CA2155866A1 - Method of enhancing hair growth and hair revitalizing compositions comprising lutzomyia protein - Google Patents

Abstract

The present invention relates to a method for the enhancement of hair growth and to topical compositions suitable for use in the method.
Peptides that can be found in the salivary glands of the sand fly Lutzomyia longipalpis, exemplified by maxadilan, are utilized for the enhancement of hair growth and the prevention of epilation and trichogenous effects.

Description

WO 94/17778 215 5 8 6 6 PCTrUS94/01202 Description METHOD OF ENHANCIN~ HAIR GROWTH AND HAIR REVITALIZING
COMPOSITIONS COMPRISING LUTZOMYIA PROTEIN
1. Field of the Invention The present invention relates to a hair revitalization composition having excellent hair revitalizing effects, such as the prevention of epilation, and excellent trichogenous effects. The invention is particularly useful in the preparation of drugs, quasi-drugs, and in the field of cosmetics. The composition comprises a peptide found in the sand fly Lutzo~r~ria longipalpis.

2. Backqround of the Invention Several possible causes of baldness and epilation have been explored as investigators have attempted to better understand and to develop means of alleviating these conditions. Abnormalities of the scalp, such as activation of androgen in the organs, for example, hair roots and sebum glands, have been considered. In addition, decrease in the blood flowing to hair follicles, hypersecretion of sebum, and the formation of peroxides have all been considered as possible causes of baldness and epilation. Conventional hair revitalizing compositions, therefore, have been formulated with the aim of alleviating or reducing the occurrence of these events.
Formulations using vit~m' n~, such as B vitamins and vitamin E; amino acids, such as serine and methionine;
vasodilators, such as swertiae extract and acetylcholine derivatives; anti-inflammatory agents, such as lithosperiradix extract and hinokitiol; female hormones, such as estradiol; and skin-hyperactive agents, such as cepharanthine, have been developed and utilized for prophylaxis and as a cure for alopecia.
- Recently, compositions containing chemical inhibitors for glycanases have been disclosed in U.S. Patent No.
5,015,470, compositions containing growth factors such as transforming growth factors alpha and beta have been disclosed in U.S. Patent No. 5,037,643, and compositions containing mixtures of nicotinates and hydrochloride have WO94/17778 2 ~ S S ~ 6 5 PCT~S94/0~202 -been disclosed in U.S. Patent No. 5,043,162. These compositions have a certain efficacy in promoting hair growth.
In spite of these various attempts, the conventional hair revitalizing/enhancement compositions have not been satisfactory in the prevention or tre;atment of alopecia, epilation and trichogenous effects. This is likely due to the fact that revitalization of hair and enhancement of hair growth are very complicated processes and may involve numerous or interrelated causes.
Consequently, there remains a need in the art for more diverse and effective means of revitalizing hair or enhancing hair growth. Such means would have beneficial effects in several applications. For example, hair growth enhancement or revitalization could be used in the cosmetic industry for increasing hair growth rates, in the ph~r~ceutical industry for the treatment of baldness, and in the animal breeding industry for stimulating wool growth and fur growth in commercially important animals, such as sheep and rabbits.

sTn~M~T~ OF ln~ INVENTION
The present inventors have researched substances effective for the treatment of epilation and possessing other excellent hair revitalizing effects, such as the prevention of trichogenous effect. As a result, it has been discovered that specific peptides that can be found in the salivary glands of the sand fly Lutzomyia longipalpis exhibit characteristics useful for revitalizing hairs, thereby making this invention possible.
Accordingly, this invention aids in fulfilling the need in the art by providing a hair revitalizing composition comprising, as effective components, peptides that can be fcund in the salivary glands of the sand fly Lutzomyia longipalpis. The peptides exhibit vasodilation activity in mammals. The hair revitalizing composition according to the present invention provides excellent trichogenous effects and WO94/17778 21~ ~ 8 6 6 PCT~S94/01202 hair revitalization effects by topical application and subcutaneous injection.
More particularly, this invention provides a method for inducing ~ maintA i n ing or increasing hair growth in a mammal.
The method comprises administering a hair growth inducing, maint~i n i ng or increasing amount of Lutzomyia protein to the skin or hair of the mammal. In a preferred embodiment of the invention, the Lutzomyia protein is topically applied to the mammal. A preferred Lutzomyia protein is maxadilan.
This invention also provides a composition suitable for topical application to mammalian skin or hair. The composition comprises Lutzomyia protein in a hair growth inducing, maint~i n i ng or increasing amount in a pharmaceutically acceptable vehicle.
The present invention is useful for the enhancement of hair growth utilizing compounds belonging to a category different from the group of substances hitherto used. The present invention is also useful for increasing production of wool or fur in animal breeding industries. The invention is thus beneficial for inducing, maint~ining or increasing hair growth in mammals.

BRIEF DESCRIPTION OF THE DRAWING
This invention will be described in greater detail with reference to the drawing in which:
The Figure shows a graph in which the hair growth of organ cultures of mouse whiskers is plotted against cultivation day to show the action of Lutzomyia longipalpis on these cultures.

DE~ATT.~n DESCRIPTION OF THE INVENTION
The present invention resulted from research efforts directed to the identification of substances which effectively prevent epilation and the trichogenous effect.
Applicants' efforts led to the discovery that specific peptides that can be found in the salivary glands of the sand fly Lutzomyia WO94/17778 PCT~S94101202 2~S5866 longipalpis exhibit strong effects, which result in the enhancement of hair growth.
1. LutzomYia Proteins.
Peptides from salivary gland lysates o`f the sand fly were previously identified and shown to be capable of vasodilation and of temporary immune suppression in mammals.
(See, International Patent Publication No. WO91/00293 of Lerner E. A., et al.; and Ribeiro et al., Science, Vol. 243 pp. 212-214 (1989)). These peptides are exemplified by the ~Lutzomyia protein" (LP), which, in one embodiment, has been referred to as "maxadilan". Maxadilan has a molecular weight of about 6800 daltons, and elutes prior to calcitonin gene-related peptide in an acetonitrile-H2O-trifluoracetic acid-reverse phrase column. (See, International Patent Pub-lication No. WO91/00293 of Lerner E. A., et al.). LP can be obtained by conventional purification chromatography from surgically excised salivary glands of Lutzomyia longipalpis.
One pair of salivary glands contains about 10-15 ng LP. The Lutzomyia protein constitutes about 1% of the total protein in the salivary glands of the sand fly Lutzomyia longipalpis.
Applicants have discovered new properties possessed by Lutzomyia protein, exemplified by maxadilan. In addition to their vasodilator activity and their ability to suppress the immune system of mammals, percutaneous injection or topical administration of the protein results in the enhancement of hair growth.
Peptides suitable for use in the methods and compositions of the present invention include those peptides extracted and purified from the lysate of salivary glands of sand fly, active analogs and active fragments thereof, described in International Patent Publication No. WO91/00293 or recombinantly produced peptides. The term Lutzomyia protein as used herein refers to all such peptides, active analogs, and active fragments. Maxadilan or active fragments thereof are examples of suitable Lutzomyia proteins for use in the present invention. Maxadilan will be used as a WO94/17778 21 5 ~ 8 6 6 PCT~S94/01202 representative peptide in the following description of the invention.
There are at least two va-iants of Lutzomyia protein.
The nucleotide sequence of the cDNA and the deduced amino acid sequence of one form of mature Lutzomyia protein is as follows:
TGT GAT GCA ACA TGC CAA TTT CGC AAG G(T)CC ATA GAT GAC

Cys Asp Ala Thr Cys Gln Phe Arg Lys Ala Ile Asp Asp Cys Gln Lys CAG GCG CAT CAT AGC AAT GTT TTG CAG ACT TCT GTA CAA ACA

Gln Ala His His Ser Asn Val Leu Gln Thr Ser Val Gln Thr Thr Ala ACA TTC ACA TCA ATG GAT ACC TCC CAA CTA CCT GGA AAT AGT
GTC TTC l44 Thr Phe Thr Ser Met Asp Thr Ser Gln Leu Pro Gly Asn Ser Val Phe AAA GAA TGT ATG AAG CAG AAG AAA AAG GAA TTT AAG GCA GGA
AAG TAA l92 Lys Glu Cys Met Lys Gln Lys Lys Lys Lys Phe Lys Ala Gly Lys (SEQ ID NO:2) AATGATTGAA GAAAATTGTA GCCGAGGAGA GAAAGAAAGA AAGTCCCATA

~ ~llAATT GTAACGAATT TTCCGAAAAA ATAA~AATATT ATGCACTCAA
TT~AAAAAAA 3l2 AAA (SEQ ID NO:l) 315 The genomic Lutzomyia protein DNA sequence is as follows:
ATG AAA TAT TCT TTA AAT AAT CTC CAT TTT CTT GTA GAC GTT

Met Lys Tyr Ser Leu Asn Asn Leu His Phe Leu Val Asp Val Ala Glu GGC TGT GAT GCA ACA TGT CAA TTT CGC AAG GCC ATA GAA GAC

Gly Cys Asp Ala Thr Cys Gln Phe Arg Lys Ala Ile Glu Asp Cys Arg AAG AAG GCG CAT CAT AGC GAT GTT TTG CAG ACT TCT GTA CAA
ACA ACT l44 Lys Lys Ala His His Ser Asp Val Leu Gln Thr Ser Val Gln Thr Thr WO94/17778 215 5 ~ 6 6 PCT~S94/01202 GCA ACA TTT ACA TCA ATG GAT ACC TCC CAA CTA CCT GGA AGT
GGT GTT l92 Ala Thr Phe Thr Ser Met Asp Thr Ser Gln Leu Pro Gly Ser Gly Val TTC AAA GAA TGC ATG AAG GAG AAA GCT AAG GAA TTT AAG GCA

Phe Lys Glu Cys Met Lys Gln Lys Ala Lys Lys Phe Lys Ala Gly Lys (SEQ ID NO:4) TAG (SEQ ID NO:5) ~c 243 The genomic Lutzomyia protein~DNA sequence varies somewhat from the Lutzomyia protein cDNA sequence and is believed to represent a variant Lutzomyia protein gene. The genomic Lutzomyia protein DNA sequence includes the DNA
sequence and deduced amino acid sequence of a l7 amino acid leader peptide. The signal sequence of Lutzomyia protein is also given in the genomic DNA sequence (nucleotides 1-51).
Compositions rich in Lutzomyia protein or its active analogs and fragments can be used in carrying out this invention. The active analogs and fragments of Lutzomyia protein are typically proteins or peptides comprising an amino acid sequence sufficiently duplicative of the sequence of the active portion of the Lutzomyia protein such that the proteins or peptides are capable of inducing, maintaining or increasing hair growth in a mammal. The peptides of the present invention need not be identical to those disclosed in International Patent Publication No. WO9l/00293. Variations can be attributable to local mutations, which do not substantially detract from the hair revitalizing properties of the peptide. Such variations can be found in peptides isolated from lysates as well as chemically or recombinantly constructed peptides.
Knowledge of the Lutzomyia protein sequence enables the skilled artisan to produce large quantities of the protein for therapeutic use. The Lutzomyia protein can be synthesized using conventional chemical solid or solution phase peptide synthesis techniques. In addition, knowledge of the sequence permits expression of the DNA encoding Lutzomyia protein in various types of host cells, including WO94/17778 2 1 5 5 8 6 6 PCT~S94/01202 both prokaryotes and eucaryotes, to produce large quantities of the protein.
Instead of its primary amino acid structure, Lutzomyia protein suitable for use in this invention can be characterized by its chemical and biological properties.
Specifically, the Lutzomyia protein suitable for use in this invention is capable of inducing vasodilation or temporary immune suppression in a mammal. The vasodilatory activity of the Lutzomyia protein is shown by relaxation of at least about 100% of a constricted rabbit aortic ring as described in International Patent Publication No. WO91/00293.
Temporary immune suppression using salivary gland extract is demonstrated by the assay of H2O2 production by macrophages as a marker of immune stimulation as described in International Patent Publication No. WO91/00293 such that H2O2 production is depressed by at least about 75~ in the assay.
In one embodiment of the invention, the Lutzomyia protein has a molecular weight of about 6839 daltons as determined by mass spectrometry. In another embodiment, the Lutzomyia protein employed in this invention is characterized by reference to calcitonin gene-related peptide (CGRP). That is, the Lutzomyia protein is characterized by elution prior to CGRP in an acetonitrile-H2O-trifluoroacetic acid elution in a reverse-phase high performance liquid chromatography column. The Lutzomyia protein can also be characterized as having vasodilation activity as measured by erythema induction in animal skin of at least about 80-100 times that of CGRP as measured by the assay described in International Patent Publication No. WO91/00293.
An example of expression of the Lutzomyia protein by recombinant techniques can be found in the article entitled ~Expression of Recombinant Maxadilan", "Maxadilan, Cloning and Functional Expression of the Gene Encoding This Potent Vasodilator Peptide", Journal of Biological Chemistry, Vol.
267, No. 2, Issue of January 15, pp. 1063, Ethan Lerner and Charles Shoemakers authors.

W094/17778 215 5 8 6 G ~ PCT~S94/01202 There is a difference in the maxadilan peptide synthesized chemically, the peptide produced by recombinant techniques, and the natural peptide. The differences can be summarized as follows.

WO94/17778 2 1 5 5 8 6 6 PCT~S94/01202 MAXADILAN
SYnthetic Recombinant Natural - Amino 63 aa's 63 aa's + 2 or 3 61 aa's + C
acid aa's at N- terminus is sequence terminus (whether amidated 2 or 3 is dependent upon plasmid used) biological 2 - 5 x more properties active in vasodilation than synthetic (probably because of folding of peptides) It will be understood that each of these peptides can be employed in practicing this invention.
2. Enhancement of Hair Growth The present invention also provides a method for inducing, maintAin;ng or increasing hair growth comprising administering Lutzomyia protein at the desired site of hair growth. The Lutzomyia protein can be applied to the desired site by topical application or by subcutaneous injection.
The Lutzomyia protein can be applied to the hair of a m~mm~ 1 the skin of a mammal, or both the hair and the skin to induce, maintain, or increase hair growth in the mammal. It is preferable that the subject is a human, although the compositions and method of the invention can be employed with hair and fur bearing animals, such as sheep, rabbits, mice, rats and other mammalia. The composition of the invention can be applied to a location proximate hair pellicles in order to obtain the beneficial effects of the invention.
(a) ComPosition for Topical APPlication. The composition of the invention can be formulated into a form suitable for topical application by incorporating the Lutzomyia protein in a suitable vehicle. The vehicle can be a solid, semi-solid or liquid vehicle that is cosmetically acceptable, pharmaceutically acceptable, or physiologically acceptable, and which enables the Lutzomyia protein to be WO94/17778 PCT~S94/0~202 2~5s866 conveyed to the skin at an appropriate dilution. The nature of the vehicle will depend upon the method chosen for topical administration of the composition. The vehicle can itself be inert or it can impart physiological or pharmaceutical benefits to the composition. i The vehicle for topical application is a substance that acts as a diluent, dispersant, or æ~olvent for the Lutzomyia protein and other reagents in the ~omposition so that the composition can be applied to and distributed substantially evenly over the hair, the scalp or both the hair and scalp at an appropriate concentration. The vehicle is preferably one that aids penetration of the Lutzomyia protein into the skin to reach the immediate environment of hair pellicles.
The vehicle for topical application of the composition of this invention can be based on water or at least one cosmetically acceptable vehicle other than water. It will be understood that non-aqueous cosmetically acceptable vehicles can also be combined with water to provide a composition suitable for topical application. Vehicles that can be employed in the composition of the invention include solids or liquids, such as emollients, solvents, humectants, thickeners, and powders.
Lutzomyia protein can be utilized in the methods and compositions of the present invention either alone, or in combination with other known treatment agents. For example, peptides of the present invention can be used in combination with one or more of the following agents: vasodilators, such as carpronium chloride, swertiae extract and acetylcholine derivatives; amino acids, such as serine and methionine;
vit~mi ~s, such as vitamin B6, vitamin E and derivatives thereof, and biotin, pantothenic acid and derivatives thereof; gly~ylrhetinic acid and derivatives thereof;
nicotinates, such as benzyl nicotinate; skin-hyperactive agents, such as female sex hormones, such as estradiol;
cepharanthine and minoxidil.
Moreover, other additives that are typically used in the hair revitalizing compositions can be combined with the WO94117778 PCT~S94/01202 Lutzomyia protein according to the present invention. For example, anti-inflammatories such as hinokitiol, hexachlorophene, benzalkonium chloride, cetylpyridinium chloride, undecylenic acid, trichlorocarbanilide, and bithionol; refrigerants such as menthol; agents such as salicylic acid, zinc, and derivatives thereof, lactic acid and alkyl esters thereof, organic acids (such as citric acid), amino acids (such as arginine), oils (such as olive oil), squalene, liquid paraffin, isopropyl myristate, higher fatty acids and higher alcohols, polyhydric alcohols (such as glycerol), propylene glycol, as well as surfactants, perfumes, antioxidants, ultraviolet absorbers, pigments, ethanol, water, humectants, propellants, and thickeners, can optionally be employed in the methods and compositions of this invention to the extent that they do not deteriorate the effect of the invention.
Although the hair revitalizing composition according to the present invention can be incorporated with an extremely low level of the Lutzomyia protein, generally, the protein is employed in the amount of about l x lO 5 to about 5% by weight per total weight of the composition, and preferably in an amount of about l x lO 5 to about 3% by weight per total weight of the composition, in topical application.
The hair revitalizing composition according to the invention can be either directly applied or sprayed on the skin or applied by percutaneous injection. The dosage of the hair revitalizing composition according to the invention varies depending on age, individual differences, symptoms, etc., but in the case of an adult, it is generally in the range of 500 fg to 500 mg, and preferably in the range of lO0 pg to lO0 mg, Lutzomyia protein per kg of body weight per day.
In one embodiment, maxadilan can be applied to humans in an amount ranging from lO0 fg to lO0 mg, and preferably in the range from lO0 pg to lO0 mg, per square cm of surface area. Maxadilan is present in the topical composition of the present invention in an amount of about 5.0 x lO lO to about WO94/17778 21 S 5 8 6 6 PCT~S94/01202 5.0 x lO l percent by weight, preferably about 5.0 x lO 8 to about 5.0 x lO 3 percent by weight. Generally, to stimulate and enhance hair growth the composition is administered to an adult in an amount of about lO0 fg to about lO0 mg, preferably about lO0 pg to about lOO~mg, per kg of host body weight per day. ~ -Preferably, the hair pellicle~of the mammal is treateddaily between l and 4 times pe ~ ay. The exact regime used will depend on several factors such as the condition of the individual, but are readily determin~hle by the treating physician. Treatment should be continued at least until the desired effect is achieved.
The composition of this invention can be in any form that it can be applied to the hair pellicle. For example, the composition can be formulated as a shampoo, a conditioner, a hair spray, a hair gel product, or a moisturizing lotion.
(b) ComPosition for Subcutaneous Iniection. In addition to topically applying the composition of this invention to the subject, the composition of the invention can also be administered to the subject by subcutaneous injection to induce, maintain or increase hair growth.
Lutzomyia protein has an extremely good hair revitalizing effect so that it has an efficacy dose in animals of at least l pg per kg of host body weight per day by subcutaneous injection. The Lutzomyia protein will not be used in an amount exceeding about l mg per kg of body weight per day.

WO94/17778 21 5 5 8 6 6 PCT~S94/01202 * * *
= Maxadilan was found to be an effective hair revitalizing agent preventing epilation and trichogenous effects. Both in vitro and in vivo results showed that maxadilan enhances hair growth. Organ cultures of hair follicles from mouse whisker were used to demonstrate the effectiveness of the present invention in vitro. -Topical application on mice and humans, and subcutaneous injections of maxadilan, demonstrated the effectiveness of the present invention in vivo.
The present invention will now be described in greater detail by way of the working examples, which however, do not in any way limit the invention. All of the amounts to be formulated are expressed as % by weight, but Lutzomyia protein is used in a 1 mg/10 ml strength aqueous solution as a stock solution in Examples 1 to 5, and Lutzomyia protein is used in the form of purified powders thereof.

EXAMPLE I
PREPARATION OF HAIR ~R~ATMENT

Lutzomyia protein and an ethylene oxide (40 mol) adduct of hardened castor oil were added to 95% ethanol, and dis-solved by stirring. Deionized water was added and mixed to obtain the liquid hair revitalizing composition according to Ex. 1 below. Hair tonics according to Ex. 2-5 and Comparative Ex. 1 were obtained similarly. The proportion of the formulation in each case is shown below in Table 1.

W094/17778 2 ~ 5 5 8 6 6 PCT~S94/01202 Hair Treatment Compoæitions Cont~i n i n~ MA~A~ i 1 An As LutzomYia Protein Ex. 1 Ex. 2 Ex~ 3 Ex. 4 Ex. 5 Comp.
` Ex. 1 L.P. * 0.01% 0.001% 0.5% 0.5% 2.5% 0%
95% Ethanol 61.0 61.9 57.0 61.5 59.5 62.0 Hardened 2.0 2.0 2.0 2.0 2.0 2.0 castor oil-ethylene oxide (40 mol) adduct * L.P. represents Lutzomyia Protein, which was maxadilan in each Ex.
** The formulation made to 100% by deionized water.

WO94/17778 2 1 5 5 8 6 6 PCT~S94/01202 EXANPLE II
DEMONSTRATION OF ~ATR GRO~T~
STTMT~.A~ION IN A TRI~UL 'CUS TEST
The hair growth-stimulating effects of compositions prepared as described in Example I were determined in a test termed a trichogenous test. The trichogenous tests for the hair revitalizing compositions of Example I were carried out using C3H/HeNCrJ mice whose hair cycle was at a resting stage. The method used was that of Ogawa et al. ("Normal and Abnormal Epidermal Differentiation", M. Seiji and I.A.
Berstein eds. (1982) pp. 159-170, Todal Press.) More specifically, each test group was comprised of 10 mice. One test group received no application, while other groups were treated with the formulations of Ex. 1 to 5 and Comparative Ex. 1. The backs of the mice were shaved by means of a pair of hair clippers and a shaver. Each sample in an amount of 0.1 ml was daily topically applied. The trichogenous (hair growth) portions at the backs of the mice were measured, and the trichogenous effects of respective samples were compared with each other by means of proportion of area. The time required for 50% trichogenous rate in each sample is shown in Table 2.

wo 94~17778 ~55~ ~ ~ PCT~S94/01202 TABL~ 2 ~air Growth St ,ulation as Determined in Mouse Trichoqenous Test Sample as Described Number of~Dàys Number of in Example I, supra Confirming 50% Accelerated Days Trichogenous Action No aPPlication 60 Ex. 1 40 20 Ex. 2 50 lO
Ex. 3 30 30 Ex. 4 45 15 Ex. 5 35 25 Comparative 60 0 Ex. l The "number of accelerated days" in Table 2 is the difference between the number of days confirming 50%
trichogenous action for "no application" and the measured value for the respective Ex., and is a measure of the hair growth-stimulating effect of the composition. The greater the number of accelerated days, the more effective the composition is in stimulating hair growth in the trichogenous test. Thus, with reference to Table 2, the stimulation of hair growth was the same in test groups in which no application was made and in the Comparative Ex. l using a composition that did not contain Lutzomyia protein. In Ex. 1 to 5, which utilized compositions of the invention containing Lutzomyia protein, hair growth was stimulated as evidenced by the increase in the "number of accelerated days" when compared with the group that received "no application" and the group that received the composition of Comparative Ex. 1, which did not contain Lutzomyia protein.

W094/17778 21 S 5 8 6 6 PCT~S94/01202 EXANPLE III
DEMONSTRATION OF HAIR
REVITALIZATION IN A TRIr~AM TEST

In order to ex~ine the hair revitalizing activities, such as preventing epilation and trichogenous effect, trichogram tests were carried out for humans. The tests were conducted for hair revitalizing compositions of Ex. 1-5 and Comparative Ex. 1 prepared as described in Example I.
Trichoqram Test. The hair roots of removed hairs were microscopically observed before and after the use of the hair revitalizing compositions, and the number of hair roots at the resting stage was counted by observing the form of the hair roots. The hair revitalizing activities of the compositions were compared by the increase or decrease in the rate of growth. The "hair roots" at the resting stage are hair roots of the hairs whose growth has stopped, and the proportion of the hair roots at the resting stage is confirmed to be higher in the person suffering from epilation than in a person not suffering from epilation.
Each of the hair revitalizing compositions according to Ex. 1-5 and Comparative Ex. 1 was continuously applied twice daily on the scalps of ten male subjects in an amount of 2 ml over a period of 6 months. One hundred hairs were removed per subject immediately before the application and 6 months after the application, and each of the hair roots was ex~;ned. The results are shown below in Table 3.

WO94/17778 PCT~S94/01202 2~ss~66 HATR REVITALIZATION AS
DE~RRMTN~n IN TRTc~or-~AM TEST

Variation of [Proportion of Rested Hair Root After the Application]/[Proportion of non-rested Hair Root at the Resting State] Unit [Person] Evalu-Sample as ation of Described Effect in 30 15-30% 15-30% 30% or Example I or more decrease +15% increase more decrease increase Ex. 1 1 5 3 1 0 Yes Ex. 2 1 3 5 1 0 Yes Ex. 3 3 4 2 1 0 Yes Ex. 4 1 4 4 1 0 Yes Ex. 5 2 5 2 1 0 Yes Comp. 0 1 7 2 0 No Example 1 From the above, it is clear that the present invention is effective in the enhancement of hair growth.

EXAMPLE TV
OIL-IN-WATER EMnLSION HAIR TONIC
An emulsion hair tonic was prepared from phase A, phase B, phase C, phase D, and phase E having the following compositions:

WO94/17778 215 5 8 6 6 PCT~S94/01202 Phase A
Lutzomyia protein (maxadilan) 1.0 Polyoxyethylene (60 mol) adduct hardened castor oil 2.0 Glycerol lO.0 Dipropylene glycol lO.0 1,3-Butylene glycol 5.0 Polyethylene glycol 1500 5.0 Phase B
Cetylisooctanate 10.0 Squalene 5.0 Vaseiine 2.0 Propylparaben 2.0 Phase C
Aqueous 1% solution of carboxyvinyl polymer 30.0 Sodium hexametaphosphate 0.03 Deionized water 8.35 Phase D
Deionized water 4.5 Phase E
Potassium hydroxide 0.12 Deionized water 5.0 Phase A and phase B were separately and thermally melted at 60C and then mixed with the homomixer to produce a gel.
Phase D was gradually added thereto and dispersed by means of the homomixer.
Subsequently, the dispersed phase C was added thereto, and finally E phase was added followed by homomixer treatment to obtain an oil-in-water type emulsion hair tonic.
This hair tonic was applied to a person as set forth in Example II. The hair tonic was confirmed to have excellent hair revitalizing effects.

WO 94/17778 PCTtUS94tO1202 2~ss866 E2~A~D?LE: V
CREAN EIAIR TONIC
A cream hair tonic was prepared from phase A and phase B
having the following compositions:
Phase A
Lutzomyia protein (maxadilan) 5.0 Liquid paraffin 5.0 Cetostearyl alcohol 5.5 Glyceryl monostearate 3.0 EO (20 mol)-2-octyldodecyl ether 3.0 Propylparaben 0.3 Perfume 0.1 Phase B
Glycerol 8.0 Dipropylene glycol 20.0 Polyethylene glycol 4000 5.0 Sodium hexametaphosphate 0.005 Deionized water 49.095 Phase A and phase B were each separately and thermally dissolved and were then mixed together. The mixture was emulsified by means of a homomixer to obtain a creamy hair tonic. Using this hair tonic, a practical test was carried out on a person as described in Example II. The hair tonic was confirmed to have excellent hair revitalizing effects.

E~AMPLE VI
CREAM HAIR TONIC
A cream hair tonic was prepared by mixing two phases having the following compositions:
Phase A
Lutzomyia protein (maxadilan) 0.5 Liquid paraffin 10.5 Glyceryl monostearate 3.0 EO ( 20 mol)-2-octyldodecyl ether 3.0 Propylparaben 0.3 Perfume 0.1 Phase B
Glycerol 10.0 Dipropylene glycol 18.0 Polyethylene glycol 4000 5.0 Sodium hexametaphosphate 0.005 Deionized water 54.595 Phase A and phase B were separately and thermally dissolved. After combining the phase A and the phase B, the mixture was emulsified by means of a homomixer to obtain a creamy hair tonic. Using this hair tonic, a practical test was carried out for person as described in Example II. The hair tonic was confirmed to have excellent hair revitalizing effects.

2lss866 E~AMPLE VII
HAIR GEL
A hair gel was prepared by-combining five phases having the following compositions: ~' Phase A
Lutzomyia protein (maxadilan) 3.0 Polyoxyethylene (60 mol) adduct hardened castor oil 2 . 0 Glycerol 10.0 1,3-Butylene Glycol 5.0 Polyethylene glycol 1500 5.0 Phase B
Cetylisooctanate 10.0 Vaseline 8.0 Propylparaben 2.0 Phase C
Aqueous 1~ solution of carboxyvinyl polymer30.0 Sodium hexametaphosphate 0.03 Deionized water 8.35 Phase D
Deionized water 4.5 Phase E
Potassium hydroxide 0.12 Deionized water 12 . 0 Phase A and phase B were separately and thermally melted at 60C and then mixed with homomixer to produce a gel.
Phase D was gradually added thereto and dispersed by means of homomixer. Subsequently, the dispersed phase C was added thereto, and finally phase E was added followed by the homomixer treatment to obtain an oil-in-water type emulsion hair tonic.
Using this hair tonic, a practical test was carried out for person as described in Example II. The hair tonic was confirmed to have excellent hair revitalizing effects.

O~ ;R rrr~ OF LUT8t~IA PROTEIN

WO94/17778 -~ 215 5 8 6 6 PCT~S94/01202 Effect on Oraan Culture of Hair Follicle of Mouse Whisker. The Figure shows plots of the results, from which each sample prepared from dissolving l mg of Lutzomyia protein into physiological saline, followed by dilution as described below, was added to the following culture medium and hair growth was measured from incubation day by an inserted microscope.
Orqan Culture of Hair Follicle of Mouse Whiskers. The tissue of the whisker portion of a C3H mouse was aseptically extracted and a hair follicle at a growth stage was isolated by means of a microblade under a stereoscopic microscope.
Using a 24 well micro-plate and placing a membrane filter produced by Nucleopore in l ml of a culture (DMEM:Ham Fl2 =
l:l), the isolated hair follicle was cultured in a CO2 incubator. Here, DMEM is Dulbecco's Modified Eagle Medium, and Ham Fl2 is culture broth of Ham Fl2.
Promotion of hair growth by Lutzomyia protein depends substantially on the amount applied. A significant effect is achieved if the amount is over 6 ~g.
Trichoqenous Effect of Maxadilan b~ Percutaneous Iniection. Each sample prepared by dissolving 5 ~g of the Lutzomyia protein maxadilan into 5 ml of physiological saline was percutaneously injected in C3H mice as described below.
Control sample was physiological saline without Lutzomyia protein.
The backs of 58 days old C3H mice were shaved by means of a pair of hair clippers. Each test group was comprised of 5 animals. The samples to be tested were intradermally or percutaneously injected daily in an amount of 0.05 ml per day, and similar injections were continued over a period of 20 days (except for Saturdays and Sundays). Thereafter, the hair growth on the mice was observed every week over a period of about 40 days.
The results of the evaluation are summarized in Table 4, in which (-) means that there is no hair gro~,7th, (+) means that there is less than 50% of the hair growth, and (+) means that there is more than 50% of the hair growth.

WO94/17778 PCT~S94/01202 2lss866 Table 4 Trichogenous Effect of Lutzomyia Protein bY ~el~uLaneous Iniection (1) Reference ~ 2) Lutzomyia Protein -;-` (maxadilan) Day after Injection 43 57Day after Injection 43 57 (DaY) (DaY) Sample No. 1 - +Sample No. 11 + +
2 - - 12 + +

3 - - 13 + +

4 _ _ 14 - +
_ +

The foregoing examples, clearly show that the present invention has excellent trichogenous effects and hair revitalization effects.
* * *
While the foregoing invention has been described in some detail for purposes of clarity and understAn~ing, it will be clear to one skilled in the art from a reading of this dis-closure that various changes in form and detail can be made without departing from the true scope of the invention.

i~l 21SS866 W094/17778 PCT~S94/01202 SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: Ribeiro, Jose M.
Lerner, Ethan A.
Remold, Heinz G.
Titus, Richard G.
Tsuji, Yoshiharu Hansawa, Chika Uzuka, Makota (ii) TITLE OF lNv~NlION: Method for Enhancing Hair Growth and Hair Revitalizing Compositions Comprising Lutzomyia Protein (iii) NUMBER OF SEQ~NC~:S: 4 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Finnegan, Henderson, Farabow, Garrett &
Dunner (B) STREET: 1300 I Street, N.W., Suite 700 (C) CITY: Washington (D) STATE: D.C.
(E) COUNTRY: USA
(F) ZIP: 20005-3315 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COM~Ul~:~: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/017,061 (B) FILING DATE: February 12, 1993 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Meyers, Kenneth J.
(B) REGISTRATION NUMBER: 25,146 (C) REFERENCE/DOCKET NUMBER: 05136-0002-00000 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 202-408-4000 (B) TELEFAX: 202-408-4400 W094/17778 PCT~S94/01202 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 315 base pairs (B) TYPE: nucleic acid ~.
(C) STRANDEDNESS: single ~.
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TGTGATGCAA CATGCCAATT TCG~AAG~CC AT~Ar~A~GAcT GCGAGAAGCA GGCGCATCAT 6 AGCAATGTTT TGCAGACTTC TG~ACAAAC~ ACTGCAACAT TCACATCAAT GGATACCTCC 12 CAACTACCTG GAAA~AGTGT CTTCAAA~-A~ TGTATGAAGC AGA~r-AAAAA GGAATTTAAG 18 TACCATATTT l~-LllGllAA TTG~AAC~A~ TTTTCCGAAA AAA~AAAATA TTATGCACTC 30 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Cys Asp Ala Thr Cys Gln Phe Arg Lys Ala Ile Asp Asp Cys Gln Lys Gln Ala His His Ser Asn Val Leu Gln Thr Ser Val Gln Thr Thr Ala Thr Phe Thr Ser Met Asp Thr Ser Gln Leu Pro Gly Asn Ser Val Phe Lys Glu Cys Met Lys Gln Lys Lys Lys Lys Phe Lys Ala Gly Lys WO94/17778 21 5 5 8 6 6 PCT~S94/01202 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 243 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQU~C~: DESCRIPTION: SEQ ID NO:3:
ATGAAA~TT CTTTAAA~AA TCTCCATTTT CATGTAGACG TTGCTGAGGG CTGTGATGCA 61 ACATGTCAAT TTCGCAAGGC CATAr-AAGAC TGCAGGAAGA AGGCGCATCA TAGCGATGTT 12J
TTGCAGACTT CTGTACAAAC AACTGCAACA TTTACATCAA TGGATACCTC CCAACTACCT 18i GGAAGTGGTG TTTTCA~r~ ATGCATGAAG GAGAAAGCTA AGGAATTTAA GGCAGGAAAG 241 TAG 24:
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Lys Tyr Ser Leu Asn Asn Leu His Phe Leu Val Asp Val Ala Glu Gly Cys Asp Ala Thr Cys Gln Phe Arg Lys Ala Ile Glu Asp Cys Arg Lys Lys Ala His His Ser Asp Val Leu Gln Thr Ser Val Gln Thr Thr Ala Thr Phe Thr Ser Met Asp Thr Ser Gln Leu Pro Gly Ser Gly Val Phe Lys Glu Cys Met Lys Gln Lys Ala Lys Lys Phe Lys Ala Gly Lys

Claims (18)

WHAT IS CLAIMED IS:

1. A method for inducing, maintaining or increasing hair growth in a mammal, wherein the method comprises administering a hair growth inducing, maintaining or increasing amount of Lutzomyia protein to the skin or hair of the mammal.

2. The method as claimed in claim 1, wherein the Lutzomyia protein is maxadilan.

3. The method as claimed in claim 2, wherein the maxadilan is administered to a human in an amount of about 25 fg to about 2.5 mg per kg of host body weight per day.

4. The method as claimed in claim 2, wherein the maxadilan is administered to a human in an amount of about 1 pg to about 1 mg per kg of host body weight per day.

5. A method for inducing, maintaining or increasing hair growth in a mammal, which comprises topically applying a composition comprising maxadilan to the mammal in a hair growth inducing, maintaining or increasing amount, wherein the composition contains maxadilan in an amount of about 5.0 x 10-10 to about 5.0 x 10-1 percent by weight.

6. The method as claimed in claim 5, wherein the composition contains maxadilan in an amount of about 5.0 x 10-8 to about 5.0 x 10-3 percent by weight.

7. The method as claimed in claim 5, wherein the maxadilan is applied to the human in an amount of about 100 fg to about 100 mg per kg of body weight per day.

8. The method as claimed in claim 5, wherein the human is treated by applying maxadilan to hair pellicles of the human from one to four times per day.

9. The method as claimed in claim 8, wherein the maxadilan is applied to the human in an amount of about 100 pg to about 100 mg per kg of body weight per day.

10. A method for inducing, maintaining or increasing hair growth in a mammal, which comprises percutaneously injecting into the mammal proximate hair pellicles a composition comprising Lutzomyia protein in a hair growth inducing, maintaining or increasing amount.

11. The method as claimed in claim 10, wherein the Lutzomyia protein is maxadilan.

12. The method as claimed in claim 11, wherein the maxadilan is applied to the human in an amount of about 1 pg to about 1 mg per kg of body weight per day.

13. A composition suitable for topical application to mammalian skin or hair, wherein the composition comprises Lutzomyia protein in a hair growth inducing, maintaining or increasing amount in a pharmaceutically acceptable vehicle.

14. The composition as claimed in claim 14, wherein the Lutzomyia protein is maxadilan.

15. The composition as claimed in claim 14, wherein the composition comprises about 1 x 10-5 to about 5 percent by weight maxadilan based on the total weight of the composition.

16. The composition as claimed in claim 14, wherein the composition comprises about 1 x 10-5 to about 3 percent by weight maxadilan based on the total weight of the composition.

17. The composition as claimed in claim 14, wherein said composition comprises maxadilan in a pharmaceutically acceptable vehicle selected from the group consisting of squalene, liquid paraffin, a fatty acid, a monohydric alcohol, a polyhydric alcohol, propylene glycol, or water.

18. The composition as claimed in claim 14, wherein said composition comprises maxadilan in a cosmetically acceptable vehicle selected from the group consisting of polyoxyethylene adduct of hardened castor oil, glycerol, dipropylene glycol, 1,3-butylene glycol, polyethylene glycol, cetylisooctanate, squalene, vaseline, propylparaben, water, liquid paraffin, cetosterearyl alcohol, glyceryl monostearate, and ethylene oxide alkyl ether.