WO2001066702A1

WO2001066702A1 - Cosmetic or pharmaceutical composition useful in inhibiting or delaying human alopecia by means of topical application of the composition

Info

Publication number: WO2001066702A1
Application number: PCT/IT2001/000106
Authority: WO
Inventors: Ugo Raffaello Citernesi
Original assignee: Ira Istituto Ricerche Applicate S.R.L.; Hauspix S.R.L.; Mediavending S.R.L.
Priority date: 2000-03-08
Filing date: 2001-03-06
Publication date: 2001-09-13
Also published as: EP1175488A1; BR0105926A; IT1318379B1; ITMI20000459A1; ITMI20000459A0; AU4452801A

Abstract

Cosmetic or pharmaceutical composition administered topically on the scalp, useful for the treatment of alopecia containing an enzyme that causes the decomposition by hydrolysis of the dihydrotestosterone thus preventing the latter causing the atrophy and successive death of the hair.

Description

Title

"COSMETIC OR PHARMACEUTICAL COMPOSITION USEFUL IN

INHIBITING OR DELAYING HUMAN ALOPECIA BY MEANS OF TOPICAL

APPLICATION OF THE COMPOSITION-' Tecnical Field

The present invention relates to a product that has applications in cosmetic or pharmaceutical treatment in order to fight alopecia or hair loss.

More particularly, the present invention refers to an enzymatic composition that allows the causes that determine hair loss to be fought with success. Hair loss in men is due to various reasons and one of the most accepted attributes hair loss to the action of particular enzymes that prevent the development of pilipheric bulbs.

In men over a certain age testosterone is transformed into dihydrotestosterone, due to

the action of the enzyme 5-α-reductase,.

Testosterone + 5-α-reductase > Dihydrotestosterone

The latter moves from the prostate or testicles, where the testosterone and the enzyme 5-α-reductase are normally localized and goes to localize in the micro-capillaries of

the pilipheric bulb, causing the atrophy and death of the bulb with consequent loss of the hair.

Background art There are two possible

s to oppose this phenomenon: - inhibit the 5-α- reductase or destroy the dihydrotestosterone as it is formed.

Since the α-reductase is found, as was stated above, localized in the testicles and prostate, it is obvious that a topical treatment in these regions is difficult to carry out. It is, therefore, an objective of the present invention to provide a composition which is applied topically on the scalp and prevents localization of the dihydrotestosterone in the pilipheric bulbs. Disclosure of the invention The objective of the present invention is achieved by bringing the dihydrotestosterone into contact with an enzyme that causes it to decompose by oxidation, thus preventing the dihydrotestosterone from causing the atrophy and the successive death of the hair. Enzymes capable of hydrolysing the dihydrotestosterone are those defined as oxidoreductases, eventually assisted by an oxidoreduction coenzyme, e.g., from NADP (H) NAD(H). One enzyme among those able to decompose dihydrotestosterone by oxidoreduction. has been demonstrated to be particularly

active: 3-α-Hydroxysteroid oxidoreductase or 3-α HSOR.

Without constituting a limitation of the ambit of the invention, we maintain that the decomposition of the dihydrotestosterone is a reversible reaction that depends on the ratio of NADP(H) NAD(H). The greater the concentration of the NADP(H), the more

the reaction goes towards the formation of the 3-α-adiolo. degrading the dihydrotestosterone and transforming the NADP(H) nucleotide into NAD(H) nucleotide. Viceversa. when the concentration of the NAD(H) exceeds the concentration of the NADP(FI). the same enzyme behaves in the inverse way and catalyzes the dihydrotestosterone-forming reaction according to the outline:

DHT+ NADP(H) > α-HSOR > 3-α-ADIOLO+NAD(H)

DHT^- NADP(H) < α-HSOR < 3-α-ADIOLO÷NAD(H)

The 3-α-hydroxystεroid oxidoreductasε can be applied in the form of a lotion.

shampoo, or in an}' other form suitable for a topical treatment. It could be mixed with other compuonds well known in the cosmetic art which would favor penetration into the lower layers of the skin of the scalp.

Best mode to cam" out the invention The present invention will be better understood, and the advantages of the same will be belter appreciated, from reading of some examples of its embodiments which are provided by way of example but which should not be interpreted as limitating the application of the invention.

DOSAGE Of the DIHYDROTESTOSTERONE the dosage of the dihydrotestosterone is an important stage in the verification of the activity of the enzyme. Determining ana lically the reduction of the concentration of the dihydrotestosterone in culture medium is sufficient to verify the effectiveness of the same system.

The methodology developed was taken from the standard methodolgy for establishing the dosage of steroids.

High concentrations of dihydrotestosterone are present In culture medium, so dosage by HPLC is advantageous. Vice versa, for a similar determination on plasma or, worse still at the level of the pilipheric bulb, where the levels of dihydrotestosterone are necessarily very low. such methodology is insufficient. For our purposes. however. the methodology developed has been shown to be sufficiently precise.

Below are reported the details of the methodology used for the determination by high resolution liquid chromatography. HPLC:

Column: LiChroART 125-4 Purospher RP 18 and. 5μm

Mobile phase: A: Water

B: Acetonitrile

Gradient: 0 minutes 75%A-25%B

20 minutes 50%A-50%D

30 minutes 50'%A -50% B

Flow-rate: 1 ml/min.

Detector: UV 220nm

Temperature: 28 °C

Injection: l Oμm

Retention time: 15 mm.

VERIFICATION Of the DEGRADATION Of DHT by 3-a-HSOR - hydroxytestosterone dehydrogenase catalyzes the interco 'ersion reaction of the carboxyilic and hydroxyilic group of the hydrotestosterone. It is a typical oxidative reaction and by doing so reduces NAD. It is therefore possible to measure the oxidation of dehvdrotestosterone in the presence of NAD, estimating the reaction kinetics by measuring an increment of absorbance at 340 nm due to the reduction of the same NAD. Moreover it is possible quantitatively to verify the presence of Dihydroxitestosterone in the enzyme solution, before and after the same reaction. Reagents: - 0.03 M tris-HCl buffered to pH7.2 with 0.001 M EDTA - 0.166 M sodium p\ rophosphate buffered to pH 9

- 0.0043 of NAD in purified water for HPLC. The NAD can vary in salt form depending on the degree of oxidation.

- 0.015% Dihydrotestosterone. preparing 15 mg of dihydrotestosterone in 100 g of absolute alcohol for HPLC.

Enzyme:

Dissohe the enzyme to a concentration of Img ^'ml in 0.03 M tris.HCl pH7.2 with 0.001 M EDTA. the successπ e dilutions can be earned out with the same buffer solution. Procedure:

Calibrate the spectrophotometer to 340 nm and 25~C - Each quartz cuvette contains the follow ing amounts:

- 0.166 M of sodio pyrophosphate 0.6 ml

- 0.0043 M NAD 0.2 ml - Water purified for HPLC 2 ml -

- Enzyme 0.1ml

One leaves all to incubate in the spectrophotometer until a temperature equilibrium is reached so as to to be able to establish the \ alue of absorbance of the blank.

At time 0, i.e. at the beginning, add 0.1 ml of the dihy drotestosterone solution, and

after a few seconds collect an aliquot of 20 μl to inject into the HPLC previously

prepared for the dosing of the dih^y drotestosterone.

The reaction is followed for 5 minutes and the \ ariations of absorbance of the solution at 340 nm are noted. After

e minutes, take another aliquot of 20 μl of solution and inject it into the HPLC again in Older to 's erify the residual amount of drotestosterone remainiι _> after the me reaction. Calculation of the amount of enzyme:

The amount of enzyme expressed in unifmg of substance is estimated as follows:

Δ A340/min L7mg= -

6.22 x mg Enzyme / ml

here μA340 = \ ariation of the absorbance to 340 nm. min = minute g = milligrammi ml = mis of the reaction solution

6.22 = conversion factor Results:

The results of the dosage of the dihydrotestosterone are shown in table 1 TABLE 1

The first column shows values relating to the absorbance at 340 mn of solution during the enzymatic kinetics phase, while the second column reports time expressed in minutes and seconds, a 1.2 0

1.34 30

1.5 60

1.65 90

1.81 120 1.94 150

2.1 180

2.24 210 2.4 240

2.55 270

2.66 300

TABLE 2 Results relating to the dosage of DHT the before and after enzyme kinetics with

HSOR

Theoretical Concentration of DHT in medium: 0.015 mg/ml

Concentration shown instru entally

At time 0: 0.012 mg/ml At time 100": 0.009 mg/ml

At time 200" : 0.007 mg/ml

At time 300": not determinab'e

EVALUATION OF THE ANTI-DHA ACTIVITY AND THE INCREASE AND

DEVELOPMENT OF HAIR IN SUBJECTS AFFECTED BY ALOPECIA ANDROGENETICA

Materials and methods

The study wascarried out on thirty males aged between 25 and 40 years clearly affected by alopecia androgenetica and at various stages of alcpecianal .

The group was divided into two homogenous subgroups, each of 15 people. Treatment

Two series of samples in phials were prepared containing 10ml of the following solutions in distilled water:

Solution A

Hydrolized Cheratin 2% Tween 20.8% Capsico resin oil 0.1 %

Preserving system pH 7.5

Solution B

Solution A with addition of 2.5 mg ^'lOml hydroxysteroid dehydrogenase before the phial was used.

In order to make the phials with enzyme unrecognizable to the patients. 25 mg' of powder was added to the phials. The A phials contained only sodium bicarbonate, the

3 sodium bicarbonate added to the enzyme.

The treatment continued for a period of six months, during which the patients used one 10 ml phial in the morning and one in the evening. Assessment

The assessment was earned out by selecting a surface of 25cm^" of the scalp for every subject on which the following measurements were carried out at intervals of 15 days: hairs counted and length measured. In this area the hairs were cut to the same length before the test began. Results

The treatment earned out with the of the type B phials, in which the enzyme hydrosteroid dehydrogenase (HSOR) was present demonstrated good effectiveness in regrowing hair; in fact, the average regrowth length after six months of treatment was 7.5mm.

Moreover beyond the greater average regrowth, there was also an increase in the averge number of hairs, from the 100 initially to 185 finally in the group treated with with phial B. while those treated with the solution A w ent from the 100 initially to the

75 finallv.

Claims

1. Cosmetic or pharmaceutical composition administered topically on the scalp, useful for the treatment of alopecia characterized by containing an enzyme that causes the decomposition by hydroh sis of the dihydrotestosterorie thus preventing the latter causing the atrophy and successive death of the hair.

2. Cosmetic or pharmaceutical composition administered topically on the scalp, useful for the treatment of alopecia, according to Claim 1. characterized by the enzyme being an oxidoreductase.

3. Cosmetic or pharmaceutical composition administered topically on the scalp, useful for the treatment of alopecia, according to Claim lor 2. characterized by the enzyme

being 3-α-hydroxysteroid oxidoreductase

4. Cosmetic or pharmaceutical composition administered topically on the scalp, useful for the treatment of alopecia, according to Claims from 1 to 3, characterized by being used together ith an oxidoreductase coenzyme.

5. Cosmetic or pharmaceutical composition administered topically on the scalp, useful for the treatment of alopecia, according to Claim 1. characterized by the coenzyme being the NADP(H)'ι\^τAD(H) system.

6. Cosmetic or pharmaceutical composition administered topically on the scalp, useful for the treatment of alopecia according to one or more of the previous Claims. characterized by comprising surfactants, cheratin and preservative.

7. The use of 3-α-hydroxysteroid oxidoreductase for the preparation of compositions

administered topically for the treatment of alopecia

8. The use of 3 -α-hydrox> steroid oxidoreductase in a mixture with the

me NADP(H)/NAD(Η) for the preparation of cosmetic or pharmaceutical compositions administered topicalh" for the treatment of alopecia due to the localization of dihydrotestosterone in the capilaries of the pilipheric bulbs.