EP0683660A1

EP0683660A1 - Method of enhancing hair growth and hair revitalizing compositions comprising lutzomyia protein

Info

Publication number: EP0683660A1
Application number: EP94909527A
Authority: EP
Inventors: Jose M. C. Ribeiro; Ethan A. Lerner; Heinz G. Remold; Richard G. Titus; Yoshiharu The Shiseido Research Center Tsuji; Chika The Shiseido Research Center Hansawa; Makoto The Shiseido Research Center Uzuka
Original assignee: Shiseido Co Ltd; Harvard College
Current assignee: Shiseido Co Ltd; Harvard College
Priority date: 1993-02-12
Filing date: 1994-02-10
Publication date: 1995-11-29
Also published as: AU6234694A; CA2155866A1; JPH09500090A; WO1994017778A1

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

METHOD OF ENHANCING 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 Lutzomyia longipalpis .

2. Background 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, an the formation of peroxides have all been considered as possible causes of baldness and epilation. Conventional hai revitalizing compositions, therefore, have been formulated with the aim of alleviating or reducing the occurrence of these events.

Formulations using vitamins, 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, suc 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 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 treatment 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 enhancin 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 pharmaceutical 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.

SUMMARY OF THE INVENTION

The present inventors have researched substances effective for the treatment of epilation and possessing othe 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 makin 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 found 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 an hair revitalization effects by topical application and subcutaneous injection.

More particularly, this invention provides a method for inducing, maintaining or increasing hair growth in a mammal. The method comprises administering a hair growth inducing, maintaining or increasing amount of Lutzomyia protein to the skin or hair of the mammal. In a preferred embodiment of th 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, maintaining 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 o wool or fur in animal breeding industries. The invention is thus beneficial for inducing, maintaining 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.

DETAILED 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 san fly Lutzomyia longipalpis exhibit strong effects, which result in the enhancement of hair growth. 1. Lutzomyia Proteins.

Peptides from salivary gland lysates of 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 weigh of about 6800 daltons, and elutes prior to calcitonin gene- related peptide in an acetonitrile-H₂0-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. W091/00293, 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 representative peptide in the following description of the invention.

There are at least two variants 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

TGC GAG AAG 4

Cys Asp Ala Thr Cys Gin Phe Arg Lys Ala lie Asp Asp

Cys Gin Lys

CAG GCG CAT CAT AGC AAT GTT TTG CAG ACT TCT GTA CAA ACA ACT GCA 9

Gin Ala His His Ser Asn Val Leu Gin Thr Ser Val Gin Thr Thr Ala

ACA TTC ACA TCA ATG GAT ACC TCC CAA CTA CCT GGA AAT AGT GTC TTC 14

Thr Phe Thr Ser Met Asp Thr Ser Gin Leu Pro Gly Asn Ser Val Phe

AAA GAA TGT ATG AAG CAG AAG AAA AAG GAA TTT AAG GCA GGA AAG TAA 19

Lys Glu Cys Met Lys Gin Lys Lys Lys Lys Phe Lys Ala Gly Lys (SEQ ID NO:2)

AATGATTGAA GAAAATTGTA GCCGAGGAGA GAAAGAAAGA AAGTCCCATA CCATATTTTG 25

TTTGTTAATT GTAACGAATT TTCCGAAAAA ATAAAATATT ATGCACTCAA TTTAAAAAAA 31

AAA (SEQ ID N0:1) 31

The genomic Lutzomyia protein DNA sequence is as follows:

ATG AAA TAT TCT TTA AAT AAT CTC CAT TTT CTT GTA GAC GTT GCT GAG 4

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 TGC AGG 9

Gly Cys Asp Ala Thr Cys Gin Phe Arg Lys Ala lie Glu Asp Cys Arg

AAG AAG GCG CAT CAT AGC GAT GTT TTG CAG ACT TCT GTA CAA ACA ACT 14

Lys Lys Ala His His Ser Asp Val Leu Gin Thr Ser Val Gin Thr Thr GCA ACA TTT ACA TCA ATG GAT ACC TCC CAA CTA CCT GGA AGT GGT GTT 192

Ala Thr Phe Thr Ser Met Asp Thr Ser Gin Leu Pro Gly Ser Gly Val

TTC AAA GAA TGC ATG AAG GAG AAA GCT AAG GAA TTT AAG GCA GGA AAG 240

Phe Lys Glu Cys Met Lys Gin Lys Ala Lys Lys Phe Lys Ala Gly Lys (SEQ ID NO:4)

TAG (SEQ ID NO:5) 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 17 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. WO91/00293. 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 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 o 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 H^C production by macrophages as a marker of immune stimulation as described in International Patent Publication No. WO91/00293 such that H 0₂ 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 characterize by reference to calcitonin gene-related peptide (CGRP). Tha is, the Lutzomyia protein is characterized by elution prior to CGRP in an acetonitrile-H₂0-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. 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.

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, maintaining 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 mammal, 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 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.

The vehicle for topical application is a substance that acts as a diluent, dispersant, or solvent for the Lutzomyia protein and other reagents in the composition 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; vitamins, such as vitamin B_β, vitamin E and derivatives thereof, and biotin, pantothenic acid and derivatives thereof; glycyrrhetinic 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 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 a 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 th 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 i

_5 employed in the amount of about 1 x 10 to about 5% by weight per total weight of the composition, and preferably i

_5 an amount of about 1 x 10 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 th 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 10 pg to 100 mg, Lutzomyia protein per kg of body weight per day.

In one embodiment, maxadilan can be applied to humans i an amount ranging from 100 fg to 100 mg, and preferably in the range from 100 pg to 100 mg, per square cm of surface area. Maxadilan is present in the topical composition of th present invention in an amount of about 5.0 x 10^" to about 5.0 x 10—^"1 percent by weight, preferably about 5.0 x 10—8 to

_3 about 5.0 x 10 percent by weight. Generally, to stimulate and enhance hair growth the composition is administered to an adult in an amount of about 100 fg to about 100 mg, preferably about 100 pg to about 100 mg, per kg of host body weight per day.

Preferably, the hair pellicle of the mammal is treated daily between 1 and 4 times per day. The exact regime used will depend on several factors such as the condition of the individual, but are readily determinable 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 Injection. 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 1 pg per kg of host body weight per day by subcutaneous injection. The Lutzomyia protein will not be used in an amount exceeding about 1 mg per kg of body weight per day.

Maxadilan was found, to be an effective hair revitalizin agent preventing epilation and trichogenous effects. Both i vitro and in vivo results showed that maxadilan enhances hai 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 i used in the form of purified powders thereof.

EXAMPLE I

PREPARATION OF HAIR TREATMENT COMPOSITIONS CONTAINING LϋTZQUΥIA PROTEIN

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 o the formulation in each case is shown below in Table 1. TABLE 1

Hair Treatment Compositions Containing Maxadilan As Lutzomyia Protein

* L.P. represents Lutzomyia Protein, which was maxadilan in each Ex. ** The formulation made to 100% by deionized water.

EXAMPLE II DEMONSTRATION OF HAIR GROWTH STIMULATION IN A TRICHOGENOUS 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 E . 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 eac sample is shown in Table 2.

TABLE 2

Hair Growth Stimulation as Determined in Mouse Trichogenous Test

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. 1 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. EXAMPLE III

DEMONSTRATION OF HAIR REVITALIZATION IN A TRICHOGRAM TEST

In order to examine 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.

Trichogram 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 examined. The results are shown below in Table 3.

TABLE 3

HAIR REVITALIZATION AS DETERMINED IN TRICHOGRAM TEST

From the above, it is clear that the present invention is effective in the enhancement of hair growth.

EXAMPLE TV

OIL-IN-WATER EMULSION HAIR TONIC

An emulsion hair tonic was prepared from phase A, phase B, phase C, phase D, and phase E having the following compositions: Phase A

Lutzomyia protein (maxadilan) 1.0

Polyoxyethylene (60 mol) adduct hardened castor oil 2.0

Glycerol 10.0

Dipropylene glycol 10.0

1,3-Butylene glycol 5.0

Polyethylene glycol 1500 5.0

Phase B

Cetylisooctanate 10.0

Squalene 5.0

Vaseline 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 60°C 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 . EXAMPLE V

CREAM HAIR TONIC

A cream hair tonic was prepared from phase A and phase 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 .

EXAMPLE 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 0.0

Deionized water 54.59

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.

EXAMPLE 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

Potassium hydroxide 0.12

Deionized water 12.0

Phase A and phase B were separately and thermally melted at 60°C 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.

OTHER EFFECTS OF LUTZOMYIA PROTEIN Effect on Organ Culture of Hair Follicle of Mouse Whisker. The Figure shows plots of the results, from which each sample prepared from dissolving 1 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.

Organ 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 icroblade under a stereoscopic microscope. Using a 24 well micro-plate and placing a membrane filter produced by Nucleopore in 1 ml of a culture (DMEM:Ham F12 = 1:1), the isolated hair follicle was cultured in a C0₂ incubator. Here, DMEM is Dulbecco's Modified Eagle Medium, and Ham F12 is culture broth of Ham F12.

Promotion of hair growth by Lutzomyia protein depends substantially on the amount applied. A significant effect is achieved if the amount is over 6 μg .

Trichogenous Effect of Maxadilan b Percutaneous Injection. Each sample prepared by dissolving 5 μg 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 growth, (+_) means that there is less than 50% of the hair growth, and (+) means that there is more than 50% of the hair growth. Table 4

Trichogenous Effect of Lutzomyia Protein by Percutaneous Injection

(1) Reference (2) Lutzomyia Protein (maxadilan)

Day after Injection 43 57 Day after Injection 43 57 ( Ωav) Day

Sample No. 1 2 3 4

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 understanding, 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.

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 INVENTION: Method for Enhancing Hair Growth and Hair Revitalizing Compositions Comprising Lutzomyia Protein

(iii) NUMBER OF SEQUENCES: 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) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln 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 (2) INFORMATION FOR SEQ ID NO:l:

(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:l:

TGTGATGCAA CATGCCAATT TCGCAAGKCC ATAGATGACT GCGAGAAGCA GGCGCATCAT

AGCAATGTTT TGCAGACTTC TGTACAAACA ACTGCAACAT TCACATCAAT GGATACCTCC

CAACTACCTG GAAATAGTGT CTTCAAAGAA TGTATGAAGC AGAAGAAAAA GGAATTTAAG

GCAGGAAAGT AAAATGATTG AAGAAAATTG TAGCCGAGGA GAGAAAGAAA GAAAGTCCCA

TACCATATTT TGTTTGTTAA TTGTAACGAA TTTTCCGAAA AAATAAAATA TTATGCACTC

AATTTAAAAA AAAAA (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 Gin Phe Arg Lys Ala lie Asp Asp Cys Gin Ly 1 5 10 15

Gin Ala His His Ser Asn Val Leu Gin Thr Ser Val Gin Thr Thr Al 20 25 30

Thr Phe Thr Ser Met Asp Thr Ser Gin Leu Pro Gly Asn Ser Val Ph 35 40 45

Lys Glu Cys Met Lys Gin Lys Lys Lys Lys Phe Lys Ala Gly Lys 50 55 60 (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) SEQUENCE DESCRIPTION: SEQ ID NO:3:

ATGAAATATT CTTTAAATAA TCTCCATTTT CATGTAGACG TTGCTGAGGG CTGTGATGCA

ACATGTCAAT TTCGCAAGGC CATAGAAGAC TGCAGGAAGA AGGCGCATCA TAGCGATGTT

TTGCAGACTT CTGTACAAAC AACTGCAACA TTTACATCAA TGGATACCTC CCAACTACCT

GGAAGTGGTG TTTTCAAAGA ATGCATGAAG GAGAAAGCTA AGGAATTTAA GGCAGGAAAG

TAG (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 Gl 1 5 10 15

Gly Cys Asp Ala Thr Cys Gin Phe Arg Lys Ala lie Glu Asp Cys Ar 20 25 30

Lys Lys Ala His His Ser Asp Val Leu Gin Thr Ser Val Gin Thr Th 35 40 45

Ala Thr Phe Thr Ser Met Asp Thr Ser Gin Leu Pro Gly Ser Gly Va 50 55 60

Phe Lys Glu Cys Met Lys Gin Lys Ala Lys Lys Phe Lys Ala Gly Ly 65 70 75 80

Claims

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~ to about 5.0 x 10^" 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

_5 composition comprises about 1 x 10 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

_5 composition comprises about 1 x 10 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.