Hair-growing agent

Details Hair-growing agent Hair-growing agent

1997-03-24

2003-01-14

Low, Christopher S. F. (Department: 1653)

Drug, bio-affecting and body treating compositions

Live hair or scalp treating compositions

C424S070110, C530S319000, C514S002600, C514S075000, C514S131000, C514S139000, C514S557000, C514S558000, C514S765000

Reexamination Certificate

active

06506370

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a hair-growing agent comprising a protein kinase C-specific inhibitor.
BACKGROUND OF THE INVENTION
There is disclosed that substances with a protein kinase-inhibiting activity, 3-amino/hydroxy-4-[4-benzoyl-phenyl carbonylamino/oxy]azepanes stimulate the growth of hair (EP663393 A1); but said proteinkinase inhibitors have a protein kinase A (hereinafter referred to as PKA)-inhibiting activity along with a protein kinase C (hereinafter referred to as PKC)-inhibiting activity.
In hair-follicle organ culture systems, a PKC inhibitor, H-7 is known to release the hair growth-retarding activity of a PKC-activating substance, 12-O-tetragalloyl-phorbol-13-acetate (British Journal of Dermatology, 133, 5, 686-693, 1995). Said H-7 is further known to have a PKA-inhibiting activity along with a PKC-inhibiting activity (BIO/TECHNOLOGY, 8, 732, 1990).
In hair-follicle organ culture systems, the PKC inhibitor, H-7 releases the hair growth-inhibiting activity of 12-O-tetragalloyl-phorbol-13-acetate, but said PKC inhibitor has not been known to have a hair growth-promoting activity (British Journal of Dermatology, 133, 5, 686-693, 1995).
Protein kinase inhibitors having both a PKC-7inhibiting activity and a PKA-inhibiting activity could not always be expected to produce satisfactory hair growth-promoting results. H-7 and 3-amino/hydroxy-4-[4-benzoyl-phenyl carbonylamino/oxy]azepanes could not always be expected to produce satisfactory hair growth-promoting results, because such compounds have both a PKC-inhibiting activity and a PKA-inhibiting activity.
The present inventors have first found that PKC-specific inhibitors produce satisfactory hair growth-promoting results.
SUMMARY OF THE INVENTION
The object of the present invention is to provide a safe and effective ahir-growing agent comprising, as an active ingredient, a PKC-specific inhibitor, and pharmaceutically acceptable vehicles.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
The protein kinase C-specific inhibitor as referred to herein is a protein kinase inhibitor having a PKC-inhibiting activity while having a PKA-inhibiting activity as little as possible. Specifically, any inhibitor can be used so long as the ratio of its 50% PKA-inhibiting constant (hereinafter referred to as PKA-IC
50
) to its 50% PKC-inhibiting constant (hereinafter referred to as PKC-IC
50
), PKA-IC
50
/PKC-IC
50
, is not smaller than 3.0 when determining PKC-IC
50
and PKA-IC
50
according to the following PKC-inhibiting, activity measuring method and according to the following PKA-inhibiting activity measuring method, respectively. For example, usable herein are protein kinase inhibitors having PKA-IC
50
/PKC-IC
50
/PKC-IC
50
of from 10 to 10
9
.
For determination of PKA-inhibiting activity of protein kinase inhibitors having a low PKA-inhibiting activity, the following method requires a large amount of such a protein kinase inhibitor to be determined therein, in which, therefore, the protein kinase-inhibiting activities of the protein kinase inhibitor could be measured only up to its uppermost soluble concentration in the system. Accordingly, if such a protein kinase inhibitor with low PKA-inhibiting activity is used, the value of PKA-IC
50
/PKC-IC
50
capable of being numerically determined in said method will be about up to 10
9
. The protein kinase inhibitors usable: in the present invention have its value of PKA-IC
50
/PKC-IC
50
of not smaller than 3, without being defined by its upper value of PKA-IC
50
/PKC-IC
50
(10
9
) capable of numerically determined by said method.
Preferred protein kinase inhibitors for use are those having a value of PKA-IC
50
/PKC-IC
50
of from 10 to 10
9
.
(1) Method for Measuring PKC-inhibiting Activity:
To measure the PKC-inhibiting activity of a protein kinase inhibitor, referred to is the Kikkawa et al's method (Journal of Biological Chemistry, 257, 13341, 1982).
Precisely, 10 &mgr;l of a sample to be tested for determining its activity is added to 250 &mgr;l of a solution comprising 2.5 &mgr;mols of magnesium acetate, 50 &mgr;g of histone Type IIIS (produced by Sigma Co.), 20 &mgr;g of phosphatidylserine, 0.8 &mgr;g of diolein, 25 nmols of calcium chloride, 5 &mgr;g of a crude enzyme (as partially purified from a rat brain according to the Kikkawa et al's method) and 5 &mgr;mols of Tris-HCl buffer (pH 7.5), and incubated therein at 30° C. for 3 minutes.
After the completion of the incubation, 1.25 nmols of [g-
32
P]ATP (from 5×10
3
to 10×10
3
cpm
mol) is added to the system, and phosphorylation reaction is carried out at 30° C. for 3 minutes, and thereafter the reaction is terminated by adding 25% trichloroacetic acid thereto.
The resulting reaction mixture is filtrated through a cellulose acetate membrane (pore size: 0.45 &mgr;m) (produced by Toyo Filter Co.), and the membrane is washed four times with 5% trichloroacetic acid, and thereafter the radioactivity of the residue remained on the membrane is measured to be a value of the sample.
On the other hand, the same process as described above is repeated without adding the sample to the system, and the radioactivity is obtained to be a control value.
The molar concentration of the sample having the value of radioactivity which is 50% of the control value is obtained to be the 50% PKC-inhibiting constant (PKC-IC
50
) of the sample.
(2) Method for Measuring PKA-inhibiting Activity:
To measure the PKA-inhibiting activity of a protein kinase inhibitor, referred to is the Kuo et al's method (see Biochemistry, 6, 1349, 1969).
Precisely, 10 &mgr;l of a sample to be tested for determining its activity is added to 250 &mgr;l of a solution comprising 5 &mgr;mols of Tris-HCl buffer. (pH 6.8), 2.5 &mgr;mols of magnesium acetate, 100 &mgr;g of histone Type IIS (produced by Sigma Co.), 0.25 nmols of c-AMP and 200 &mgr;g of a crude enzyme (as partially purified from a calf heart according to the Kuo et al's method), and incubated therein at 30° C. for 3 minutes.
After the completion of the incubation, 1.25 nmols of [g-
32
P]ATP (from 5×10
3
to 10×10
3
cpm
mol) is added to the system, and phosphorylation reaction is carried out at 30° C. for 3 minutes, and thereafter the reaction is terminated by adding 25% trichloroacetic acid thereto.
The resulting reaction mixture is filtrated through a cellulose acetate membrane (pore size: 0.45 &mgr;m) (produced by Toyo Filter Co.), and the membrane is washed four times with 5% trichloroacetic acid, and thereafter the radioactivity of the residue remained on the membrane is measured to be a value of the sample.
On the other hand, the same process as described above is repeated without adding the sample to the system, and the radioactivity is obtained to be a control value.
The molar concentration of the sample having the value of radioactivity which is 50% of the control value is obtained to be the 50% PKA-inhibiting constant (PKA-IC
50
) of the sample.
Specific examples of the protein kinase C-specific inhibitor for use in the invention may include polymyxin B, calphostin C, palmitoyl-DL-carnitine and hexadecylphosphocholine (militefosine, produced by Sigma Co.), and also their pharmaceutically-acceptable salts.
The pharmaceutically-acceptable salts may include, for example, hydrochlorides, hydrobromides, sulfates, nitrates, formates, acetates, benzoates, maleates, fumarates, succinates, tartrates, citrates, oxalates, methanesulfonates, toluenesulfonates, aspartates, and glutamates.
The hair-growing agent of the present invention may be taken any form, provided that it properly contains the protein kinase C-specific inhibitor of the invention. For example, a liquid or solid hair-growing agent comprising the protein kinase C-specific inhibitor of the invention along with pharmaceutically acceptable vehicles is used.
The liquid or solid hair-growing agent may include liquid-type preparations such as hair liquids, hair tonics and hair lotions; and solid-type preparations such as ointments and hair creams.

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