WO1985003637A1

WO1985003637A1 - Methods and pharmaceutical compositions for the treatment of hyperplastic diseases of the skin

Info

Publication number: WO1985003637A1
Application number: PCT/US1984/000291
Authority: WO
Inventors: Kenneth M. Halprin; Kenji Adachi
Original assignee: Halprin Kenneth M; Kenji Adachi
Priority date: 1984-02-24
Filing date: 1984-02-24
Publication date: 1985-08-29
Also published as: AU2690684A

Abstract

Method of using and pharmaceutical compositions containing polyoxygenated labdane derivatives, such as Forskolin, which are utilized in the treatment of hyperplastic diseases of the skin, such as psoriasis.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF HYPERPLASTIC DISEASES OF THE SKIN

This invention relates to novel methods of use for and pharmaceutical compositions containing polyoxygenated labdane derivatives. The methods of use and pharmaceutical compositions are useful in the treatment of skin diseases.

More particularly, the present invention provides methods of use for and pharmaceutical compositions containing polyoxygenated labdane derivatives of the formula I

in which

R₁, R₂, R₃ and R₄, are ide and each represents oxygen or the group

in which R₆ is hydrogen, alkyl, alkenyl or alkynyl of 1 to 6 carbon atoms, cycloalkyl of 5 to 8 carbon atoms, dialkylaminoalkyl or aralkyl in which the alkyl are at most 4 carbon atoms, acyl of 1 to 20 carbon atoms, alkoxycarbonyl, or arylaminocarbonyl of 2 to 10 carbon atoms, and R₅ is ethyl or vinyl.

R₇ is hydroxy, hydrogen, or together with R₁ forms the cyclic sulfide ester It is preferred that R₇ is hydroxy.

The acid addition salts are also included for derivatives having basic groups. Also included are stereoisomers and mixtures thereof. The polyoxygenated labdane derivatives may be utilized alone or in combination with one or more receptor stimulators, such as epinephrine and histamine.

Preferable alkyl, alkenyl or alkynyl for R₆ are methyl, allyl and propynyl, while a more preferred cycloalkyl for R₆ is cyclohexyl.

When R₆ is dialkylaminoalkyl, the diethylaminoethyl group is preferred and a suitable acid addition salt is, for example, one derived from an inorganic or organic acid, for example, the hydrochloride, hydrobromide, sulfate, phosphate, acetate, oxalate, tartrate, citrate, maleate or fumarate.

A suitable aralkyl for R₆ is phenylalkyl, for example, the benzyl group which is unsubstituted or substituted by one or more substituents in the phenyl radical. The substituents may be a halogen such as fluorine, chlorine or bromine, an alkyl or alkoxy of 1 to 3 carbon atoms, such as methyl, ethyl, methoxy and ethoxy, a haloalkyl, such as trifluoromethyl, a nitro, an amino or a hydroxy.

A suitable acyl group for R₆ is alkanoyl, alkenoyl, alkynoyl, aroyl, aralkanoyl or heteroaroyl of up to 10 carbon atoms and at most 3 heteroatoms, such as oxygen, nitrogen or sulfur. Examples of alkanoyl groups are formyl, acetyl, trifluoroacetyl, propionyl, butyryl, isobutyryl, valeryl, palmitoyl, stearoyl and bromoisobutyryl. The alkanoyl group may also be derived from a dicarboxylic acid, for example, oxalic acid or succinic acid. The alkenoyl groups may contain one or more double bonds, such as in acryloyl and oleoyl. The alkynoyl groups may contain one or more triple bonds, for example propiolyl, and additionally one or more double bonds. Suitable aroyl groups are especially the benzoyl group, the phenyl radical being unsubstituted or substituted by one or more substituents, for example, halogen, alkyl, alkoxy, trifluoromethyl, nitro, amino or hydroxy. Examples of aralkanoyl or heteroaroyl are the phenylacetyl and pyridine-3-carbonyl groups.

When R₆ is an alkoxycarbonyl or arylaminocarbonyl, examples are ethoxycarbonyl and anilinocarbonyl.

Preferred representatives of compounds of formula I are those in which R₇ is hydroxy, R₅ stands for -CH = CH₂ or -CH₂CH₃, R₁, R₂ and R₃ are

and R₄ represents oxygen, or R₁ and R₄ represent oxygen and R₂ and R₃ are

or R₁, R₂ and R₄ represent oxygen and R₃ is

or R₁, R₂, R₃ and R₄ represent

wherein the radicals

have the meanings specified above.

Especially preferred are compounds of formula I in which R₇ is hydroxy, R₅ is -CH = CH₂ or -CH₂CH₃, R₁ is

R₄ represents oxygen or

, and R₂ and R₃ both are

R₂ is and R₃ is or

R₂ is and R₃ is

R₆ being an acyl radical as defined above. Even more preferred are compounds in which R₁ and R₂ are

R₃ is R₄ is 0, and R₅ is CH=CH₂.

When R₇ is hydrogen, a preferred compound is one in which R₁ and R₂ are R₃ is is 0

and R₅ is CH=CH₂.

Another preferred compound is one in which R₁ together with R₄ form a cyclic sulfide ester

R₂ is R₃ is and R₅ is CH=CH₂.

In the following Table polyoxygenated labdane derivatives of formula I are listed together with their melting points.

Ta

The compounds utilized in the invention are prepared according to the methods described in U. S. Patent Nos. 4,088,659, 4,118,508 and 4,134,986. These methods are summarized below. Starting compounds are compounds of the following formula II

(II)

(cf. compounds 30 and 31 of Table I) in which in the case of compound 30, R' is hydroxy and R'₃ is O-acyl and in the case of compound 31 R'₂ is O-acyl and R'₃ is hydroxy. A. Compound 30 of Table I, which is disclosed in U.S. Patent No. 4,088,659, has a melting point of 228°-230°C. and is generically referred to as Forskolin. It is obtained by extracting Coleus forskohlii, a plant of the Labiatae family, with a halogenated hydrocarbon solvent, for example, one having 1 to 3 carbon atoms and up to 6 halogen atoms, removing the solvent from the extract to leave a residue, dissolving the residue in an alkanol, for example, one having 1 to 6 carbon atoms, removing undissolved material from the alkanolic solution, and isolating the compound from the alkanolic solution, for example, by evaporation of the alkanol. If desired, the plant may be extracted with a hydrocarbon solvent and the isolated compound may be purified further by column chromatography and crystallization.

B. Compound 31 of Table I has a melting point of 208°-211°C. and is obtained by extracting Coleus forskohlii with a solvent such as an aromatic hydrocarbon, aromatic and aliphatic halogenated hydrocarbon, dialkyl ether, dialkyl ketone, alkanol, carboxylic acid or ester, dimethylformamide, dioxane, tetrahydrofurane, or dimethylsulfoxide. The extract is then concentrated to a residue. The residue is treated in one of a number of ways, described below in more detail, with a solvent or solvent mixture to obtain a crude product which is treated with a base, for example, an alkali alkoxide in a solvent such as an ether. On acidification to a pH of 5-7, concentration, and dilution with water, a mixture of crude terpenoids is filtered off. Other basic treatments involve an alkali carbonate or bicarbonate in alcohol or aqueous alcohol, or a basic metal oxide such as basic aluminum oxide in a solvent such as an aromatic hydrocarbon or an ether, in each case with formation of a crude terpenoid product. This crude product may be chromatographed to obtain a semi-pure terpenoid. The crude terpenoid product may be recrystallized prior to chromatography, or the semi-pure terpenoid may be recrystallized after chromatography, from solvents such as ethyl acetate, chloroform or benzene, optionally in admixture with an aliphatic hydrocarbon having 5-7 carbon atoms, or preferably from petroleum ether, hexane, or pentane, to obtain the product which is generically referred to as Coleforsin.

If an aromatic hydrocarbon such as benzene or toluene, or an aliphatic halohydrocarbon such as chloroform, is used as an extractant for the plant material, the crude product is obtained from the resulting residue by repeated solution and precipitation by addition of a material in which the residue is partly or entirely insoluble.

In an alternative process, the residue obtained by extraction of the plant with an aromatic hydrocarbon or a halohydrocarbon is first extracted with several portions of a lower alkanol having 1 to 6 carbon atoms. The alkanol extracts are combined, filtered and evaporated to dryness. This residue is then recrystallized to form the crude product.

In a third embodiment, the residue obtained from the first extraction is extracted with an alkanol and this extract is distributed between the immiscible solvents, for example, benzene and water, in only one of which the desired material is soluble. The organic layer is separated, dried, and evaporated to form a residue which is then recrystallized.

C. Compounds of the formula III

(III)

in which R₁, R₂ and R₄ are oxygen or

R₆ being hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, dialkylaminoalkyl, or aralkyl, and R₃ represents

are prepared by subjecting to an acid or basic hydrolysis the compound 30 in Table I or from other compounds of formula III in which R₁, R₂ and R₄ have the above meaning and R₃ stands for

wherein R₆ is acyl, alkoxycarbonyl or arylaminocarbonyl. For hydrolysis an inorganic acid, for example, hydrochloric acid, or organic acid, for example, trifluoroacetic acid, an alkali metal hydroxide, for example, sodium hydroxide, or an alkali metal carbonate, for example, potassium carbonate, may be used.

The reaction may be carried out in the presence of a solvent, for example, an aqueous alkanol having at most 6 carbon atoms, for example, methanol or ethanol, or a mixture of water with an ether miscible therewith, for example, dioxane or tetrahydrofuran. The reaction may be accelerated or completed by heating up to the boiling temperature of the solvent used.

D. Compounds of formula III in which R₁, R₃ and R₄ are oxygen or

R₆ being hydrogen or alkyl, alkenyl, alkynyl, cycloalkyl, dialkylaminoalkyl, or aralkyl, are prepared in analogous manner from the compound 31 of Table I or from corresponding compounds of formula III in which R₁, R₃, and R₄ have the above meaning and R₂ stands for

wherein R₆ represents acyl, alkoxycarbonyl or arylaminocarbonyl.

E. Compounds of formula III in which R₁, R₂ and R₄ have the above meaning and R₃ stands for

wherein R₇ has the meaning defined below, are prepared by reacting the compound 31 or a compound of the formula III in which R₁, R₂, and R₄ are oxygen or

R₆ has the above meaning, and R₃ represents

with a compound of the formula R₇X, R₇ being alkyl, alkenyl, alkynyl, cycloalkyl, dialkylaminoalkyl or aralkyl as defined above and X is a halogen atom such as chlorine, bromine or iodine, for example, methyl iodide, allyl bromide, benzyl bromide and diethylaminoethyl chloride, or with a compound of the formula (R₇)₂SO₄ in which R₇ stands for an alkyl radical as defined above, for example, (CH₃)₂SO₄. The reaction is carried out according to known methods in the presence of a base, for example, an anhydrous alkali metal carbonate, such as potassium carbonate. The reaction is suitably carried out in the presence of a dry solvent, for example, a ketone, such as acetone, or an aromatic hydrocarbon such as benzene. To accelerate or complete the reaction, the reaction mixture may be heated to the boiling temperature of the solvent used.

F. Compounds of formula III in which R₁, R₂ and R₄ have the above meaning and R₃ stands for

wherein R₇ is a dialkylaminoalkyl group may be transformed into their acid addition salts by known methods, for example, simply by adjusting the pH of a solution of the compound with an acid to approximately neutral. Suitable acids are, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, citric acid, maleic acid and fumaric acid.

When R₇ is an acyl group as defined above, the compound of formula III may be treated with an acid anhydride, for example formic-acetic anhydride, acetic anhydride, trifluoroacetic anhydride, propionic anhydride, or butyric anhydride, or with an acyl halide wherein acyl has the above meaning and the halogen is chlorine or bromine, for example, α -bromoisobutyryl bromide, in the presence of a base such as a nitrogen-containing compound, for example, pyridine, by a known method at a temperature in the range of from room temperature to 0°C.

When R₇ is an alkoxycarbonyl group, the compound of formula III may be treated with a halocarbonic acid ester, preferably a chlorocarbonic acid ethyl ester, or a dialkylcarbonic anhydride, in the presence of a base and in known manner.

When R₇ is an arylaminocarbonyl group, the compound of formula III may be treated with an organic isocyanate, for example phenyl isocyanate, according to a known method. The reaction may be carried out in the presence of a dry solvent, for example, benzene or toluene. It can be accelerated or completed by heating, for example, up to the boiling temperature of the solvent used.

G. Compounds of formula III in which R₂ and R₄ are oxygen or

R₆ having the above meaning, with the exception that it does not stand for hydrogen, and R₁ and R₃ are

R₇ having the above meaning, are prepared by treating compounds 30 or 31 or other compounds of formula III in which R₂ and R₄ have the same meaning as defined above and

R₁ and R₃ are

with a reagent as defined above depending on the nature of R₇. Some of the reactions may be facilitated by adding one of the described bases. By heating, for example, up to the boiling temperature of the solvent used, the reactions may be accelerated or completed.

H. Compounds of formula III in which R₁ and R₄ have the above meaning, R₂ is

with R₆ being an acyl group and R₃ is

are prepared by treating with a base the compound 30 or another compound of formula III in which R₁ and R₄ have the above meaning, R₂ is

and R₃ is

wherein R₆ is an acyl group. As base there can be used an alkali metal alkoxide, for example, sodium methoxide or ethoxide. In this case, the treatment may be carried out in the presence of a solvent, for example, an ether, preferably dioxane, tetrahydrofurane or diethyl ether. The base may also be an alkali metal carbonate, for example, sodium or potassium carbonate, or an alkali metal bicarbonate, for example, potassium bicarbonate. In this case, an alkanol having from 1 to 6 carbon atoms, for example, methanol or ethanol, either with or without the addition of water, may be used as solvent. As base a basic metal oxide is suitable, for example, basic alumina, which may be used in the presence of a solvent, for example, an aromatic hydrocarbon such as benzene or toluene, an ether such as diethyl ether, dioxane or tetrahydrofurane. In all cases of a treatment with a base the reaction may be accelerated or completed by heating, for example, up to the boiling point of the solvent used. I. Compounds of formula III in which R₁ is oxygen and R₂, R₃ and R₄ have the meaning defined above, with the exception that R₃ and R₄ cannot stand for

are prepared by oxidizing by a known method the compound 30 or another compound of formula III wherein R₁ is

and R₂, R₃ and R₄ have the above meanings, for example, with Jones reagent. The oxidation may be carried out in a solvent, for example, a ketone such as acetone, at a temperature in the range of from room temperature to 0°C. J. Compounds of formula III in which R₁ and R₂ are oxygen and R₃ and R₄ have the meaning defined above, with the exception that they cannot stand for

are prepared by oxidizing the compound 30 or another compound of formula III in which R₁ and R₂ are

and R₃ and R₄ have the above meaning by a known method using as oxidant, for example, Jones reagent, Collins reagent or Sarrett reagent.

K. Compounds of formula III in which R₁, R₂ and R₃ have the same meaning as defined above, with the exception that they cannot stand for oxygen or

with R₆ being an acyl group, and R₄ is

are prepared by reducing the compound 30 or 31 or another compound of formula III in which R₁, R₂ and R₃ are as defined above and R₄ is oxygen by a known method using a complex metal hydride, for example, lithium-aluminum hydride. The reaction may be carried out in the presence of a solvent, for example, an ether such as dialkyl ether, preferably diethyl ether, or a cyclic ether such as dioxane or tetrahydrofurane. The reaction may be carried out at a temperature below room temperature and down to 0°C.

L. Compounds of formula I in which R₁ , R₂, R₃ and R₄ have the above meaning and R₅ is -CH₂CH₃ are prepared by catalytically hydrogenating a compound 30 or 31 or another compound of formula I in which R₁, R₂, and R₃ and R₄ have the indicated meaning and R₅ is -CH=CH₂, using as a catalyst metal, for example, Raney nickel or a platinum metal, such as platinum, palladium, rhodium, and ruthenium, or the oxides and sulfides thereof. The platinum metal catalysts may be used in the form of the finely divided metals or supported on a carrier, such as asbestos, activated carbon, alumina, barium sulfate, or barium carbonate. The hydrogenation may be carried out in the presence of a solvent, for example, an alkanol such as ethanol, an ether such as diethyl ether, a carboxylic acid, such as acetic acid, or an ester, such as ethyl acetate. The hydrogenation may be accelerated or completed by the application of pressure, for example, up to 10 atmospheres, or heat, for example, by heating to the boiling point of the solvent used.

The polyoxygenated labdane derivatives of the invention are particularly useful in the treatment of hyperplastic skin diseases such as psoriasis. The compounds may also be used for therapy of dysplastic skin diseases such as lichen planus, pityriasis rubra pilaris, ichthyosis, seborrheic dermatitis, actinic keratoses, mycosis fungoides, and skin cancers such as basal cell and squamous cell carcinomas. In addition, these compounds may be utilized to stimulate hormone and pigment production in the treatment of pigmentary diseases such as vitiligo.

The most preferred compound of these labdane derivatives is compound 30, Forskolin, which has the formula

This compound is the subject of U. S. Patent No. 4,088,659. Forskolin has been found to activate epidermal adenylate cyclase and inhibit epidermal cell mitosis and is especially useful in the treatment of psoriasis vulgaris. The action of Forskolin on the adenylate cyclase system of certain cells and tissues was previously known but its action on the epidermal adenylate cyclase system was not known until now.

Since the β-adrenergic-cyclic adenosine monophosphate (cyclic AMP or cAMP) system is defective in psoriasis lesions, the increase of the cyclic AMP level in skin using polyoxygenated labdane derivatives has been found to have therapeutic value in the treatment of psoriasis vulgaris.

The polyoxygenated labdane derivatives may be administered in a number of ways including topically as, for example, an aerosol, cream, paste, jelly, ointment, oil, powder or solution.

As an aerosol, the formulation may contain the product concentrate and a propellant. The product concentrate may include the active ingredient along with other agents such as a solvent like alcohol, ethanol or water, an antioxidant like ascorbic acid, a surfactant like polyglycerol oleate or glycerol monostearate, a preservative like methyl paraben or propyl paraben and an absorption enhancer like lanolin or petrolatum. The propellant may be a fluorinated hydrocarbon like propellant 11, 12 or 114, a compressed gas such as nitrogen, carbon dioxide or nitrous oxide or a hydrocarbon such as butane, propane or isobutane.

As a cream, the formulation may be an oil-in-water or a water-in-oil emulsion which contains the active ingredient together with an emulsifier such as polyethylene glycol 400 monolaurate or sorbitan sesquioleate, a preservative, an antioxidant, an absorption enhancer and a vehicle such as water. The cream may also include a surfactant to reduce the water tension and a buffer agent such as citric acid. An example of an emollient cream, that is, a water-in-oil emulsion, is one containing 1 percent of the active ingredient such as forskolin, 3 percent woolwax alcohol, .25 percent cetostearylic alcohol, 47 percent white petrolatum and 48.5 percent water. An aqueous cream, that is, an oil-in-water emulsion may, for example, include 1 percent of the active ingredient such as forskolin, 10 percent of cetostearylic alcohol, 4 percent mineral oil, 4 percent isopropyl myristate, 4 percent polysorbate 60, 1 percent sorbitan monostearate 60, 1 percent benzyl alcohol, and 75 percent water.

As a paste, the formulation may include 20 to 50 percent of the active ingredient in an aqueous base and as a jelly the active ingredient may be dispersed in a natural gum or a cellulose containing compound such as methyl cellulose.

As an ointment, the active ingredient may be dispersed in a water insoluble base such as petrolatum or mineral oil, a preservative, a surfactant such as sodium lauryl sulfate and a antioxidant. An ointment may contain, for example, 1 percent of an active ingredient such as forskolin, together with 20 percent mineral oil and 79 percent petrolatum. An oil, rather than an ointment, may be formed by using liquid petrolatum or mineral oil.

The active ingredient may also be formulated in a solution, for example, containing 1 percent of an active ingredient like forskolin, 10 percent 1,2-propylene glycol, 10 percent polyethylene glycol 400, 50 percent ethanol (94%) and 29% water.

The active ingredient may also be impregnated in a sponge or bandage and applied to the affected area. In such a case, the active ingredient may be impregnated in the sponge or bandage with a base such as silicon.

The active ingredient may also be incorporated in a talc like base and used as a powder. The formulation is applied directly to the affected area as needed, for example, once a day for a five-day period. However, the amount of the formulation, the concentration of the formulation, the number of applications per day and the length of treatment will depend upon the degree of the affliction.

The following examples illustrate the stimulation of epidermal adenylate cyclase in the incubation of pig skin squares. In the examples below, skin slices of approximately 80% epidermis were taken from the backs of domestics pigs using a keratome with the cutting blade adjusted to 0.2mm to 0.3mm. The skin slices were cut into 5 x 5mm squares at 4°C. and floated keratin layer up in Hank's balanced salt solution. After a preincubation of 30 minutes at 37°C., the squares were incubated in Forskolin and, as indicated below, in certain other chemicals. After the incubation period, the squares were frozen between pieces of dry ice to stop the reaction and the cAMP contents were measured by a radioimmunoassay in accordance with the methods described by Steiner et al., J. Biol. Chem. 247, 1106-1113 (1972) with micromodification according to the methods described in Adachi et al., J. Invest. Dermatol. 74, 74-76 (1980). When the cAMP levels in the epidermis were high, the acetylation step of the radioimmunoassay was omitted.

Crude membrane preparations were obtained by homogenization with an all glass homogenizer for 3 minutes in 50 mM of a HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer having a pH of 8.0 with 0.1M of EDTA and ImM of MgCl₂. The homogenate was centrifuged at 20,000 g for 20 minutes and the membrane preparation was washed three times by recentrifugation. The adenylate cyclase activity was assayed by Salomon's method according to the procedure set forth in Salomon, Adv. Cyclic Nucleotide Res. 10 35-55 (1979) with the slight modification described in Takeda et al., J. Invest. Dermatol. 81, 131-136 (1983). This assay is based on the 2-step chromatographic separation of [³²P]cAMP. The protein was measured by the method described in Lowry et al., J. Biol. Chem. 193, 265-275 (1951) with human serum albumin as a standard.

The basic procedures for the explant culture of pig skin were those described for mouse skin in Halprin et al., J. Invest. Dermatol. 72, 88-98 (1979) with an adaptation to pig skin. The procedure involved cutting the keratome-slice epidermis into 2mm squares which were attached to glass coverslips in air for 15 minutes. An

RPMI medium containing antibiotics and a 10% fetal calf serum was then added to each culture and the cultures were allowed to grow for four days. Cultures having a good growth, that is greater than 300 μm from the explant on at least three sides were then transferred to the RPMI medium containing 5% dialyzed fetal calf serum, antibiotics, and nucleotides for testing the effects of the Forskolin concentrations. The cultures were grown for 4 hours in the presence of 0.1 μg/ml of colchicine, fixed in alcohol and then stained with hematoxylin. The number of mitoses were then counted.

Example 1

Using the procedure described above, the effect of

150 μM of Forskolin on the cAMP accumulation in both the epidermal tissue and the medium was tested over a period of 60 minutes. The results are described below in Table

II and are plotted in Figure 1, incubation versus cAMP level. The intracellular results are represented by the circles (o) in Figure 1 and the medium results are represented by the squares (□) . The intracellular cAMP levels represent an average of four determinations ± the standard error (SE) . The medium cAMP levels are the averages of two determinations.

Example 2

The effect of different concentrations of Forskolin on the cAMP accumulation in both the epidermal tissue and the medium was tested according to the method described above. The incubation time for Forskolin activation was 10 minutes and the results are described below in Table III. The data is an average of two different skin slices in duplicate for each assay. Figure 2 plots the cAMP level in the tissue versus the concentration of Forskolin utilized.

Example 3 In order to determine if the activation by Forskolin was reversible, epidermal slices were incubated for 45 minutes with 150 μM of a Forskolin and then transferred to plain Hanks' balanced salt solution medium at 37°C. and the cAMP level in the epidermis decreased very quickly. However, even at two hours after the transfer, the cAMP level of 6 Pmol/mg protein was still higher than the basal level of 1 Pmol/mg. The results of the test are reported below in Table IV. The data is an average of two different skin slices in duplicate for each assay. Figure 3 plots the cAMP level versus the time. The first 45 minutes represent the Forskolin stimulation. After 45 minutes, the time represents the period after washing in the Hanks' balanced salt solution.

Example 4

The effects of Forskolin were also tested in combination with H₂ and/or β₂-receptor aganonists using the procedure described above. In the test, skin slices were incubated for three minutes and the concentrations of the stimulators were at maximum stimulatory doses to examine additive effects. The stimulators utilized were a 50 μM concentration of epinephrine (a β₂-receptor aganonist) and a 550 μM concentration of histamine (a H₂- receptor aganonist). The Forskolin utilized had a concentration of 150 μM. In the test, Forskolin and each of the stimulators were tested separately and in combination. Combinations of Forskolin with epinephrine, with histamine, and with epinephrine and histamine had multiplicative, and not merely additive, effects in stimulating the adrenylate cyclase. The results are reported below in Table V. The values shown represent averages ± the standard error (SE) of four determinations.

Example 5

The effects of a four-hour exposure to Forskolin on epidermal cell mitoses was also determined. In the test, different concentrations of Forskolin were utilized and the number of colchicine-arrested mitoses seen per thousand cells in the culture was determined to give the mitotic index. The results are reported below in Table VI and each result represents an average from ten outgrowth cultures ± the standard error (SE). The results of the test are plotted in Figure 4 which shows the mitotic index versus the concentration of Forskolin.

The use of a polyoxygenated labdane derivative, Forskolin was also used to treat psoriasis vulgaris in human patients. The patients received no therapy for eight days and then a 1 percent Forskolin cream with an occlusive bandage was applied once a day for five days. The cream contained 1 percent Forskolin, 3 percent woolwax alcohol, .25 percent cetostearylic alcohol, 47 percent white petrolatum and 48.75 percent water. Example 6

The right knee of a patient, a 25 year old female, who had psoriasis for five years was treated for five days with the 1 percent emollient cream containing Forskolin. The high efflorescence of the right knee receded to a residual erythema after five days. When therapy was discontinued the knee returned to the original condition.

The same patient was treated without the occlusive bandage for two weeks on her right elbow and the psoriasis which had exhibited a high efflorescence receded to a residual erythema. Example 7

The sole of the right foot of a 38 year old female was treated with the Forskolin cream and after five days the condition healed to a slight erythema. There was a dramatic cessation of itching which had not previously been achieved through the use of any topical corticoids for this psoriatic condition which she had for the past seven years. Example 8

A 32 year old female having psoriasis for 18 years was treated on the left thigh with the Forskolin cream. After five days of treating the condition, which was very severe, there was only some minor erythema visible. Example 9

A 41 year old male having a mild psoriasis on his right elbow was treated with the Forskolin cream for five days and achieved a full recession which lasted for at least ten days after the therapy was discontinued.

Claims

The Claims 1. A method for treating a hyperplastic disease of the skin in a patient suffering therefrom, which comprises topically administering an effective amount therefor of a compound of the formula

in which

R₁ , R₂, R₃ and R₄ , are identical or different and each represents oxygen or the group

in which R₆ is hydrogen, alkyl, alkenyl or alkynyl of 1 to 6 carbon atoms, cycloalkyl of 5 to 8 carbon atoms, dialkylaminoalkyl or aralkyl in which the alkyl are at most 4 carbon atoms, acyl of 1 to 20 carbon atoms, alkoxycarbonyl, or arylaminocarbonyl of 2 to 10 carbon atoms,

R₅ is ethyl or vinyl, and

R₇ is hydroxy, hydrogen, or together with R₁ forms the cyclic sulfide ester

2. The method, as claimed in claim 1, wherein the skin disease is a dysplastic skin disease.

3. The method, as claimed in claim 1, wherein the skin disease is a pigmentary skin disease.

4. The method, as claimed in claim 1, wherein the skin disease is psoriasis.

5. The method, as claimed in any one of claims 1 to 4, wherein in the compound R₁ , R₂ , and R₃ are

R₄ is oxygen, R₅ is -CHCH₂ or -CH₂CH₃ , and R₇ is hydroxy.

6. The method, as claimed in any one of claims 1 to 4 , wherein in the compound R₁ and R₄ are oxygen and R₂ and R₃ are R₅ is -CHCH₂ or -CH₂CH₃, and R₇ is hydroxy.

7. The method, as claimed in any one of claims 1 to

4 , wherein in the compound R₁ , R₂ and R₃ are oxygen and R₃ is R₄ is oxygen, R₅ is -CHCH₂ or -CH₂CH₃ , and R₇

is hydroxy.

8. The method, as claimed in any one of claims 1 to 4 , wherein in the compound R₁ , R₂, R₃ and R₄ are R₅

is -CHCH₂ or -CH₂CH₃, and R₇ is hydroxy.

9. The method, as claimed in any one of claims 1 to 4, wherein in the compound R₁ and R₂ are ,

R₃ is R₄ is oxygen, R₅ is -CHCH₂ and R₇ is

hydroxy.

10. The method, as claimed in any one of claims 1 to 4, wherein in the compound R₁ and R₃ are

R₂ is R₄ is oxygen, R₅ is -CHCH₂ and R₇ is

hydroxy.

11. The method, as claimed in any one of claims 1 to 4, wherein in the compound R₁ and R₂ are ,

R₃ is , R₄ is oxygen , R₅ is -CHCH₂ and R₇ is

hydroxy.

12. The method, as claimed in any one of claims 1 to 4 , wherein in the compound R₁ and R₂ are ,

R₃ is , R₄ is oxygen, R₅ is -CHCH₂ and R₇ is

hydroxy

.

13. The method, as claimed in any one of claims 1 to 4, wherein in the compound R₁ and R₂ are

R₃ is , R₄ is oxygen, R₅ is -CHCH₂ and R₇ is

hydroxy.

14. The method, as claimed in any one of claims 1 to 4, wherein in the compound R₁ and R₂ are ,

, R₄ is oxygen, R₅ is -CHCH₂ and R₇ is

hydroxy.

15. The method, as claimed in any one of claims 1 to

4, wherein in the compound R₁, R₂ and R₃ are

R₄ is oxygen, R₅ is -CHCH₂ and R₇ is hydroxy.

16. The method, as claimed in any one of claims 1 to 4, wherein in the compound R₁ and R₂ are ,

R₃ is , R₄ is oxygen, R₅ is -CHCH₂ and R₇ is

hydrogen.

17. The method, as claimed in any one of claims 1 to 4 , wherein in the compound R₁ and R₇ taken together are R₂ is , R₃ is , R₄ is oxygen, R₅

is -CHCH₂ and R₇ is oxygen.

18. The method, as claimed in any one of claims 1 to 4, wherein in the compound R₁ oxygen, R₂ is

R₃ is , R₄ is oxygen, R₅ is -CHCH₂ and R₇ is

hydroxy.

19. The method, as claimed in any one of claims 1 to 4, wherein in the compound R₁ and R₂ are ,

R₃ is , R₄ is , R₅ is -CHCH₂ and R₇ is

hydroxy.

20. The method, as claimed in any one of claims 1 to 3, wherein in the compound R₁ and R₂ are ,

R₃ is , R₄ is oxygen, R₅ is -CHCH₂ and R₇ is

hydroxy.

21. A method for treating psoriasis vulgaris in a patient suffering therefrom, which comprises topically administering an effective amount therefor of Forskolin.

22. A pharmaceutical composition for the treatment of a hyperplastic disease of the skin comprising, in combination with a pharmaceutical carrier, an effective amount therefor of a compound of the formula

in which

R₁ , R ₂ , R₃ and R₄ , are identical or different and each represents oxygen or the group

in which R₆ is hydrogen, alkyl, alkenyl or alkynyl of 1 to 6 carbon atoms, cycloalkyl of 5 to 8 carbon atoms, dialkylaminoalkyl or aralkyl in which the alkyl are at most 4 carbon atoms, acyl of 1 to 20 carbon atoms, alkoxycarbonyl, or arylaminocarbonyl of 2 to 10 carbon atoms, R₅ is ethyl or vinyl, and R₇ is hydroxy, hydrogen, or together with R₁ forms the cyclic sulfide ester P

23. The pharmaceutical composition, as claimed in claim 22, wherein the skin disease is a dysplastic skin disease.

24. The pharmaceutical composition, as claimed in claim 22, wherein the skin disease is a pigmentary skin disease.

25. The pharmaceutical composition, as claimed in claim 22, wherein the skin disease is psoriasis.

26. The pharmaceutical composition, as claimed in any one of claims 22 to 25, wherein in the compound R₁, R₂, and R₃ are R₄ is oxygen, R₅ is -CHCH₂ or -CH₂CH₃ ,

and R₇ is hydroxy.

27. The pharmaceutical composition, as claimed in any one of claims 22 to 25 , wherein in the compound R₁ and R₄ are oxygen and R₂ and R₃ are R₅ is -CHCH₂ or

-CH₂CH₃, and R₇ is hydroxy.

28. The pharmaceutical composition, as claimed in any one of claims 22 to 25, wherein in the compound R₁, R₂ and R₃ are oxygen and R₃ is R₄ is oxygen, R₅ is

-

-CHCH₂ or -CH₂CH₃, and R₇ is hydroxy.

29. The pharmaceutical composition, as claimed in any one of claims 22 to 25, wherein in the compound R₇ is hydroxy, R₅ is -CHCH₂ or -CH₂CR₃, and R₁ , R₂, R₃ and R₄ are

30. The pharmaceutical composition, as claimed in any one of claims 22 to 25, wherein in the compound R₁ and R₂ are , R₃ is R₄ is oxygen, R₅ is -CHCH₂

and R₇ is hydroxy.

31. The pharmaceutical composition, as claimed in any one of claims 22 to 25, wherein in the compound R₁ and R₃ are , R₂ is R₄ is oxygen, R₅ is

₃

-CHCH₂ and R₇ is hydroxy.

32. The pharmaceutical composition, as claimed in any one of claims 22 to 25, wherein in the compound R₁ and R₂ are , R₃ is , R₄ is oxygen, R₅ is

-CHCH₂ and R₇ is hydroxy.

33. The pharmaceutical composition, as claimed in any one of claims 22 to 25, wherein in the compound R₁ and R₂ are , R₃ is , R₄ is oxygen, R₅ is

-CHCH₂ and R₇ is hydroxy.

34. The pharmaceutical composition, as claimed in any one of claims 22 to 25, wherein in the compound R₁ and R₂ are , R₃ is , R₄ is oxygen, R₅ is

-CHCH₂ and R₇ is hydroxy.

35. The pharmaceutical composition, as claimed in any one of claims 22 to 25, wherein in the compound R₁ and R₂ are , R₃ is , R₄ is oxygen , R₅ is

^

-CHCH₂ and R₇ is hydroxy.

36. The pharmaceutical composition, as claimed in any one of claims 22 to 25 , wherein in the compound R₁ , R₂ and R₃ are , R₄ is oxygen, R₅ is -CHCH₂ and R₇ is

hydroxy.

37. The pharmaceutical composition, as claimed in any one of claims 22 to 25, wherein in the compound R₁ and R₂ are , R₂ is , R₄ is oxygen, R₅ is

-CHCH₂ and R₇ is hydrogen.

38. The pharmaceutical composition, as claimed in any one of claims 22 to 25, wherein in the compound R₁ and R₇ taken together are R₂ is , R₃ is

R₄ is oxygen, R₅ is -CHCH₂ and R₇ is oxygen.

39. The pharmaceutical composition, as claimed in any one of claims 22 to 25, wherein in the compound R₁ oxygen, R₂ is , R₃ is , R₄ is oxygen, R₅ is

R₄ is oxygen, R₅ is -CHCH₂ and R₇ is hydroxy.

40. The pharmaceutical composition, as claimed in any one of claims 22 to 25, wherein in the compound R₁ and R₂ are ,, R₃ is ,, R₄ is : , R₅ is

-CHCH₂ and R₇ is hydroxy.

41. The pharmaceutical composition, as claimed in any one of claims 22 to 24, wherein in the compound R₁ and R₂ are , R₃ is , R₄ is oxygen, R₅ is

-CHCH₂ and R₇ is hydroxy.

42. A pharmaceutical composition for the treatment of psoriasis vulgaris comprising, in combination with a pharmaceutical carrier, an effective amount therefor of Forskolin.

43. A method for treating psoriasis vulgaris in a patient suffering therefrom which comprises topically administering an effective amount thereof of a substance comprising Forskolin and a β₂-receptor aganonist.

44. A method for treating psoriasis vulgaris in a patient suffering therefrom which comprises topically administering an effective amount of a substance comprising Forskolin and an H₂-receptor aganonist.

45. The method, as claimed in claim 43, wherein the substance comprises Forskolin, a β₂-receptσr aganonist and an H₂-receptor aganonist.

46. The method, as claimed in claim 43 or 45, wherein the β₂-receptor aganonist is epinephrine.

47. The method, as claimed in claim 44 or 45, wherein the H₂-receptor aganonist is histamine.

48. A pharmaceutical composition for the treatment of psoriasis vulgaris comprising, in combination with a pharmaceutical carrier, an effective amount therefor of Forskolin and a β₂-receptor aganonist.

49. A pharmaceutical composition for the treatment of psoriasis vulgaris comprising, in combination with a pharmaceutical carrier, an effective amount therefor of Forskolin and an H₂-receptor aganonist.

50. The pharmaceutical composition, as claimed in claim 48, further including an H₂-receptor aganonist.

51. The pharmaceutical composition, as claimed in claim 48 or 50 wherein the β₂-receptor aganonist is epinephrine.

52. The pharmaceutical composition, as claimed in claim 49 or 50, wherein the H₂-receptor aganonist is histamine.