WO1996027384A2

WO1996027384A2 - Composition for treating skin conditions comprising an inhibitor of skin iso-phosphodiesterase

Info

Publication number: WO1996027384A2
Application number: PCT/IB1996/000176
Authority: WO
Inventors: Georges Cehovic; Patrice Andre
Original assignee: Parfums Christian Dior
Priority date: 1995-03-08
Filing date: 1996-03-06
Publication date: 1996-09-12
Also published as: EP0813418A2; WO1996027384A3; JPH11501311A

Abstract

The present invention relates to a composition for improving skin conditions containing as active ingredient a specific inhibitor of human skin iso PDE and a dermatological acceptable carrier. It also relates to a method for improving skin conditions comprising the application of this composition.

Description

COMPOSITION FOR TREATING SKIN CONDITIONS COMPRISING AN INHIBITOR OF SKIN ISO- PHOSPHODIESTERASE

FIELD OF INVENTION

The present invention generally relates to a method for treating inflammation and irritation of the skin by topical application. This composition is also useful in cosmetics for improving skin conditions related to damages produced by weather, sun, chemicals or aging.

BACKGROUND OF THE PRIOR ART

The role of cyclic AMP or cyclic 3 '5' adenosine monophosphate (cAMP) as a second messenger and regulator of numerous cellular functions is now universally accepted. It has been established that the level of cAMP in the cell depends on the speed of its formation in the cell under the direction of adenyl cyclase and its hydrolysis by the enzyme phosphodiesterase (PDE) (R.W. Butcher and al. J. Biol. Chem. 241 , 1651 , 1966). Because all the earlier studies were conducted using saturating substrates, only later was it established that kinetically different forms of PDE activity were present in most tissues (VV.J. Thompson, Biochem. 10, 31 , 1 71 ).

In earlier studies caffeine, theophilline and some other xanthines were found to inhibit PDE at high concentrations. The discovery of multiple forms of PDE, or so called iso PDEs, lead to an attempt to obtain selective inhibitors of different iso PDE's with the idea of possible therapeutic applications (G. Cehovic and al. Adv. Cyclic. Nucleotides Res. vol. 1 , 521 , 1 72, Raven Press, N.Y. and Stefanovich in Cyclic Nucleotides and Therapeutic Applications, Ed. A.G. Robison and G. Cehovic Pergamon Press Oxford 197S).

It was also established that certain hormones and anti-inflammatory agents acts in vivo by regulation of the character and intensity of inflammation and immune responses. This regulation is mediated by a general inhibitory action of cyclic AMP. Also, it was reported that the intracellular level of cyclic AMP can be decreased during the inflammation process by a decrease of adenyl cyclase activity or increaed PDEs level ( M. Hitchcock, J. or Immunol. 188, 557, 1977).

It has previously been established that cyclic AMP with large amounts of non specific PDE inhibitors theophylline or caffeine were able to reduce some symptoms of inflammation. Also, by using topically some very potent cyclic AMP derivatives (namely dibutyryl or monobutyryl cyclic AMP) one obtained a prevention and/or improvement of skin inflammation (USA Patent 4 208 406).

The present invention relates to an improved composition based on the isolation of very active and specific inhibitors of iso PDEs from plants.

Several reports and experimental data showed that cyclic AMP decreases with age, whereas PDE increases with age (S.K. Puri and L. Volicer, Mechanisms of Aging and Dev. 15, 239, 1981. A. Birkenfeld and A. Ben-Zvi, Clinical Experimental Immunology, 55, 651 , 1984. . Tobise and al. Circ. Res. 74, 596, 1994). The addition of iso PDE inhibitors potentiates the action of cyclic AMP and also improves normal skin functions which deteriorate with ase.

SUMMARY OF THE INVENTION

The invention relates to compositions for the treatment of skin infllamation and prevention and improvement of skin conditions related to the damage produced by weather, chemicals or aging.

Extensive studies of PDEs in human skin, led as to the isolation and purification of a new iso PDE from human skin. This isolation was obtained with a new method of isoelectrofocussing (Rotofor Bio Rad) . We have found that iso PDE in human skin is different in men and in women. The iso PDEs in females is around pH 5.5 and in males pH 6.5. This difference has permitted the separation of these two PDEs and their use to select specific iso PDE inhibitors. By using these new iso PDEs isolated from human skin as a substrate we have tested several different plant extracts. The most active on human skin iso PDE were flavonoid extracts from Ginkgo Biloba namely Amentoflavone, Bilobetine and Ginkgo Biloba Biflavonoid, and also extracts from Green Tea and Orthosiphon or their active ingredient.

Some of these extracts, purified by HPLC, or pure products, were tested for their potency to inhibit human skin iso PDE.

The Bilobetine and Amentoflavone showed similar potency in inhibiting PDE as the crude Ginkgo Biloba extract. This Ginkgo Biloba extract was active in concentration of 1 - 10 μg/ml. This exceptionally high potency indicates the possibility of its use without further purification.

By using some specific PDE inhibitors, we have established that this new human skin iso PDE belongs to the group No 4 or cAMP specific iso enzyme family.

The present invention deals with composition for improving skin conditions containing as active ingredient a specific inhibitor of human skin iso PDE and a dermatological acceptable carrier and also with method for improving skin conditions comprising the appl ication of said composition.

The composition according to the invention can be under a form of an emulsion, a cream, a gel, a foam or a lotion.

Especially, the invention deals with composition comprising specific inhibitor of female human skin iso PDE or male human skin iso PDE

By "specific inhibitor of human skin iso PDE" we intend an inhibitor which gives a better inhibition on said iso PDE than iheophylline which is known to be a non specific PDE inhibitor.

Said composition may also contain cyclic AMP or one of its analogs or derivatives as described in U.S. Patent 3 978 213 and U.S. Patent 4 208 406. In vivo studies with Ginkgo Biloba extracts were performed on guinea pigs irradiated with UN. Ginkgo Biloba extract at the concentration of 0.01 % applied topically clearly decreased UN. produced erythema. The addition of 0.1 % of cyclic AMP potentiated the effect.

Extremely low concentrations of Ginkgo Biloba extract in vitro and m vivo indicates a relative inhibitory specificity on skin iso PDE.

Said compositions are especially useful for treatment of inflammation of skin but also for improving skin conditions caused by wind, cold, chemicals, sunshine, insect bites or other irritating substances as poison oak or poison , air conditioning, polution and others (croton oil, mustard oil, etc ) Said compositions are also useful to improve normal physiological function in

A topical application of a cream, gel, lotion, emulsion, foam or spray with Ginkgo Biloba extract or other specific inhibitors at the concentration of 0 01 % with O 1 % of cyclic AMP is recommended for mild skin inflammation or irritation produced b\ cold, wind, sunshine, chemicals, air conditioning, polution and other skin stressing conditions and aging

BRIEF DESCRIPTION OF THE DRAWINGS

Tigure 1 show s the results of phosphodiesterase assav on Rotofor for fractions of female human skjn using the Thompson PDE assav method The x-avis gives the fraction number

The gives the radioactivity of each fraction, expressed in 103 cpm, on the left side, and it gives the pH of each fraction on the right side

Figure 2 is analogous to figure 2 except that it corresponds to male human

Figure 3 show s the inhibitory action of some plant extracts on iso PDE from human skin The extracts are the following I - Ginkgo Biloba biflavonoϊds

I I - Licorice

I I I - Amentoflavone

IV - Chalcone The x-axis gives the concentration of the extract (μg/ml) . The y-axis gives the percentage of inhibition.

Figure 4 shows the results of the study of UV induced Erythema in Guinea Pigs following topical application of creams with : - 0.1 % cAMP, (cAMP),

0.01 % Ginkgo Biloba (GB),

0.01 % Ginkgo Biloba and 0.1 % cAMP (GB + cAMP), Placebo (PL), The x-axis represents the time after the application, expressed in hour. The y-axis represents the ratio of erythema to corresponding 0-hour measurement, expressed in %.

STUDIES OF ISOLATION AND PURIFICATION OF DIFFERENT ISO PHOSPHODIESTERASES (iso PDE) FROM HUMAN SKIN

Many important functions of the skin are under control of adenyl cyclic system. Functional activity of the cells depends on the level of cyclic AMP. Its level, i.e. cAMP, depends on the speed of synthesis of cyclic nucleotide versus its hydrolysis by phosphodiesierases ( PDE) . In recent years important progress was made in the identification of several types of iso PDEs. However, many problems remain unsolved, e.g. the iso PDEs are relatively large protein molecules ( MW around 50 to 1 10 kD), they are not stable and lose their biological activity very quickly even when frozen at -20°C, also, many laboratories are using different methods of separation and isolation and it is still very difficult to make a clear classification of different iso PDEs obtained from various laboratories.

No PDE isolations from human skin were previously reported.

For several years we have studied different methods of isolation and purification of iso PDE. We have found that the isoelectrofocussing method by the Rotofor apparatus (Bio Rad) presents a particular advantage. Separation of proteins by isoelectrofocussing based on the fact that all proteins have a pH dependent net charge. When a protein undergoes electrophoresis through an established pH gradient, it will migrate until it reaches the pH where the net charge of protein is zero. At that point, il will stop migrating and is said to be focused. The presence of ampholites maintain the gradient for longer periods allowing the slower migrating proteins to focus. Most iso PDE enzymes have a very close molecular weight (60-70 kD) and most methods of separation of PDE are based on differences of molecular weight which present difficulties in separation.

In a Rotofor apparatus there are twenty compartments separated by membranes. At the end of electrophoresis (2 - 3 hours) the solution in these 20 compartments are pumped (by vacuum) into 20 tubes. After measuring pH, obtained fractions are assayed for their hv drolysing activities on different substrates (cAMP, cGMP, in presence of CA+/Calmoduhn or cGMP) using the Thompson PDE assay method (W. J. Thompson, Biochem. 10, 3 1 , 1971 ) Usual.) 50 to 100 μl of obtained fractions are used for each assay. The total volume of each fraction varies from 1 9 to 3.0 ml The remaining solution is stored at -20'C or lyophilized in a l iophi zer freeze dryer 4.5 ( Labconco) .

After Rotofor purification and concentration, iso PDEs w ere used to study the action of different PDE inhibitors and particular} some plant extracts Studies of acti ity show the specific activ uv of new iso PDE

The most important and unexpected discov er} was that the major cAMP iso PDE from male skin was different from that from w omen's skin. A major peak of cAMP iso PDE in females is located in fractions 6 and 7 by Rotofor separation corresponding to pH 5.0 to 6.0, especially pH 5.2 - 5.8 (figure 1 ) In the extracts of male sk n the major cAMP iso PDE peaks are in fractions 9 and 10, corresponding to pl l 6.0 to 7 0, especiall} pH 6 0 - 6.5 (figure 2) The total spectrum of cAMP and iso PDE is clearl different in males and females

The purification and the separation of these different iso PDEs from human skin allow us to study the action of different PDC inhibitors from plant extracts or some potent xanthine derivatives for their potency as inhibitors on a new iso PDE from human skin STUDY OF DIFFERENT PDE INHIBITORS OF HUMAN SKIN ISO PDE

By using different PDE (Standard PDE from beef heart (Sigma)) or from rat or guinea pig brain PDE we observed variable potency depending on which PDE was used. We have also studied a large variety of plant extracts (mostly extracts from leaves or roots). We have also observed differences in response if the iso PDE used was from women's skin or from male. Table 1 shows the inhibition of various plant extracts on iso PDE from human female skin.

Among the most active inhibitors of female skin iso PDE were the flavonoids especially extracted from Ginkgo Biloba. Several fractions we purified by HPCL and studied those potency on human skin iso PDE. The most active were Ginkgo Biloba Biflavonoid and Amentoflavone (figure 3 ).

The Ginkgo Biloba Biflavonoid showed extremely high inhibitory activity on human skin iso PDE. It was active at the concentration of 0.001 mg/ml. Fifty percent (50 %) inhibition was obtained in vitro at the concentration of 10 μg/ml.

We have reported previously (and patented) the anti-inflammatory effect of cAMP in combination with some xanthine derivative ( theophylline, aminophylline). The anti-PDE potency of Ginkgo Biloba (over 40 times more potent as the xanthines) surpassed all other compounds. Ginkgo Biloba and its analogs appears to be specific inhibitors of the human skin iso PDE.

For this reason, additional studies were performed to test the anti- inflammatory action by topical application in the form of creams on guinea pigs.

The existence of multiple forms of PDEs has led to the classifications of five distinct families ( I - V). Based on kinetic properties and sensitivi ty to pharmacological and physiological mediators (G.A. Beavo and R. Reifsnyder, Trends Pharmacol. Sci. 1 1 , 150, 1990). Recently some isozyme selective inhibitors were developed (CD. Nicholson and M. Shahid, Pulmonary Pharmacol., 7, 1 , 1994). We have studied the effect of some of these selective inhibitors on our new iso PDE isolated from human skin and compared with their action on PDE from beef heart (Sigma). It was found that a very high inhibitory effect was produced by specific inhibitors for the N° IV family, namely Rolipram and RO-20-174, other inhibitors were less active. By using as a substrate beef heart PDE (Sigma) no substantial inhibition was observed with Rolipram and RO-20-174. The Bilobetin (Ginkgo Biloba fraction) produced a very high inhibitory effect on skin iso PDE. Our human skin iso form of PDE based on this result can be classified into PDE N" IV group with the sensitivity to the Rolipram and Ginkgo Biloba and Bilobetine.

ULTRAVIOLET ( U.V. ) INDUCED ERYTHEMA IN GUINEA PIGS FOLLOWING TOPICAL APPLICATION IN VIVO

The anti-inflammatory action of cyclic AMP alone and in combination with Ginkgo Biloba was studied on ultraviolet light (UN.) induced erythema.

Method :

In each experiment, ten female guinea pigs were used. Prior to testing, the dorsal hair was clipped and the remaining hair removed using a commercial depilatory. A minimum of two hours later the guinea pigs were exposed to UN. for 5 minutes from a distance of 1 3- 15 cm in a restraining holder.

The dorsal skin was covered with half cylindrical bakelite shield containing four holes ( 1.3 cm) in diameter symmetrically oriented. In order to minimize site to site viarations in skin reactivity, the administration of test articles was alternated on the test sites. The UN. lamp G. E. model RSK 6 was used immediately following the U.V. exposure, the creams were topically applied (0.1 ml/site) and gently dispersed by gloved fingers on the appropriate test sites. Sixty minutes following the application, the remaining cream was gently removed with a tissue. The cream application was repeated at 60, 120 and 180 minutes. The test sites were evaluated at 0, 60, 120, 180 and 240 minutes and 24 hours following topical application. A negative (placebo) control site was included for each animal.

All test sites were graded as described below : O = no erythema; 1 = skin appears pink (barely perceptible); 2 = red, well defined erythema; 3 = beet red, moderate to severe erythema; 4 = beet red, severe erythema to slight eschar formation. It was established in several experiments that cAMP alone reduced UN. erythema, Ginkgo Biloba had the same effect in the first 3 hours.

In this study, the placebo consisted of a cream formulation, (table 2 for composition) which was used as the base for all other test articles. The 0.1 % cAMP cream alone reduced erythema after 3 hours of application (table 3 ), whereas the Ginkgo Biloba cream alone significantly decreased the erythema after 2 hours of application with enhanced activity over a period of 4 hours. Enhanced activity was evident with the combination of 0. 1 % cAMP and 0.01 % of Ginkgo Biloba. More important is the fact that the above results are obtained after acute administration of each cream while expected use of a product would be chronic. Thereby the expectation would be potentiation effect with a combination of 0.1 % cAMP and 0.01 % Ginkgo Biloba, the combination of 0.1 % cAMP and 0.01 % Ginkgo Biloba completly reduced the erythema by 100 % after 3 hours. Figure 4 shows the decrease in erythema following topical application of creams with cAMP and Ginkgo Biloba.

Recently (G. Dent and al., Lung, 172; 129, 1994) reported "low Km AMP PDE activity of lung fibroblast was increased after exposure of intact cells to IBMX or other cyclic AMP elevating agents. This apparent feedback mechanism may be involved in maintaining cyclic AMP homeostasis in cells. " Our observation of a weaker effect in using PDE inhibitors alone (without cAMP) in chronical experiments is in agreement with this report.

TABLE 1

INHIBITION OF VARIOUS PLANT EXTRACTS ON iso PDE

FROM HUMAN FEMALE SKIN*

(expressed in percentage)

CONCENTRATIONS

Extract 0.25 mg/mi 0.50 mg/ml 0.75 mg/ml

Green tea 52.0 67.2=0.7 89.0=2.0

Orthosiphon 84.9=0.5 93.5=0.5 98.0±1.0

Theophylline 80.0=3.0 81.0=0.3 81.7=0.7

Female human skin iso PDE obtained by extraction and isoelectrofocusing separation by Rotofor method

TABLE 2

FORMULATION OF CREAM FOR 100 g

Stearic acid 5.0 g

PEG-6 stearate (and glyceryl stearate and ceteth-20) 10.0 g

Decyl oleate 2.0 g

Methylparaben 1.5 g

Propylparaben 1.5 g

Propylene glycol 10.0 g

Demineralized water 68.7 g TABLE 3

PERCENT REDUCTION IN ULTRAVIOLET INDUCED ERYTHEMA IN GUINEA PIGS

1 h 2 h 3 h 4 h

Placebo 21 39 55 68

0.1% cAMP 29 40 60 86

0.01% Ginkgo Biloba 47 72 88 94

0.1% cAMP + 0.01% Ginkgo Biloba 42 69 97 100

Claims

1) Composition for improving skin conditions containing as active ingredient a specific inhibitor of human skin iso PDE and a dermatological acceptable carrier.

2) Composition according to claim 1 wherein the specific inhibitor is a specific inhibitor of female human skin iso PDE.

3) Composition according to claim 1 wherein the specific inhibitor is a specific inhibitor of male human skin iso PDE.

4) Composition according to claim 1 wherein the composition also contains cyclic AMP or its derivatives

5) Composition according to claim 1 which contains as specific inhibitor a compound or extract selected among an effective Ginkgo Biloba extract or its active ingredient, - an effective Green Tea extract or its active ingredient, and an effective Orthosiphon extract or its active ingredient

6) Composition according to claim 1 which contains as specific inhibitor Amentoflavone or Bilobetine.

7) Composition according to claim 1 under a form of an emulsion, a cream, a gel, a foam or a lotion.

8) Method for improving skin conditions, among which improving skin conditions caused by wind, cold, chemicals, sunshine, insect bites or other irritating substances as poison oak or poison iv y, air conditioning, polution or others (croton oil, mustard oil, etc ), improving physiological function m aged skin and treating inflammation of skin, comprising the application of a composition according to claim 1

9) Method according to claim 8, comprising the application of a composition according to claim 2 10) Method according to claim 8, comprising the application of a composition according to claim 3.

11) Method according to claim 8, comprising the application of a composition according to claim 4.

12) Method according to claim 8, comprising the application of a composition according to claim 5.

13) Method according to claim 8, comprising the application of a composition according to claim 6.

14) Method according to claim 8, comprising the application of a composition according to claim 7.

15) Process for selection of specific inhibitor of human skin iso PDE comprising the use of said iso PDE.

16) Female human skin iso PDE which can be extracted from female human skin, which has a isoelectric point between 5.0 and 6.0, and which may be classified among the cAMP specific iso enzyme.

17) Male human skin iso PDE which can be extracted from male human skin, which has a isoelectric point between 6.0 and 7.0, and which may be classified among the cAMP specific iso enzyme.