-D-alanine.sup.3] [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
cyclosporin A (Compound 3), having an excellent hair restoring effect
without immunosuppresive activity, in the carrier such as a liposome,
microcapsule, microsphere, composite particle or emulsion.
[0035] In accordance with another aspect of the invention, there
is provided a composition of a carrier particle with a high degree
of penetration to the follicle and the skin, and thus excellent
hair restoring effects, and a method of preparing the same. The
composition of the carrier particle of the invention is prepared
by encapsulating [.gamma.-hydroxy-N-methyl-L-leucine4] [.gamma.-hydroxy-N-methyl-L-leucine-
9] cyclosporin A (Compound 4), having an excellent hair restoring
effect without immunosuppresive activity, in the carrier such as
a liposome, microcapsule, microsphere, composite particle or emulsion.
[0036] In accordance with another aspect of the invention, there
is provided a composition of a carrier particle with a high degree
of penetration to the follicle and the skin, and thus excellent
hair restoring effects, and a method of preparing the same. The
composition of the carrier particle of the invention is prepared
by encapsulating [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] [alanine
thiomide.sup.7] cyclosporin A (or [.sup.7.PSI..sup.8 CS--NH] cyclosporin
A) (Compound 5), having an excellent hair restoring effect without
immunosuppresive activity, in the carrier such as a liposome, microcapsule,
microsphere, composite particle or emulsion.
[0037] In accordance with another aspect of the invention, there
is provided a composition of a carrier particle with a high degree
of penetration to the follicle and the skin, and thus excellent
hair restoring effects, and a method of preparing the same. The
composition of the carrier particle of the invention is prepared
by encapsulating [L-threonine].sup.2[L-leucine].sup.5 [.gamma.-hydroxy-N-methyl-L-leucine.-
sup.4] [D-hydroxyisovaleric acid].sup.8[L-leucine].sup.10 cyclosporin
A (Compound 6), having an excellent hair restoring effect without
immunosuppresive activity, in the carrier such as a liposome, microcapsule,
microsphere, composite particle or emulsion.
[0038] In accordance with yet another aspect of the invention,
there is provided a composition of a carrier particle with a high
degree of penetration to the follicle and the skin, and thus excellent
hair restoring effects, and a method of preparing the same. The
composition of the carrier particle of the invention is prepared
by encapsulating [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] [D-serine.sup.8]
cyclosporin A, having an excellent hair restoring effect without
immunosuppresive activity, in the carrier such as a liposome, microcapsule,
microsphere, composite particle or emulsion.
[0039] With regard to the composition of the carrier particle loading
a [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cyclosporin derivative,
the non-immunosuppressive cyclosporin derivative, which has a high
degree of penetration to the follicle and the skin, and thus excellent
hair restoring effect, and the method of preparing the same, the
present invention is more directed to a liposome composition and
a method of preparing the same. Preferably, the liposome is prepared
as follows. Amphiphilic molecules and a cyclosporin derivative are
dissolved in organic solvent. The organic solvent is evaporated
at ambient temperature, giving a mixture of dry lipid film consisting
of the amphiphilic molecules and the cyclosporin derivative. Then,
a certain amount of an aqueous solution is added to hydrate the
dry phospholipid film. The resultant film is homogenized at 500
bar using a mechanical dispersion instrument.
[0040] With regard to the composition of the carrier particle loading
a [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cyclosporin derivative,
the non-immunosuppressive cyclosporin derivative, which has a high
degree of penetration to the follicle and the skin, and thus excellent
hair restoring effect, and the method of preparing the same, the
present invention is more directed to a microcapsule composition
and a method of preparing the same. Preferably, the microcapsule
is prepared as follows. A cyclosporin derivative is dissolved in
an oil phase. The oil phase is emulsified in an aqueous solution.
Then, the capsule wall materials in the aqueous phase of the emulsion
are subjected to a chemical reaction.
[0041] With regard to the composition of the carrier particle loading
a [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cyclosporin derivative,
the non-immunosuppressive cyclosporin derivative, which has a high
degree of penetration to the follicle and the skin, and thus excellent
hair restoring effect, and the method of preparing the same, the
present invention is more directed to a microsphere composition
and a method of preparing the same. Preferably, the microsphere
is prepared as follows. A cyclosporin derivative and a polymer are
dissolved in an oil phase. The oil phase is dispersed in a second
immiscible phase. The oil phase is then evaporated.
[0042] With regard to the composition of the carrier particle loading
a [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cyclosporin derivative,
the non-immunosuppressive cyclosporin derivative, which has a high
degree of penetration to the follicle and the skin, and thus excellent
hair restoring effect, and the method of preparing the same, the
present invention is more directed to a composite particle composition
and a method of preparing the same. Preferably, the composite particle
is prepared as follows. A cyclosporin derivative and surfactant
are mixed in an aqueous phase. The solution is forcibly dispersed
using a mechanical dispersion instrument.
[0043] With regard to the composition of the carrier particle loading
a [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cyclosporin derivative,
the non-immunosuppressive cyclosporin derivative, which has a high
degree of penetration to the follicle and the skin, and thus excellent
hair restoring effect, and the method of preparing the same, the
present invention is more directed to an emulsion composition and
a method of preparing the same. Preferably, the emulsion is prepared
as follows. A cyclosporin derivative in an oil phase is emulsified
in an aqueous phase containing an emulsifying agent.
[0044] With regard to the composition of the carrier particle loading
a [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cyclosporin derivative,
the non-immunosuppressive cyclosporin derivative, which has a high
degree of penetration to the follicle and the skin, and thus excellent
hair restoring effect, and the method of preparing the same, the
present invention is more directed to compositions for use on hair
and methods of preparing the same. The liposomes, microcapsules,
microspheres, composite particles or emulsions carrying the cyclosporin
derivatives may be applied in preparing the compostions for use
on hair.
[0045] In the invention, [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
cyclosporin derivatives, the non-immunosuppressive hair-restoring
agent, are encapsulated in carrier particles such as liposomes,
microcapsules, microspheres, composite particles and emulsions,
thereby improving penetration to the follicle and the skin. As revealed
in hair growth tests, these encapsulated cyclosporin derivatives
showed an excellent hair restoring effect.
[0046] Liposomes of the invention can be prepared by dispersing
a cyclosporin derivative and amphiphilic molecules such as phospholipids
or other bilayer-forming molecules in an aqueous phase. As for phospholipids,
phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine
(PS), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol
(PI), and derivatives thereof may be used. The other amphiphilic
molecules which form bilayers include surfactants such as acyl N.sub.n,N.sub.n-dimethyl-1,n-diamino
alkyl (n=2-10, 8-30 carbon atoms in the alkyl group), dialkyl dimethyl
ammonium salt (12-18 carbon atoms in the alkyl chain, anion is Cl.sup.-
or Br.sup.-) and monoalkyl trimethyl ammonium salt (12-22 carbon
atoms in the alkyl chain, anion is Cl.sup.- or Br.sup.-), and fatty
acids (8-30 carbon atoms in the alkyl chain). As for instruments
for dispersion, a colloid mill depending on the friction force of
a grinding stone which is rotated at high speed, a roll mill depending
on the shear forces generated between adjacent rolls, in which several
metal rolls are successively rotated, and a sonicator depending
on mechanical disruption by ultrasonic energy may be used. Also,
a high-pressure dispersion instrument (microfluidizer, Microfluidics
Corp., USA) can be used. With this instrument, dispersion is achieved
by shear stress, collision energy and cavitation, generated by flowing
a liquid through a narrow orifice under high pressure. Other available
instruments for homogenization may include Ultra Turrax (Janke and
Kunkel, Germany), Nanoget (Nanoget Engineering, Germany) and Brogli
(Italy). For preparing liposomes, a weight ratio of a cyclosporin
derivative to amphiphilic molecules is 1/100 to 1/1, preferably
1/10 to 7/10, most preferably 1/5 to 1/2. If the cyclosporin derivative
is employed at a weight ratio lower than these ranges, its hair
restoring effect cannot be obtained. On the other hand, if the ratio
is higher, liposomes fail to be formed. The weight % of amphiphilic
molecules in a liposome suspension is 1 to 40%, preferably 2 to
30%, most preferably 3 to 20%. If the amphiphilic molecules are
employed at a weight % lower than these ranges, it is hard to apply
the cyclosporin derivative at the amount which exhibits its hair
restoring effect into a composition for use on human hair. On the
other hand, if the content is higher, a liposome is not likely to
be formed since a dispersed state is poor upon liposome preparation.
[0047] Microcapsules of the invention can be prepared as follows.
In the microcapsules are encapsulated [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
cyclosporin derivatives which are non-immunosuppressive hair-restoring
agents. A cyclosporin derivative in an oil phase is emulsified in
an aqueous phase in which wall materials of a microcapsule are dissolved.
The wall materials in the aqueous phase are subjected to cross-linking
or a condensation reaction, preparing the microcapsule. As for the
wall materials, gelatin, carboxymethyl-cellulose (CMC), alginate,
Arabic gum, acacia gum, methylol melamine, methylol urea, and derivatives
thereof may be used. A cross-linking agent may include formaldehyde
and glutaraldehyde. For oil phase, silicone oil such as polydimethyl
siloxane (viscosity: 1.5.times.10.sup.-6 m.sup.2/s-2.5.times.10.sup.-1
m.sup.2/s; Trade name: DC Fluid 200 (Dow Corning Co.), Abil 10,
Abil K03 (Goldschmidt), Rhodorsil 47 V 10, Silbione 70047 V 10,
Silbione 70047 V 100, Silbione 70047 V 300 (Rhone Poulenc), and
Siliconee Oil L45 (Union Carbide)), plant or animal oil such as
sweet almond oil, avocado oil, castor oil, olive oil, jojoba oil,
sunflower oil, wheat germ oil, sesame oil, ground nut oil, raisin
seed oil, sova oil, rape seed oil, safflower oil, coconut oil, corn
oil, hazelnut oil, palm oil and apricot-kernel oil, mineral oil
such as fluid paraffin, synthetic oil such as caprylic/capric triglycerides
and triglycerides (C.sub.10-C.sub.18), and fatty acid triglyceride
may be used.
[0048] Microspheres of the invention can be prepared as follows.
In the microspheres are encapsulated [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
cyclosporin derivatives which are non-immunosuppressive hair-restoring
agents. An oil phase in which a polymer is dissolved is emulsified
in an aqueous phase in which an emulsifying agent is dissolved,
thereby giving an O/W emulsion. A spontaneous reciprocal diffusion
between the oil phase and the aqueous phase occurs, making the polymer
insoluble. As a result, a polymer microsphere is formed, and the
organic solvent is evaporated. As for the polymer, poly(lactic acid)
(PLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid)
(PLGA), poly(E-caprolactone) (PECL), and cellulose-acetate phthalate
may be used. For the oil phase, dichloromethane, chloroform, acetone,
a mixture of dichloromethane/acetone, and a mixture of acetone/propylene
glycol dicaprylate dicaprate may be used. The emulsifying agent
includes polyvinyl alcohol and gelatin.
[0049] Composite particles of the invention, that is, [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
cyclosporin derivative/surfactant composite particles can be prepared
as follows. The cyclosporin derivative, a surfactant and distilled
water are measured out at respective desired amounts, and they are
mixed. The solution was homogenized using a mechanical dispersion
instrument, thereby preparing a fine composite particle. The surfactant
serves as a stabilizer for dispersion of the cyclosporin derivative.
[0050] As for the surfactant, anionic, cationic, nonionic or amphiphilic
surfactants can be used. The anionic surfactant includes alkyl sulphates,
alkyl ether sulphates, alkaryl sulphonates, alkanoyl isethionates,
alkyl succinates, alkyl sulphosuccinates, N-alkoylsarcosinates,
alkyl phosphates, alkyl ether phosphates, alkyl ether carboxylates,
alpha-olefin sulphonates, especially sodium, magnesium, and ammonium
salts thereof, and mono-, di-, and tri-ethanolamine salts thereof.
In these surfactants, alkyl and acyl groups have generally 8 to
18 carbon atoms, and are unsaturated. Alkyl ether sulphates, alkyl
ether phosphates, and alkyl ether carboxylates have respectively
1 to 10 ethylene oxide or propylene oxide groups. Particular examples
of the anionic surfactants include sodium oleyl succinate, ammonium
lauryl sulphosuccinate, ammonium lauryl sulphate, sodium dodecylbenzene
sulphonate, triethanolamine dodecylbenzene sulphonate, sodium cocoyl
isethionate, sodium lauroyl isethionate, sodium N-lauryl sarcosinate.
The most preferred anionic surfactants are sodium lauryl sulphate,
triethanolamine lauryl sulphate, triethanolamine monolauryl phosphate,
sodium lauryl ether sulphate (having 1 to 3 ethylene oxide groups),
ammonium lauryl sulphate, sodium lauryl ether sulphate (having 1
to 3 ethylene oxide groups).
[0051] As for a cationic surfactant, a quaternary ammonium salt
represented by the following structure can be used. 5
[0052] In the above formula, among R.sub.1, R.sub.2, R.sub.3 and
R.sub.4, one or two is/are linear alkyl or branched alkyl or hydroxy
alkyl groups (C.sub.8-C.sub.22), and the others are alkyl groups
having 1 to 3 carbon atoms, hydroxy alkyl groups, benzyl groups
or polyoxyethylene groups. X is a halogen atom or an alkyl sulfate
group having one or two carbon atoms. Among the quaternary ammonium
salts, distearyldimethylammonium chloride, stearyl trimethylammonium
methosulfate, stearyltrimethylammoniu- m chloride, stearyldimethylbenzylammonium
chloride, docosyltrimethylammonium methosulfate, docosyltrimethylammonium
chloride, docosyldimethylbenzylammonium chloride, didocosyldimethylammonium
chloride, lauryldiethylbenzylammonium chloride, lauryltrimethylammonium
bromide, distearylmethylhydroxymethyl chloride, cetyltrimethylammonium
chloride, N-stearyl-N,N,N-tri(polyoxyethylene)ammonium chloride,
cetyltriethylammonium bromide or stearyldimethylammonium chloride
is preferably used.
[0053] As for a nonionic surfactant, a primary or secondary fatty
alcohol (C.sub.8-C.sub.18, linear or branched chain) or a product
obtained from a condensation reaction between phenol and alkylene
oxide (mainly having 6 to 30 ethylene oxide groups) can be used.
Other nonionic surfactants suitable for use in the invention include
alkyl-polyglycosides, mono-, di-alkyl alkanol amides, and coco mono-isopropanolamide.
[0054] Suitable amphiphilic surfactants used in the invention may
include alkyl amine oxides, alkyl betaines, alkyl amidoprophyl betaines,
alkyl sulphobetaines, alkyl glycinates, alkyl carboxyglycinates,
alkyl amphopropionates, alkyl amphoglycinates, and alkyl amidopropyl
hydroxylsultaines. In these surfactants, the alkyl and acyl groups
have 8 to 19 carbon atoms. For example, lauryl amine oxide or cocodimethyl
sulphopropyl betaine can be used. Lauryl betaine, cocamidopropyl
betaine or sodium cocamphopripionate is preferably used.
[0055] For preparing the composite particles of the invention,
in a mixture of [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cyclosporin
derivative/surfactant/water, a weight % of the cyclosporin derivative
is 0.01 to 35%, preferably 1 to 30%, most preferably 2 to 25%. A
weight ratio of the surfactant to the cyclosporin derivative is
1/100 to 100/1, preferably 1/50 to 50/1, most preferably 1/25 to
25/1. If the contents of cyclosporin derivative and surfactant employed
are lower than these ranges, it cannot be ensured that the final
cyclosporin derivative content is in a high range of 0.1 to 3% which
exhibits hair restoring effect, since the cyclosporin derivative/surfactant
composite particles are diluted upon preparing a composition for
use on human hair. On the other hand, if the contents are higher,
it is hard to disperse them using a mechanical dispersion instrument.
[0056] As for the dispersion instrument, a colloid mill depending
on the friction force of a grinding stone which is rotated at high
speed, a roll mill depending on the shear forces generated between
adjacent rolls, in which several metal rolls are successively rotated,
or a sonicator depending on mechanical disruption by ultrasonic
energy may be used. Also, a high-pressure dispersion instrument
(microfluidizer, Microfluidics Corp., USA) can be used. With the
instrument, dispersion is achieved by shear stress, collision energy
and cavitation, generated by flowing a liquid through a narrow orifice
under high pressure. Other available instruments for homogenization
may include Ultra Turrax (Janke and Kunkel, Germany), Nanoget (Nanoget
Engineering, Germany) and Brogli (Italy) and mechanical dispersion
instruments equivalent thereto.
[0057] Emulsions according to the invention contain cyclosporin
derivatives, and the method of preparing them is as follows. An
oil phase in which the cyclosporin derivative is dissolved is emulsified
in an aqueous phase containing an emulsifying agent. As for the
oil phase, plant or animal oil such as sweet almond oil, avocado
oil, castor oil, olive oil, jojoba oil, sunflower oil, wheat germ
oil, sesame oil, ground nut oil, raisin seed oil, sova oil, rape
seed oil, safflower oil, coconut oil, corn oil, hazelnut oil, palm
oil and apricot-kernel oil, mineral oil such as fluid paraffin,
synthetic oil such as caprylic/capric triglycerides and triglycerides
(C.sub.10-C.sub.18), and fatty acid triglyceride may be used.
[0058] For preparing the emulsions of the invention, a volume ratio
of the oil phase to the aqueous phase is 0.01/1 to 1.2/1, preferably
0.05/1 to 1/1, most preferably 0.1/1 to 0.7/1. If the volume ratio
of the oil phase to the aqueous phase is lower than these ranges,
it cannot be ensured that the final cyclosporin derivative content
is in a high range of 0.1 to 3% which exhibits hair restoring effect,
since the emulsions containing cyclosporin derivatives are diluted
upon preparing a composition for use on human hair. On the other
hand, if the volume ratio is higher, it is hard to manufacture stable
emulsions.
[0059] As for the emulsifying agent, substances which are relatively
hydrophilic and have a surface activating ability, including polyvinyl
alcohol, gelatin, polysorbate 80, sodium alginate, sodium oleate,
methyl cellulose, albumin, sodium dodecyl sulfate, sodium lauryl
sulfate, polysorbate 20 and fluroric (F68), and the cationic, anionic,
amphiphilic, nonionic surfactants which are used in preparing the
composite particles of the invention can be used. As for the dispersion
instrument, a colloid mill, a roll mill, a sonicator, a high-pressure
dispersion instrument (microfluidizer, Microfluidics Corp., USA),
Ultra Turrax (Janke and Kunkel, Germany), Nanoget (Nanoget Engineering,
Germany) and Brogli (Italy) and mechanical dispersion instruments
equivalent thereto can be used.
[0060] In accordance with the invention, the carrier particles
which are fine particles of several microns in size, such as liposomes,
microcapsules, microspheres, composite particles and emulsions,
in which a cyclosporin derivative according to the invention is
encapsulated, advantageously show higher skin penetration and better
in vivo hair restoring effect than those of free cyclosporin derivatives
dissolved in organic solvent at a molecular level. Further, the
carrier particles of the invention show good dispersion and phase
stability over time in compositions for use on hair. Accordingly,
it is advantageous that the carrier particles can prevent free forms
of cyclosporin derivatives being poorly dispersed and phase-unstable
over time, upon application to the hair cleaning compositions.
[0061] The fine carrier particles loading the cyclosporin derivatives
manufactured in the invention have the following advantages over
conventional solutions containing cyclosporin derivatives and cyclosporin
powder which is not formulated.
[0062] 1. High Degree of Penetration and Excellent Hair Restoring
Effect
[0063] The carrier particles loading the cyclosporin derivatives,
manufactured according to the methods of the invention derivatives,
which are fine particles of several microns in size, exhibit a high
degree of penetration to the skin through the follicle and excellent
in vivo hair restoring effects, compared to conventional formulations.
[0064] 2. Good Dispersion and Phase Stability Over Time
[0065] Since cyclosporin derivative powder in particle form, not
formulated, is strongly hydrophobic, its dispersion in cleaning
compositions is poor. In addition, the powder particles are likely
to agglomerate even after forcible mechanical dispersion, thereby
the powder particles are precipitated with time. That is, its phase
stability is poor. On the other hand, the carrier particles loading
cyclosporin derivatives of the invention are hydrophilic due to
their surface characteristics. Accordingly, the dispersion state
in compositions for use on human skin is good, and their phase stability
is excellent.
[0066] In summary, the carrier particles loading the cyclosporin
derivatives, manufactured according to the methods of the invention
derivatives, exhibit excellence in terms of skin penetration, in
vivo hair restoring effect, dispersion in compositions for use on
human skin, and phase stability with time, compared to conventional
solutions containing cyclosporin derivatives and cyclosporin powder
which is not formulated.
[0067] Meanwhile, the carrier particles loading the cyclosporin
derivatives, manufactured according to the methods of the invention
derivatives can be applied in manufacturing compositions for use
on hair, such as shampoos and rinses.
BEST MODE FOR CARRYING OUT THE INVENTION
[0068] Preparation of Cyclosporin Derivatives
[0069] To prepare [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cyclosporin
derivatives, which have an excellent hair growth stimulating effect
and exhibit no immunosuppressive activity, a bacterial strain, Sebekia
benihana KCTC 9173 was employed. Cyclosporin derivatives having
N-methyl-L-leucine at their amino acid No. 4 were dissolved in methanol.
Each of thus prepared cyclosporin solutions was added at an amount
of 100 mg/L to the culture medium 24 hrs after starting the main
culture. The bacteria were further cultured for 72 hrs.
[0070] After 72 hours, the total culture solution was extracted
with an equal volume of ethyl acetate, thereby collecting the sample.
The organic solvent layer was concentrated. From the concentrated
sample, [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cyclosporin deriatives,
which are a hair-restoring component with no immunosuppressive activity,
were isolated and collected by means of high performance liquid
chromatography, to serve as test samples for the following experiments.
The isolation condition was as follows. A C-18 column was used.
For the elution, a 100% solvent A was flowed for 2 min. The concentration
of the solvent was lowered to 60%, and the 60% solvent was flowed
over 4 min, and then the concentration was slowly lowered to 39%,
over 60 min. The concentration of the solvent was then returned
to 100%, and flowed for a further 5 min. The solvent A was 25% aqueous
methanol. As the diluent solvent B, 100% acetonitrile was used.
[0071] Hereinafter, the present invention will be described in
detail, in conjunction with various examples. Those skilled in the
art will realize that these examples are provided only for illustrative
purposes, and the present invention is not to be construed as being
limited to those examples.
[0072] Formulations
[0073] Formulation 1:
[0074] Preparation of a Liposome Carrying a Cyclosporin Derivative
[0075] A method of encapsulating a cyclosporin derivative into
a liposome of the invention is as follows. Amphiphilic molecules
composing the liposome used herein include phospholipids such as
phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol
(PI), cholesterol, cationic surfactants such as stearamidopropyldimethylamine
(SAPDA), distearyldimethyl ammoniumchloride (DSDAC) and stearyltrimethylammoniumchloride
(STAC), and stearic acid (SA). The amphiphilic molecules and cyclosporin
derivative were dissolved in an organic solvent such as acetone,
methanol, ethanol, isopropanol, or chloroform. The organic solvent
was evaporated at ambient temperature, giving a mixture of dry lipid
film consisting of amphiphilic molecules and the cyclosporin derivative.
Then, a certain amount of an aqueous solution was added to hydrate
the dry phospholipid film. The resultant film was homogenized at
500 bar using a microfluidizer.
[0076] In the following, the invention is described in more detail.
EXAMPLES 1 TO 7
Preparation of a Liposome Carrying [.gamma.-hydroxy-N-methyl-L-leucine.sup-
.4] Cyclosporin A, Compound 1
[0077] According to the method described in Formulation 1, liposome
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 1.
1TABLE 1 Formulation of liposome (unit: weight %) Ingredients Ex.
1 Ex. 2 Ex. 3 Ex. 4 Ex. 5 Ex. 6 Ex. 7 PC 10 8 8 8 -- -- -- PE --
2 -- -- -- -- -- PI -- -- 2 -- -- -- -- Cholesterol -- -- -- 2 --
-- -- DSDMAC -- -- -- -- 10 -- -- STAC -- -- -- -- -- 5 -- SAPDA
-- -- -- -- -- -- 5 SA -- -- -- -- -- 5 5 [.gamma.-hydroxy- 5 5
5 5 5 5 5 N-methyl-L- leucine.sup.4] cyclosporin A Water 85 85 85
85 85 85 85
EXAMPLES 8 TO 14
Preparation of a Liposome Carrying [.gamma.-hydroxy-N-methyl-L-leucine.sup-
.4] Cyclosporin C, Compound 2
[0078] According to the method described in Formulation 1, liposome
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 2.
2TABLE 2 Formulation of liposome (unit: weight %) Ex. Ex. Ex. Ex.
Ex. Ex. Ex. Ingredients 8 9 10 11 12 13 14 PC 10 8 8 8 -- -- --
PE -- 2 -- -- -- -- -- PI -- -- 2 -- -- -- -- Cholesterol -- --
-- 2 -- -- -- DSDMAC -- -- -- -- 10 -- -- STAC -- -- -- -- -- 5
-- SAPDA -- -- -- -- -- -- 5 SA -- -- -- -- -- 5 5 [.gamma.-hydroxy-
5 5 5 5 5 5 5 N-methyl-L- leucine.sup.4] cyclosporin C Water 85
85 85 85 85 85 85
EXAMPLES 15 TO 21
Preparation of a Liposome Carrying [N-methyl-D-alanine.sup.3][.gamma.-hydr-
oxy-N-methyl-L-leucine.sup.4] Cyclosporin A, Compound 3
[0079] According to the method described in Formulation 1, liposome
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 3.
3TABLE 3 Formulation of liposome (unit: weight %) Ingredients Ex.
15 Ex. 16 Ex. 17 Ex. 18 Ex. 19 Ex. 20 Ex. 21 PC 10 8 8 8 -- -- --
PE -- 2 -- -- -- -- -- PI -- -- 2 -- -- -- -- Cholesterol -- --
-- 2 -- -- -- DSDMAC -- -- -- -- 10 -- -- STAC -- -- -- -- -- 5
-- SAPDA -- -- -- -- -- -- 5 SA -- -- -- -- -- 5 5 [N-methyl-D-alanine.sup.3][.gamma.-
5 5 5 5 5 5 5 hydroxy-N-methyl-L-leucine.sup.4] cyclosporin A Water
85 85 85 85 85 85 85
EXAMPLES 22 TO 28
Preparation of a Liposome Carrying [.gamma.-hydroxy-N-methyl-L-leucine.sup-
.4] [.gamma.-hydroxy-N-methyl-L-leucine.sup.9] Cyclosporin A, Compound
4
[0080] According to the method described in Formulation 1, liposome
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 4.
4TABLE 4 Formulation of liposome (unit: weight %) Ingredients Ex.
22 Ex. 23 Ex. 24 Ex. 25 Ex. 26 Ex. 27 Ex. 28 PC 10 8 8 8 -- -- --
PE -- 2 -- -- -- -- -- PI -- -- 2 -- -- -- -- Cholesterol -- --
-- 2 -- -- -- DSDMAC -- -- -- -- 10 -- -- STAC -- -- -- -- -- 5
-- SAPDA -- -- -- -- -- -- 5 SA -- -- -- -- -- 5 5 [.gamma.-hydroxy-N-methyl-L-
5 5 5 5 5 5 5 leucine.sup.4] [.gamma.-hydroxy-N- methyl-L-leucine.sup.9]
cyclosporin A Water 85 85 85 85 85 85 85
EXAMPLES 29 TO 35
Preparation of a Liposome Carrying [.gamma.-hydroxy-N-methyl-L-leucine.sup-
.4] [alanine thiomide.sup.7] Cyclosporin A (or [.sup.7.PSI..sup.8
CS--NH] Cyclosporin A), Compound 5
[0081] According to the method described in Formulation 1, liposome
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 5.
5TABLE 5 Formulation of liposome (unit: weight %) Ingredients Ex.
29 Ex. 30 Ex. 31 Ex. 32 Ex. 33 Ex. 34 Ex. 35 PC 10 8 8 8 -- -- --
PE -- 2 -- -- -- -- -- PI -- -- 2 -- -- -- -- Cholesterol -- --
-- 2 -- -- -- DSDMAC -- -- -- -- 10 -- -- STAC -- -- -- -- -- 5
-- SAPDA -- -- -- -- -- -- 5 SA -- -- -- -- -- 5 5 [.gamma.-hydroxy-N-methyl-L-
5 5 5 5 5 5 5 leucine.sup.4] [alanine thiomide.sup.7] cyclosporin
A Water 85 85 85 85 85 85 85
EXAMPLES 36 TO 42
Preparation of a Liposome Carrying [L-threonine].sup.2[L-leucine].sup.5[-h-
ydroxy-N-methyl-L-leucine.sup.4] [D-hydroxyisovaleric Acid].sup.8[L-leucine].sup.10
Cyclosporin A, Compound 6
[0082] According to the method described in Formulation 1, liposome
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 6.
6TABLE 6 Formulation of liposome (unit: weight %) Ingredients Ex.
36 Ex. 37 Ex. 38 Ex. 39 Ex. 40 Ex. 41 Ex. 42 PC 10 8 8 8 -- -- --
PE -- 2 -- -- -- -- -- PI -- -- 2 -- -- -- -- Cholesterol -- --
-- 2 -- -- -- DSDMAC -- -- -- -- 10 -- -- STAC -- -- -- -- -- 5
-- SAPDA -- -- -- -- -- -- 5 SA -- -- -- -- -- 5 5 [L-threonine].sup.2[L-leucine].sup.5[.gamma.-
5 5 5 5 5 5 5 hydroxy-N-methyl-L-leucine.sup.4] [D-hydroxyisovaleric
acid].sup.8[L- leucine].sup.10 cyclosporin A Water 85 85 85 85 85
85 85
EXAMPLES 43 TO 49
Preparation of a Liposome Carrying [.gamma.-hydroxy-N-methyl-L-leucine.sup-
.4] Cyclosporin A, Compound 1
[0083] According to the method described in Formulation 1, liposome
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 7.
7TABLE 7 Formulation of liposome (unit: weight %) Ingredients Ex.
43 Ex. 44 Ex. 45 Ex. 46 Ex. 47 Ex. 48 Ex. 49 PC 10 8 8 8 -- -- --
PE -- 2 -- -- -- -- -- PI -- -- 2 -- -- -- -- Cholesterol -- --
-- 2 -- -- -- DSDMAC -- -- -- -- 10 -- -- STAC -- -- -- -- -- 5
-- SAPDA -- -- -- -- -- -- 5 SA -- -- -- -- -- 5 5 [.gamma.-hydroxy-N-methyl-L-
0.1 0.1 0.1 0.1 0.1 0.1 0.1 leucine.sup.4] cyclosporin A Water 89.9
89.9 89.9 89.9 89.9 89.9 89.9
EXAMPLES 50 TO 56
Preparation of a Liposome Carrying [.gamma.-hydroxy-N-methyl-L-leucine.sup-
.4] Cyclosporin C, Compound 2
[0084] According to the method described in Formulation 1, liposome
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 8.
8TABLE 8 Formulation of liposome (unit: weight %) Ingredients Ex.
50 Ex. 51 Ex. 52 Ex. 53 Ex. 54 Ex. 55 Ex. 56 PC 10 8 8 8 -- -- --
PE -- 2 -- -- -- -- -- PI -- -- 2 -- -- -- -- Cholesterol -- --
-- 2 -- -- -- DSDMAC -- -- -- -- 10 -- -- STAC -- -- -- -- -- 5
-- SAPDA -- -- -- -- -- -- 5 SA -- -- -- -- -- 5 5 [.gamma.-hydroxy-N-methyl-L-
0.1 0.1 0.1 0.1 0.1 0.1 0.1 leucine.sup.4] cyclosporin C Water 89.9
89.9 89.9 89.9 89.9 89.9 89.9
EXAMPLES 57 TO 63
Preparation of a Liposome Carrying [N-methyl-D-alanine.sup.3][.gamma.-hydr-
oxy-N-methyl-L-leucine.sup.4] Cyclosporin A, Compound 3
[0085] According to the method described in Formulation 1, liposome
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 9.
9TABLE 9 Formulation of liposome (unit: weight %) Ingredients Ex.
57 Ex. 58 Ex. 59 Ex. 60 Ex. 61 Ex. 62 Ex. 63 PC 10 8 8 8 -- -- --
PE -- 2 -- -- -- -- -- PI -- -- 2 -- -- -- -- Cholesterol -- --
-- 2 -- -- -- DSDMAC -- -- -- -- 10 -- -- STAC -- -- -- -- -- 5
-- SAPDA -- -- -- -- -- -- 5 SA -- -- -- -- -- 5 5 [N-methyl-D-alanine.sup.3][.gamma.-
0.1 0.1 0.1 0.1 0.1 0.1 0.1 hydroxy-N-methyl-L-leucine.sup.4] cyclosporin
A Water 89.9 89.9 89.9 89.9 89.9 89.9 89.9
COMPARATIVE EXAMPLE 1
[0086] Preparation of a Solution of a Cyclosporin Derivative
[0087] A cyclosporin derivative was dissolved in ethanol and acetone,
preparing a 5% cyclosporin solution. In testing a hair growth promoting
ability, a 0.1% cyclosporin solution was employed, and the solution
was prepared by dissolving a cyclosporin derivative in a 50% aqueous
ethanol solution.
[0088] Formulation 2:
[0089] Preparation of a Microcapsule Carrying a Cyclosporin Derivative
[0090] A method of encapsulating a cyclosporin derivative into
a microcapsule of the invention is as follows. A cyclosporin derivative
was dissolved in olive oil and medium chain triglyceride (MCT, Takasago
Int'l Corp.). The solution was added to a 3% aqueous gelatin solution
(Sigma, USA), and emulsified at 50.degree. C. at 2000 rpm for 10
min. Then, the solution was added with a 3% aqueous carboxylmethyl
cellulose (CMC, medium viscosity) (Sigma, USA) solution or a 3%
aqueous Arabic gum (MW 250,000) (Sigma, USA) solution, and additionally
emulsified for 10 min. The resulting solution was added with a 10%
aqueous acetic acid solution, and adjusted to pH 4.4. The emulsified
solution was cooled to a temperature of approximately 10C and stirred
for 30 min. The solution was then added with a 25% aqueous glutaraldehyde
(GA) solution and stirred at 200 rpm for 4 hrs at ambient temperature,
curing the capsule.
EXAMPLES 64 TO 69
Preparation of a Microcapsule Carrying [.gamma.-hydroxy-N-methyl-L-leucine-
.sup.4] Cyclosporin A, Compound 1
[0091] According to the method described in Formulation 2, microcapsule
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 10.
10TABLE 10 Formulation of microcapsule (unit: weight %) Ingredients
Ex. 64 Ex. 65 Ex. 66 Ex. 67 Ex. 68 Ex. 69 Olive oil 20 -- 20 --
20 -- MCT -- 20 -- 20 -- 20 [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
5 5 5 5 5 5 cyclosporin A Gelatin A (75-100 Bloom) 30 30 -- -- --
-- Gelatin A (300 Bloom) -- -- 30 30 30 30 CMC 30 30 30 30 -- --
Arabic gum -- -- -- -- 30 30 Acetic acid (10 w % aqueous solution)
1 1 1 1 1 1 GA (25 w % aqueous solution) 1.5 1.5 1.5 1.5 1.5 1.5
Water 12.5 12.5 12.5 12.5 12.5 12.5
EXAMPLES 70 TO 75
Preparation of a Microcapsule Carrying [.gamma.-hydroxy-N-methyl-L-leucine-
.sup.4] Cyclosporin C, Compound 2
[0092] According to the method described in Formulation 2, microcapsule
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 11.
11TABLE 11 Formulation of microcapsule (unit: weight %) Ingredients
Ex. 70 Ex. 71 Ex. 72 Ex. 73 Ex. 74 Ex. 75 Olive oil 20 -- 20 --
20 -- MCT -- 20 -- 20 -- 20 [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
5 5 5 5 5 5 cyclosporin C Gelatin A (75-100 Bloom) 30 30 -- -- --
-- Gelatin A (300 Bloom) -- -- 30 30 30 30 CMC 30 30 30 30 -- --
Arabic gum -- -- -- -- 30 30 Acetic acid (10 w % aqueous solution)
1 1 1 1 1 1 GA (25 w % aqueous solution) 1.5 1.5 1.5 1.5 1.5 1.5
Water 12.5 12.5 12.5 12.5 12.5 12.5
EXAMPLES 76 TO 81
Preparation of a Microcapsule Carrying [N-methyl-D-alanine.sup.3][.gamma.--
hydroxy-N-methyl-L-leucine.sup.4] Cyclosporin A, Compound 3
[0093] According to the method described in Formulation 2, microcapsule
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 12.
12TABLE 12 Formulation of microcapsule (unit: weight %) Ingredients
Ex. 76 Ex. 77 Ex. 78 Ex. 79 Ex. 80 Ex. 81 Olive oil 20 -- 20 --
20 -- MCT -- 20 -- 20 -- 20 [N-methyl-D-alanine.sup.3][.gamma.-hydroxy-N-
5 5 5 5 5 5 methyl-L-leucine.sup.4] cyclosporin A Gelatin A (75-100
Bloom) 30 30 -- -- -- -- Gelatin A (300 Bloom) -- -- 30 30 30 30
CMC 30 30 30 30 -- -- Arabic gum -- -- -- -- 30 30 Acetic acid (10
w % aqueous solution) 1 1 1 1 1 1 GA (25 w % aqueous solution) 1.5
1.5 1.5 1.5 1.5 1.5 Water 12.5 12.5 12.5 12.5 12.5 12.5
EXAMPLES 82 TO 87
Preparation of a Microcapsule Carrying [.gamma.-hydroxy-N-methyl-L-leucine-
.sup.4] Cyclosporin A, Compound 1
[0094] According to the method described in Formulation 2, microcapsule
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 13.
13TABLE 13 Formulation of microcapsule (unit: weight %) Ingredients
Ex. 82 Ex. 83 Ex. 84 Ex. 85 Ex. 86 Ex. 87 Olive oil 20 -- 20 --
20 -- MCT -- 20 -- 20 -- 20 [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
0.1 0.1 0.1 0.1 0.1 0.1 cyclosporin A Gelatin A (75-100 Bloom) 30
30 -- -- -- -- Gelatin A (300 Bloom) -- -- 30 30 30 30 CMC 30 30
30 30 -- -- Arabic gum -- -- -- -- 30 30 Acetic acid (10 w % aqueous
solution) 1 1 1 1 1 1 GA (25 w % aqueous solution) 1.5 1.5 1.5 1.5
1.5 1.5 Water 17.4 17.4 17.4 17.4 17.4 17.4
EXAMPLES 88 TO 93
Preparation of a Microcapsule Carrying [.gamma.-hydroxy-N-methyl-L-leucine-
.sup.4] Cyclosporin C, Compound 2
[0095] According to the method described in Formulation 2, microcapsule
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 14.
14TABLE 14 Formulation of microcapsule (unit: weight %) Ingredients
Ex. 88 Ex. 89 Ex. 90 Ex. 91 Ex. 92 Ex. 93 Olive oil 20 -- 20 --
20 -- MCT -- 20 -- 20 -- 20 [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
0.1 0.1 0.1 0.1 0.1 0.1 cyclosporin C Gelatin A (75-100 Bloom) 30
30 -- -- -- -- Gelatin A (300 Bloom) -- -- 30 30 30 30 CMC 30 30
30 30 -- -- Arabic gum -- -- -- -- 30 30 Acetic acid (10 w % aqueous
solution) 1 1 1 1 1 1 GA (25 w % aqueous solution) 1.5 1.5 1.5 1.5
1.5 1.5 Water 17.4 17.4 17.4 17.4 17.4 17.4
EXAMPLES 94 TO 99
Preparation of a Microcapsule Carrying [N-methyl-D-alanine.sup.3][.gamma.--
hydroxy-N-methyl-L-leucine.sup.4] Cyclosporin A, Compound 3
[0096] According to the method described in Formulation 2, microcapsule
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 15.
15TABLE 15 Formulation of microcapsule (unit: weight %) Ingredients
Ex. 94 Ex. 95 Ex. 96 Ex. 97 Ex. 98 Ex. 99 Olive oil 20 -- 20 --
20 -- MCT -- 20 -- 20 -- 20 [N-methyl-D-alanine.sup.3][.gamma.-hydroxy-N-
0.1 0.1 0.1 0.1 0.1 0.1 methyl-L-leucine.sup.4] cyclosporin A Gelatin
A (75-100 Bloom) 30 30 -- -- -- -- Gelatin A (300 Bloom) -- -- 30
30 30 30 CMC 30 30 30 30 -- -- Arabic gum -- -- -- -- 30 30 Acetic
acid (10 w % aqueous solution) 1 1 1 1 1 1 GA (25 w % aqueous solution)
1.5 1.5 1.5 1.5 1.5 1.5 Water 17.4 17.4 17.4 17.4 17.4 17.4
[0097] Formulation 3
[0098] Preparation of Microsphere Carrying a Cyclosporin Derivative
[0099] A cyclosporin derivative, and poly(lactic acid) (PLA, MW
90,000-120,000) (Sigma, USA) or poly(lactic acid-co-glycolic acid)
(50:50, MW 50,000-75,000, PLGA) were dissolved in a mixture of chloroform/acetone
or dichloromethane/acetone. The solution was added to an aqueous
phase of polysorbate 80, and emulsified at 2000 rpm for 10 min,
preparing O/W emulsion. The organic solvent was then removed by
evaporation at ambient temperature under reduced pressure. After
complete evaporation, the residue was added with an aqueous solution
at an amount equal to the weight of the solvent removed. A microsphere
carrying 5% cyclosporin derivative was thus prepared.
EXAMPLES 100 TO 103
Preparation of a Microsphere Carrying [.gamma.-hydroxy-N-methyl-L-leucine.-
sup.4] Cyclosporin A, Compound 1
[0100] According to the method described in Formulation 3, microsphere
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 16.
16TABLE 16 Formulation of microsphere (unit: weight %) Ingredients
Ex. 100 Ex. 101 Ex. 102 Ex. 103 Chloroform 30 30 -- -- Dichloromethane
-- -- 30 30 Acetone 10 10 10 10 [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
5 5 5 5 cyclosporin A PLA 5 -- 5 -- PLGA -- 5 -- 5 Polysorbate 80
0.2 0.2 0.2 0.2 Water 49.8 49.8 49.8 49.8
EXAMPLES 104 TO 107
Preparation of a Microsphere Carrying [.gamma.-hydroxy-N-methyl-L-leucine.-
sup.4] Cyclosporin C, Compound 2
[0101] According to the method described in Formulation 3, microsphere
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 17.
17TABLE 17 Formulation of microsphere (unit: weight %) Ingredients
Ex. 104 Ex. 105 Ex. 106 Ex. 107 Chloroform 30 30 -- -- Dichloromethane
-- -- 30 30 Acetone 10 10 10 10 [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
5 5 5 5 cyclosporin C PLA 5 -- 5 -- PLGA -- 5 -- 5 Polysorbate 80
0.2 0.2 0.2 0.2 Water 49.8 49.8 49.8 49.8
EXAMPLES 108 TO 111
Preparation of a Microsphere Carrying [N-methyl-D-alanine.sup.3][.gamma.-h-
ydroxy-N-methyl-L-leucine.sup.4] Cyclosporin A, Compound 3
[0102] According to the method described in Formulation 3, microsphere
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 18.
18TABLE 18 Formulation of microsphere (unit: weight %) Ingredients
Ex. 108 Ex. 109 Ex. 110 Ex. 111 Chloroform 30 30 -- -- Dichloromethane
-- -- 30 30 Acetone 10 10 10 10 [N-methyl-D-alanine.sup.3][.gamma.-hydroxy-
5 5 5 5 N-methyl-L-leucine.sup.4] cyclosporin A PLA 5 -- 5 -- PLGA
-- 5 -- 5 Polysorbate 80 0.2 0.2 0.2 0.2 Water 49.8 49.8 49.8 49.8
EXAMPLES 112 TO 115
Preparation of a Microsphere Carrying [.gamma.-hydroxy-N-methyl-L-leucine.-
sup.4] Cyclosporin A, Compound 1
[0103] According to the method described in Formulation 3, microsphere
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 19.
19TABLE 19 Formulation of microsphere (unit: weight %) Ingredients
Ex. 112 Ex. 113 Ex. 114 Ex. 115 Chloroform 30 30 -- -- Dichloromethane
-- -- 30 30 Acetone 10 10 10 10 [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
0.1 0.1 0.1 0.1 cyclosporin A PLA 5 -- 5 -- PLGA -- 5 -- 5 Polysorbate
80 0.2 0.2 0.2 0.2 Water 54.7 54.7 54.7 54.7
EXAMPLES 116 TO 119
Preparation of a Microsphere Carrying [.gamma.-hydroxy-N-methyl-L-leucine.-
sup.4] Cyclosporin C, Compound 2
[0104] According to the method described in Formulation 3, microsphere
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 20.
20TABLE 20 Formulation of microsphere (unit: weight %) Ingredients
Ex. 116 Ex. 117 Ex. 118 Ex. 119 Chloroform 30 30 -- -- Dichloromethane
-- -- 30 30 Acetone 10 10 10 10 [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
0.1 0.1 0.1 0.1 cyclosporin C PLA 5 -- 5 -- PLGA -- 5 -- 5 Polysorbate
80 0.2 0.2 0.2 0.2 Water 54.7 54.7 54.7 54.7
EXAMPLES 120 TO 123
Preparation of a Microsphere Carrying [N-methyl-D-alanine.sup.3][.gamma.-h-
ydroxy-N-methyl-L-leucine.sup.4] Cyclosporin A, Compound 3
[0105] According to the method described in Formulation 3, microsphere
suspensions carrying the cyclosporin derivative were prepared, with
varying compositions of the ingredients shown in Table 21.
21TABLE 21 Formulation of microsphere (unit: weight %) Ingredients
Ex. 120 Ex. 121 Ex. 122 Ex. 123 Chloroform 30 30 -- -- Dichloromethane
-- -- 30 30 Acetone 10 10 10 10 [N-methyl-D-alanine.sup.3][.gamma.-hydroxy-
0.1 0.1 0.1 0.1 N-methyl-L-leucine.sup.4] cyclosporin A PLA 5 --
5 -- PLGA -- 5 -- 5 Polysorbate 80 0.2 0.2 0.2 0.2 Water 54.7 54.7
54.7 54.7
[0106] Formulation 4
[0107] Preparation of a Cyclosporin/Surfactant Composite Particle
[0108] A method of preparing a cyclosporin/surfactant composite
particle is as follows. A cyclosporin derivative, and distearyl
dimethyl ammonium chloride (DSDAC), sodium lauryl sulfate (SLS),
cocodimethyl sulphopropyl betaine (CDSPB), or Tween 60 were mixed
in an aqueous phase. The solution was homogenized using an ultra
high-pressure dispersion instrument (microfluidizer) at 500 bar,
preparing a fine composite particle.
EXAMPLES 124 TO 127
Preparation of a [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] Cyclosporin
A, Compound 1/Surfactant Composite Particle
[0109] According to the method described in Formulation 4, suspensions
of the cyclosporin derivative/surfactant composite particle were
prepared, with varying compositions of the ingredients shown in
Table 22.
22TABLE 22 Formulation of composite particle (unit: weight %) Ingredients
Ex. 124 Ex. 125 Ex. 126 Ex. 127 DSDAC 1 -- -- -- SLS -- 1 -- --
CDSPB -- -- 1 -- Tween 60 -- -- -- 1 [.gamma.-hydroxy-N-methyl-L-leucine.sup-
.4] 5 5 5 5 cyclosporin A Water 94 94 94 94
EXAMPLES 128 TO 131
Preparation of a [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] Cyclosporin
C, Compound 2/Surfactant Composite Particle
[0110] According to the method described in Formulation 4, suspensions
of the cyclosporin derivative/surfactant composite particle were
prepared, with varying compositions of the ingredients shown in
Table 23.
23TABLE 23 Formulation of composite particle (unit: weight %) Ingredients
Ex. 128 Ex. 129 Ex. 130 Ex. 131 DSDAC 1 -- -- -- SLS -- 1 -- --
CDSPB -- -- 1 -- Tween 60 -- -- -- 1 [.gamma.-hydroxy-N-methyl-L-leucine.sup-
.4] 5 5 5 5 cyclosporin C Water 94 94 94 94
EXAMPLES 132 TO 135
Preparation of a [N-methyl-D-alanine.sup.3] [.gamma.-hydroxy-N-methyl-L-le-
ucine.sup.4] Cyclosporin A, Compound 3/Surfactant Composite Particle
[0111] According to the method described in Formulation 4, suspensions
of the cyclosporin derivative/surfactant composite particle were
prepared, with varying compositions of the ingredients shown in
Table 24.
24TABLE 24 Formulation of composite particle (unit: weight %) Ingredients
Ex. 132 Ex. 133 Ex. 134 Ex. 135 DSDAC 1 -- -- -- SLS -- 1 -- --
CDSPB -- -- 1 -- Tween 60 -- -- -- 1 [N-methyl-D-alanine.sup.3][.gamma.-hydr-
oxy- 5 5 5 5 N-methyl-L-leucine.sup.4] cyclosporin A Water 94 94
94 94
EXAMPLES 136 TO 139
Preparation of a [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] Cyclosporin
A, Compound 1/Surfactant Composite Particle
[0112] According to the method described in Formulation 4, suspensions
of the cyclosporin derivative/surfactant composite particle were
prepared, with varying compositions of the ingredients shown in
Table 25.
25TABLE 25 Formulation of composite particle (unit: weight %) Ingredients
Ex. 136 Ex. 137 Ex. 138 Ex. 139 DSDAC 1 -- -- -- SLS -- 1 -- --
CDSPB -- -- 1 -- Tween 60 -- -- -- 1 [.gamma.-hydroxy-N-methyl-L-leucine.sup-
.4] 0.1 0.1 0.1 0.1 cyclosporin A Water 98.9 98.9 98.9 98.9
EXAMPLES 140 TO 143
Preparation of a [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] Cyclosporin
C, Compound 2/Surfactant Composite Particle
[0113] According to the method described in Formulation 4, suspensions
of the cyclosporin derivative/surfactant composite particle were
prepared, with varying compositions of the ingredients shown in
Table 26.
26TABLE 26 Formulation of composite particle (unit: weight %) Ingredients
Ex. 140 Ex. 141 Ex. 142 Ex. 143 DSDAC 1 -- -- -- SLS -- 1 -- --
CDSPB -- -- 1 -- Tween 60 -- -- -- 1 [.gamma.-hydroxy-N-methyl-L-leucine.sup-
.4] 0.1 0.1 0.1 0.1 cyclosporin C Water 98.9 98.9 98.9 98.9
EXAMPLES 144 TO 147
Preparation of a [N-methyl-D-alanine.sup.3] [.gamma.-hydroxy-N-methyl-L-le-
ucine.sup.4] Cyclosporin A, Compound 3/Surfactant Composite Particle
[0114] According to the method described in Formulation 4, suspensions
of the cyclosporin derivative/surfactant composite particle were
prepared, with varying compositions of the ingredients shown in
Table 27.
27TABLE 27 Formulation of composite particle (unit: weight %) Ingredients
Ex. 144 Ex. 145 Ex. 146 Ex. 147 DSDAC 1 -- -- -- SLS -- 1 -- --
CDSPB -- -- 1 -- Tween 60 -- -- -- 1 [N-methyl-D-alanine.sup.3][.gamma.-hydr-
oxy- 0.1 0.1 0.1 0.1 N-methyl-L-leucine.sup.4] cyclosporin A Water
98.9 98.9 98.9 98.9
[0115] Formulation 5
[0116] Preparation of an Emulsion Containing a Cyclosporin Derivative
[0117] A method of preparing an emulsion containing a cyclosporin
derivative is as follows. To a mixture of olive oil, triglyceride
(C.sub.10-C.sub.18, TG) and octylsalicylate (OS), a cyclosporin
derivative was added and dissolved. The solution was added to an
aqueous polyvinyl alcohol (PVA, MW 30,000-70,000) (Sigma, USA) solution
or an aqueous polysorbate 20 solution. The resulting solution was
emulsified at 2000 rpm for 10 min at ambient temperature.
EXAMPLES 148 TO 153
Preparation of an Emulsion Containing [.gamma.-hydroxy-N-methyl-L-leucine.-
sup.4] Cyclosporin A, Compound 1
[0118] According to the method described in Formulation 5, emulsions
containing the cyclosporin derivative were prepared, with varying
compositions of the ingredients shown in Table 28.
28TABLE 28 Formulation of emulsion (unit: weight %) Ingredients
Ex. 148 Ex. 149 Ex. 150 Ex. 151 Ex. 152 Ex. 153 Olive oil 30 30
-- -- -- -- MCT -- -- 30 30 -- -- OS -- -- -- -- 30 30 [.gamma.-hydroxy-N-methyl-L-leucine-
.sup.4] 5 5 5 5 5 5 cyclosporin A PVA 0.5 -- 0.5 -- 0.5 -- polysorbate
20 -- 0.5 -- 0.5 -- 0.5 Water 64.5 64.5 64.5 64.5 64.5 64.5
EXAMPLES 154 TO 159
Preparation of an Emulsion Containing [.gamma.-hydroxy-N-methyl-L-leucine.-
sup.4] Cyclosporin C, Compound 2
[0119] According to the method described in Formulation 5, emulsions
containing the cyclosporin derivative were prepared, with varying
compositions of the ingredients shown in Table 29.
29TABLE 29 Formulation of emulsion (unit: weight %) Ingredients
Ex. 154 Ex. 155 Ex. 156 Ex. 157 Ex. 158 Ex. 159 Olive oil 30 30
-- -- -- -- MCT -- -- 30 30 -- -- OS -- -- -- -- 30 30 [.gamma.-hydroxy-N-methyl-L-leucine-
.sup.4] 5 5 5 5 5 5 cyclosporin C PVA 0.5 -- 0.5 -- 0.5 -- polysorbate
20 -- 0.5 -- 0.5 -- 0.5 Water 64.5 64.5 64.5 64.5 64.5 64.5
EXAMPLES 160 TO 165
Preparation of an Emulsion Containing [N-methyl-D-alanine.sup.3]
[.gamma.-hydroxy-N-methyl-L-leucine.sup.4] Cyclosporin A, Compound
3
[0120] According to the method described in Formulation 5, emulsions
containing the cyclosporin derivative were prepared, with varying
compositions of the ingredients shown in Table 30.
30TABLE 30 Formulation of emulsion (unit: weight %) Ingredients
Ex. 160 Ex. 161 Ex. 162 Ex. 163 Ex. 164 Ex. 165 Olive oil 30 30
-- -- -- -- MCT -- -- 30 30 -- -- OS -- -- -- -- 30 30 [N-methyl-D-alanine.sup.3]
[.gamma.-hydroxy- 5 5 5 5 5 5 N-methyl-L-leucine.sup.4] cyclosporin
A PVA 0.5 -- 0.5 -- 0.5 -- polysorbate 20 -- 0.5 -- 0.5 -- 0.5 Water
64.5 64.5 64.5 64.5 64.5 64.5
EXAMPLES 166 TO 171
Preparation of an Emulsion Containing [.gamma.-hydroxy-N-methyl-L-leucine.-
sup.4] Cyclosporin A, Compound 1
[0121] According to the method described in Formulation 5, emulsions
containing the cyclosporin derivative were prepared, with varying
compositions of the ingredients shown in Table 31.
31TABLE 31 Formulation of emulsion (unit: weight %) Ingredients
Ex. 166 Ex. 167 Ex. 168 Ex. 169 Ex. 170 Ex. 171 Olive oil 30 30
-- -- -- -- MCT -- -- 30 30 -- -- OS -- -- -- -- 30 30 [.gamma.-hydroxy-N-methyl-L-leucine-
.sup.4] 0.1 0.1 0.1 0.1 0.1 0.1 cyclosporin A PVA 0.5 -- 0.5 --
0.5 -- polysorbate 20 -- 0.5 -- 0.5 -- 0.5 Water 69.4 69.4 69.4
69.4 69.4 69.4
EXAMPLES 172 TO 177
Preparation of an Emulsion Containing [.gamma.-hydroxy-N-methyl-L-leucine.-
sup.4] Cyclosporin C, Compound 2
[0122] According to the method described in Formulation 5, emulsions
containing the cyclosporin derivative were prepared, with varying
compositions of the ingredients shown in Table 32.
32TABLE 32 Formulation of emulsion (unit: weight %) Ingredients
Ex. 172 Ex. 173 Ex. 174 Ex. 175 Ex. 176 Ex. 177 Olive oil 30 30
-- -- -- -- MCT -- -- 30 30 -- -- OS -- -- -- -- 30 30 [.gamma.-hydroxy-N-methyl-L-leucine-
.sup.4] 0.1 0.1 0.1 0.1 0.1 0..1 cyclosporin C PVA 0.5 -- 0.5 --
0.5 -- polysorbate 20 -- 0.5 -- 0.5 -- 0.5 Water 69.4 69.4 69.4
69.4 69.4 69.4
EXAMPLES 178 TO 183
Preparation of an Emulsion Containing [N-methyl-D-alanine.sup.3][.gamma.-h-
ydroxy-N-methyl-L-leucine.sup.4] Cyclosporin A, Compound 3
[0123] According to the method described in Formulation 5, emulsions
containing the cyclosporin derivative were prepared, with varying
compositions of the ingredients shown in Table 33.
33TABLE 33 Formulation of emulsion (unit: weight %) Ingredients
Ex. 178 Ex. 179 Ex. 180 Ex. 181 Ex. 182 Ex. 183 Olive oil 30 30
-- -- -- -- MCT -- -- 30 30 -- -- OS -- -- -- -- 30 30 [N-methyl-D-alanine.sup.3][.gamma.--
hydroxy- 0.1 0.1 0.1 0.1 0.1 0..1 N-methyl-L-leucine.sup.4] cyclosporin
A PVA 0.5 -- 0.5 -- 0.5 -- polysorbate 20 -- 0.5 -- 0.5 -- 0.5 Water
69.4 69.4 69.4 69.4 69.4 69.4
TEST EXAMPLE 1
[0124] Measurements of Size and Zeta Potential
[0125] With respect to representative particles carrying a cyclosporin
derivative prepared in Examples, their sizes and zeta potentials
were measured using Zeta Plus instrument (Brookhaven Instruments
Co.). The results are shown in Table 34.
34TABLE 34 Properties of carriers of the invention Carrier Average
size (.mu.m) Zeta potential (mV) Liposome of Ex. 1 6.0 -20.1 Liposome
of Ex. 3 5.4 -22.3 Liposome of Ex. 5 5.7 +43.4 Liposome of Ex. 7
6.3 +33.0 Microcapsule of Ex. 65 6.6 -32.2 Microcapsule of Ex. 67
5.5 -30.5 Microcapsule of Ex. 69 6.0 -28.4 Microsphere of Ex. 101
5.3 -15.3 Microsphere of Ex. 103 5.4 -17.2 Composite particle of
Ex. 124 6.2 +44.1 Composite particle of Ex. 125 7.2 -30.1 Emulsion
of Ex. 149 6.9 -20.6 Emulsion of Ex. 151 7.1 -18.5 Emulsion of Ex.
153 7.2 -17.7
[0126] It can been seen in Table 34 that as for the composite particles,
their sizes were approximately 5 to 7 .mu.m. The respective zeta
potentials indicate surface properties of the composite particles.
For example, the cationic composite particle the example 124 showed
a plus (+) zeta potential value, while the negative composite particle,
the particle of the example 125 showed a minus (-) zeta potential
value.
TEST EXAMPLE 2
[0127] Evaluation of Skin Penetration In Vitro
[0128] Female hairless SKH1 mice of ages 6 to 8 weeks were employed.
To measure in vitro skin absorption of the carrier particles of
the invention, a diffusion cell consisting of a donor chamber and
a receptor chamber was utilized. The mice skin was positioned between
the two chambers, in which the epidermis was directed to the donor
chamber while the dermis was directed to the receptor chamber. Phosphate-buffered
saline (pH 7.4, 37.degree. C.) was filled in the receptor chamber
and let stand for 1 hr, thereby the skin and the buffer solution
reaching equilibrium. Then, 300 mg of the carrier particle suspensions
(in a suspension, a cyclosporin content is 5%) were respectively
applied to the epidermis (the applied area is 3.14 cm.sup.2). The
donor chambers were sealed with parafilm. After 12 hrs, a 0.2 ml
fluid was sampled from the receptor chamber, and the amount of the
cyclosporin derivative penetrated through the skin was quantified
by means of HPLC. The data are shown in Table 35.
35TABLE 35 Skin absorption profile Amount of cyclosporin Carrier
penetrated (mg) Liposome of Ex. 1 3.78 Liposome of Ex. 3 3.66 Liposome
of Ex. 5 3.89 Liposome of Ex. 7 3.63 Microcapsule of Ex. 65 2.55
Microcapsule of Ex. 67 2.87 Microcapsule of Ex. 69 2.78 Microsphere
of Ex. 101 3.10 Microsphere of Ex. 103 3.30 Composite particle of
Ex. 124 2.88 Composite particle of Ex. 125 2.98 Emulsion of Ex.
149 3.44 Emulsion of Ex. 151 2.99 Emulsion of Ex. 153 3.01 5% [.gamma.-hydroxy-N-methy-
l-L-leucine.sup.4] 1.23 cyclosporin A in ethanol 5% [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
0.97 cyclosporin A in acetone
[0129] As shown in Table 35, the fine carrier particles of Examples,
which are fine particles of several microns in size, showed skin
penetration of cyclosporin derivatives 2 to 3 times higher than
those of free cyclosporin derivatives dissolved in ethanol or acetone
solutions, upon application to mouse skin. Thus, the carrier particles
of the invention such as liposomes, microcapsules, microspheres,
composite particles and emulsions, in which a cyclosporin derivative
is encapsulated, have an advantage of higher skin penetration than
those of free cyclosporin derivatives and dissolved in organic solvent
at a molecular level.
TEST EXAMPLE 3
[0130] Evaluation of Hair Growth Promoting Effect by Carriers Loading
[.gamma.-hydroxy-N-methyl-L-leucine.sup.4] Cyclosporin A, Compound
1
[0131] Female C57BL/6 mice of ages 6 to 7 weeks were utilized.
After removing hairs on the middle of the back with an electric
shaver, the mice were weighed and randomly assigned to the test
groups with an even distribution of weights. After one day of adaptation,
the mice were applied with respective carrier suspensions loading
0.1% [.gamma.-hydroxy-N-methyl-L-leucine.sup.4] cyclosporin A to
their hair removed areas once a day at a dose of 100 .mu.l, for
30 days. The results were determined by visual examination, in terms
of degrees of hair regrowth. With respect to respective hair-removed
areas, rates of new hair growth were examined and compared.
[0132] As can be seen in Table 36, the carrier particles of the
invention such as liposomes, microcapsules, microspheres, composite
particles and emulsions, in which the cyclosporin derivative is
encapsulated, the particles being fine particles of several microns
in size, showed a significant hair growth promoting effect, owing
to their higher penetration to the skin and follicle, than that
of the free cyclosporin derivative in organic solvent such as 50%
ethanol. Meanwhile, over the course of 30 days, comparing the appearance
of the backs, the mice of the control and all test groups showed
no specific skin irritation.
36TABLE 36 Hair regrowth in mice Carrier applied Area rate of hair
regrowth (%) Liposome of Ex. 43 90 Liposome of Ex. 45 91 Liposome
of Ex. 47 89 Liposome of Ex. 49 85 Microcapsule of Ex. 82 86 Microcapsule
of Ex. 84 90 Microcapsule of Ex. 86 91 Microsphere of Ex. 112 91
Microsphere of Ex. 114 93 Composite particle of Ex. 136 91 Composite
particle of Ex. 138 88 Emulsion of Ex. 166 90 Emulsion of Ex. 168
92 Emulsion of Ex. 170 86 0.1% [.gamma.-hydroxy-N-methyl-L- 62 leucine.sup.4]
cyclosporin A in a 50% aqueous ethanol solution
EXAMPLES 184 TO 188 AND COMPARATIVE EXAMPLE 2
[0133] Use for Formulation of Shampoo
[0134] The liposomes, microcapsules, microspheres, composite particles
and emulsions, prepared in the above Examples, and powder of the
cyclosporin derivative which is not formulated, were respectively
added to compositions for hair use. The mixtures were blended at
about 35 to 40.degree. C. in a stirrer (300 to 400 rpm), preparing
respective homogeneous hair cleaning compositions (shampoos). The
content of the cyclosporin derivative was set to 1% in the shampoo
composition. Table 37 shows the contents of individual ingredients.
37TABLE 37 Formulation of shampoo (unit: weight %) Comp. Ingredients
Ex. 184 Ex. 185 Ex. 186 Ex. 187 Ex. 188 Ex. 2 Hair cleaning Composition
Water 53 53 53 53 53 72 Betaine 1 1 1 1 1 1 SLES 20 20 20 20 20
20 SLS 5 5 5 5 5 5 CDE 1 1 1 1 1 1 Liposome of Ex. 1 (Compound 1:
5% content) 20 -- -- -- -- -- Microcapsule of Ex. 65 (Compound 1:
5% content) -- 20 -- -- -- -- Microsphere of Ex. 101 (Compound 1:
5% content) -- -- 20 -- -- -- Composite particle of Ex. 124 (Compound
1: 5% content) -- -- -- 20 -- -- Emulsion of Ex. 149 (Compound 1:
5% content) -- -- -- -- 20 -- Powder of cyclosporin derivative (Compound
1: 100% content) -- -- -- -- -- 1 Note: Betaine used herein is cocamidopropyl
betaine; SLES used herein is sodium lauryl ether sulfate, which
has two ethylene oxide groups and 12 carbon atoms; SLS used herein
is sodium lauryl sulfate, which has 12 carbon atoms; CDE represents
coconut diethanolamide; and in the first column, each of the contents
of cyclosporin parenthesized represents the content of cyclosporin
derivative carried in each formulation.
TEST EXAMPLE 4
[0135] Test of Shampoo for Phase Stability Over Time
[0136] To evaluate phase stability of hair cleaning preparations
over an elapsed time, the shampoos of Examples 184 to 188 and Comparative
Example 2 were stored for 90 days at ambient temperature and at
40.degree. C., respectively. Properties of the shampoos were observed,
and the results are shown in Table 38.
38TABLE 38 Phase stability of shampoo Ex. Ex. Ex. Ex. Ex. Temperature
184 185 186 187 188 Comp. Ex. 2 ambient temp. stable stable stable
stable stable precipitated 40.degree. C. stable stable stable stable
stable precipitated
[0137] As can be seen in Table 38, the carrier particles of the
invention such as liposomes, microcapsules, microspheres, composite
particles and emulsions, in which the cyclosporin derivative is
encapsulated, showed good phase stability over time. On the other
hand, the powder of cyclosporin derivative which is not formulated
was unstable in the hair cleaning composition. Thus, there is another
advantage in that the carrier particles of the invention in which
the cyclosporin derivative is encapsulated, can protect the free
cyclosporin derivative from being phase conversion over time, in
shampoo compositions.
EXAMPLES 189 TO 193 AND COMPARATIVE EXAMPLE 3
[0138] Use for Formulation of Rinse
[0139] The liposomes, microcapsules, microspheres, composite particles
and emulsions, prepared in the above Examples, and powder of the
cyclosporin derivative which is not formulated, were respectively
added to rinse compositions. The mixtures were blended at about
35 to 40.degree. C. in a stirrer (200 to 300 rpm), preparing respective
homogeneous compositions (rinses). Table 39 shows the contents of
individual ingredients.
39TABLE 39 Formulation of rinse (unit: weight %) Comp. Ingredients
Ex. 189 Ex. 190 Ex. 191 Ex. 192 Ex. 193 Ex. 3 Rinse Composition
Behenyl trimethyl 0.3 0.3 0.3 0.3 0.3 0.3 ammonium chloride Liquid
paraffin 1.5 1.5 1.5 1.5 1.5 1.5 Stearyl alcohol 5 5 5 5 5 5 Glycerin
1 1 1 1 1 1 Cetyl trimethyl 5 5 5 5 5 5 ammonium chloride Silicone
2 2 2 2 2 2 Water 65.2 65.2 65.2 65.2 65.2 84.2 Liposome of Ex.
1 (Compound 1: 5% content) 20 -- -- -- -- -- Microcapsule of Ex.
65 (Compound 1: 5% content) -- 20 -- -- -- -- Microsphere of Ex.
101 (Compound 1: 5% content) -- -- 20 -- -- -- Composite particle
of Ex. 124 (Compound 1: 5% content) -- -- -- 20 -- -- Emulsion of
Ex. 149 (Compound 1: 5% content) -- -- -- -- 20 -- Powder of cyclosporin
derivative (Compound 1: 100% content) -- -- -- -- -- 1 Note: Silicone
used herein is a silicone oil emulsion (Dow Corning Co., 2-1691
emulsion); and in the first column, each of the contents of cyclosporin
parenthesized represents the content of cyclosporin derivative carried
in each formulation.
TEST EXAMPLE 5
[0140] Test of Rinse for Phase Stability Over Time
[0141] To evaluate phase stability of rinse compositions with an
elapsed time, the rinses of Examples 189 to 193 and Comparative
Example 3 were stored for 90 days at ambient temperature and at
40.degree. C., respectively. Properties of the rinses were observed,
and the results are shown in Table 40.
40TABLE 40 Phase stability of rinse Ex. Ex. Ex. Ex. Ex. Temperature
189 190 191 192 193 Comp. Ex. 3 ambient temp. stable stable stable
stable stable precipitated 40.degree. C. stable stable stable stable
stable precipitated
[0142] As can be seen in Table 40, the carrier particles of the
invention such as liposomes, microcapsules, microspheres, composite
particles and emulsions, in which the cyclosporin derivative is
encapsulated, showed good phase stability over time. On the other
hand, the powder of cyclosporin derivative which is not formulated,
was unstable in the rinses. Thus, there is another advantage in
that the carrier particles of the invention in which the cyclosporin
derivative is encapsulated, can protect the cyclosporin derivative
from phase conversion over time, in rinse compositions.
TEST EXAMPLE 6
[0143] Evaluation of Hair Restoring Effect by Shampoo and Rinse
Containing Carriers Loading [.gamma.-hydroxy-N-methyl-L-leucine.sup.4]
Cyclosporin A, Compound 1
[0144] Female C57BL/6 mice of ages 6 to 7 weeks were utilized.
After removing hairs on the middle of the back with an electric
shaver, the mice were weighed and randomly assigned to the test
groups with an even distribution of weights. After one day of adaptation,
the mice were applied with respective samples of the shampoos or
rinses (content of each cyclosporin derivative was 1.0%) prepared
above at amounts of 200 .mu.l to their hair removed areas, and 3
min later, the applied areas were washed with running water for
10 sec. Starting from the day after hair removal, the treatment
of the samples was carried out 5 to 6 times per week, once a day
for 30 days. On day 30, after the experiment begins, the experimental
animals were anesthetized with phentobarbitol, and photographed.
[0145] The hair restoring effects were determined, based on the
areas in which hairs were restored. The hair restoration rates were
calculated using the area in which the hair restoring effect was
shown, measured on the 30.sup.th day after treatment, with respect
to respective hair-removed areas immediately after hair removal.
The results are shown in Table 41.
41TABLE 41 Hair restore in mice Hair Hair Carrier used Shampoo
growth Rinse growth Liposome Ex. 184 +++ Ex. 189 +++ Microcapsule
Ex. 185 +++ Ex. 190 +++ Microsphere Ex. 186 +++ Ex. 191 +++ Composite
Paricle Ex. 187 +++ Ex. 192 +++ Emulsion Ex. 188 ++ Ex. 193 +++
Cyclosporin powder Comp. Ex. 2 + Comp. Ex. 3 + (not formulated)
Evaluations are as follows: -, The hair restoration rate is 10%
or less; +, The rate is 10-30%; ++, The rate is 30-70%; and +++,
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