Topical Formulations for Use in the Treatment or Prevention of Skin Cancers
Field of the Invention
The present invention relates to topical formulations for use in the treatment or prevention of skin cancers that can be so prevented or treated by inhibiting angiogenesis and/or vasculogenesis. It also relates to the use in the treatment or prevention of skin cancers of certain steroids that inhibit angiogenesis and/or vasculogenesis.
Background to the Invention
Tumor cells require oxygen and nutrients to grow, reproduce and survive. The development of new oxygen-carrying blood vessels (angiogenesis) allows the tumor to increase in size and also to spread to other sites in the body. It has been shown (e.g. see WO-A-95/21613, WO-A-99/62890 and Wood et al, Cancer Res., 2000, 60, pp. 2178- 2189) that compounds that inhibit angiogenesis and/or vasculogenesis can be used to block the creation of new blood vessels and thus prevent tumor growth and spread. Examples of such compounds include the tyrosine kinase inhibitor PTK787/ZK 222584, which has shown promise in trials on its activity in the treatment of solid tumors such as colorectal cancer.
Skin cancer, including basal cell carcinomas, squamous cell carcinomas, melanomas and skin metastases, is one of the commonest types of cancer. Due to a variety of factors including increased exposure to UV light, the incidence of skin cancer has increased significantly in recent years. All of the anti-angiogenesis compounds so far developed such as PTK787/ZK 222584 are administered orally only and are unsuitable for the treatment of skin cancers as it is not possible to get sufficient of these compounds through the skin barrier to the required site of action. A compound that has activity in inhibiting angiogenesis and/or vasculogenesis and which can be easily delivered locally in significant amounts to the site affected by the skin cancer would be a potential candidate as a compound for the treatment or prevention of skin cancer.
Danazol is known for use in the treatment of breast cancer and endometrial cancer. Trilostane is known for use in the treatment of breast cancer and prostate cancer. However, neither is known for use in the treatment or prevention of skin cancer.
Furthermore, neither is known to have activity in inhibiting angiogenesis and/or vasculogenesis. The inventors of the present invention have now made the surprising discovery that these compounds and other related compounds have excellent activity in inhibiting angiogenesis and/or vasculogenesis and that they can be readily applied as a topical formulation to an area of skin affected by a skin tumor so as to deliver an effective amount of said compound to the affected site, thus making them excellent candidates for use in the treatment and prevention of skin cancer.
Summary of the Invention
In a first aspect of the present invention there is provided the use of a steroid selected from the group consisting of ethisterone and derivatives thereof and trilostane and derivatives thereof in the manufacture of a topical medicament for the prevention or treatment of skin cancers that can be so prevented or treated by inhibiting angiogenesis and/or vasculogenesis.
In a second aspect of the present invention, there is provided a topical formulation for the treatment or prevention of skin cancers that can be so prevented or treated by inhibiting angiogenesis and/or vasculogenesis comprising a pharmaceutically acceptable carrier or diluent and an effective amount of a steroid selected from the group consisting of ethisterone and derivatives thereof and trilostane and derivatives thereof.
In a third aspect of the present invention, there is provided a method for the treatment or prevention of a skin cancer that can be so prevented or treated by inhibiting angiogenesis and/or vasculogenesis, said method comprising the topical administration to a patient in need thereof of an effective amount of a steroid selected from the group consisting of ethisterone and derivatives thereof and trilostane and derivatives thereof.
As is explained and exemplified in greater detail below, the topical formulations of the present invention demonstrate a remarkable and unique combination of effects that make them suitable for the treatment or prevention of a wide variety of skin cancers. Specifically, the topical formulations of the present invention have been found to demonstrate the following properties:
(a) the ability to modify the proliferation of endothelial cells, and more particularly to inhibit angiogenesis and/or vasculogenesis; and
(b) the ability to deliver these compounds directly to the affected sites in effective amounts.
The fact that the formulations of the present invention have these properties makes them particularly suited to the prevention or treatment of skin cancers which can be prevented or treated by inhibiting angiogenesis and/or vasculogenesis. Because the steroids selected from the group consisting of ethisterone and derivatives thereof and trilostane and derivatives thereof can be easily delivered in significant amounts to the site affected by the skin cancer, their ability to inhibit angiogenesis and/or vasculogenesis enables them to block the creation of new blood vessels to prevent tumor growth and spread. It is possible that the known hormonal effect of the steroids of the present invention may also contribute to their particular effectiveness (e.g. see Gledhill et al, J. Steroid Biochem. Molec. Biol, 1992, Vol. 43(4), pp. 289-296 and Panahy et al, British Medical Journal, 1987, Vol. 295, pp. 464-466).
Skin cancers that can be treated or prevented by inhibiting angiogenesis and/or vasculogenesis by the topical administration to a patient in need thereof of an effective amount of a steroid selected from the group consisting of ethisterone and derivatives thereof and trilostane and derivatives thereof include, but are not limited to basal cell carcinomas, squamous cell carcinomas, melanomas and skin metastases. More preferred skin cancers include basal cell carcinomas and skin metastases, particularly skin metastases from breast cancer, lung cancer, uterine cancer, intenstinal cancer, colon cancer and melanomas.
The steroids that can be used in the topical formulations and in the method of treatment or prevention of skin cancers according to the present invention are selected from a group consisting of ethisterone and derivatives thereof and trilostane and derivatives thereof. Preferred examples of ethisterone and derivatives thereof include compounds of the following formula (I) and pharmacologically acceptable salts and esters thereof:
wherein:
R1 is an alkyl group having from 1 to 6 carbon atoms, an alkenyl group having from 2 to 6 carbon atoms or an alkynyl group having from 2 to 6 carbon atoms;
R is hydroxyl, an alkoxy group having from 1 to 6 carbon atoms, an alkanoyloxy group having from 1 to 7 carbon atoms, a group of formula (II) or a group of formula (IIl):
wherein Rπ is hydrogen, an alkyl group having from 1 to 6 carbon atoms, a hydroxyl group, an alkoxy group having from 1 to 6 carbon atoms or a group of formula -N(R14)2 wherein each group R14 is the same or different and is hydrogen or an alkyl group having from 1 to 6 carbon atoms, each of R!2 and R13 is the same or different and is hydrogen or an alkyl group having from 1 to 6 carbon atoms, and m and n are the same or different and each is 0 or an integer of from 1 to 4; each of R3 and R4 is the same or different and is hydrogen or an alkyl group having from 1 to 6 carbon atoms;
each of R5 and R6 is the same or different and is hydrogen or an alkyl group having from 1 to 6 carbon atoms or R5 and R6 together represent a single bond; each of R7, R8, R9 and R10 is the same or different and is hydrogen, an alkyl group having from 1 to 6 carbon atoms, a hydroxyl group, an alkoxy group having from 1 to 6 carbon atoms or a group of formula -N(RIS)2 wherein each group R15 is the same or different and is hydrogen or an alkyl group having from 1 to 6 carbon atoms, or
R7 and R8 and/or R9 and R10 together with the carbon atom to which they are attached represent a carbonyl group, or
R7, R8, R9 and R10 together with the carbon atoms to which they are attached represent a 5- to 9-membered heterocyclyl group, said 5- to 9-membered heterocyclyl group optionally being substituted with from 1 to 7 substituents (said substituents are the same or different and are selected from substituent group a defined below); substituent group α represents a group consisting of a halogen atom, a hydroxyl group, a cyano group, an amino group, an alkyl group having from 1 to 6 carbon atoms, an alkoxy group having from 1 to 6 carbon atoms, an alkylthio group having from 1 to 6 carbon atoms, an alkylsulfinyl group having from 1 to 6 carbon atoms, an alkylsulfonyl group having from 1 to 6 carbon atoms, a phenyl group and a group of formula -N(R16J2 wherein each group R16 is the same or different and is hydrogen or an alkyl group having from 1 to 6 carbon atoms.
Preferred examples of trilostane and derivatives thereof include compounds of the following formula (IV) and pharmacologically acceptable salts and esters thereof:
R18, R19 and R21 are the same or different and each is hydrogen or an alkyl group having from 1 to 6 carbon atoms;
R17 is hydrogen, an alkyl group having from 1 to 6 carbon atoms or an alkenyl group having from 2 to 6 carbon atoms,
R22 is hydroxyl, an alkoxy group having from 1 to 6 carbon atoms, an alkanoyloxy group having from 1 to 7 carbon atoms, a group of formula (H) as defined above for formula (I) or a group of formula (ITI) as defined above for formula (I), or
R17 and R22 together represent an oxo group, an ethylenedioxy group or a propylenedioxy group; each of R20 and R24 is the same or different and is hydrogen or an alkyl group having from 1 to 6 carbon atoms or R20 and R24 together represent a single bond; each of R23 and R29 is the same or different and is hydrogen or an alkyl group having from 1 to 6 carbon atoms or R23 and R29 together represent an epoxy linkage or a single bond; each of R25, R26, R27 and R28 is the same or different and is hydrogen, an alkyl group having from 1 to 6 carbon atoms, a cyano group, a hydroxyl group, an alkoxy group having from 1 to 6 carbon atoms or a group of formula -N(R30)2 wherein each group R30 is the same or different and is hydrogen or an alkyl group having from 1 to 6 carbon atoms, or
R25 and R26 and/or R27 and R28 together with the carbon atom to which they are attached represent a carbonyl group, or
R25, R26, R27 and R28 together with the carbon atoms to which they are attached represent a 5- to 9-membered heterocyclyl group, said 5- to 9-membered heterocyclyl group optionally being substituted with from 1 to 7 substituents (said substituents are the same or different and are selected from substituent group α defined above).
Where R7, R8, R9 and R10 together with the carbon atoms to which they are attached represent an optionally subsitituted 5- to 9-membered heterocyclyl group or R25, R26, R27 and R28 together with the carbon atoms to which they are attached represent an optionally substituted 5- to 9-membered heterocyclyl group, said heterocyclyl group is a
5- to 9-membered heterocyclic group containing from 1 to 4 atoms selected from a group consisting of a nitrogen atom, an oxygen atom and a sulfur atom and may be, for example, an unsaturated heterocyclic group such as a furyl group, a thienyl group, a pyrrolyl group, an azepinyl group, a pyrazolyl group, an imidazolyl group, an oxazolyl group, an isoxazolyl group, a thiazolyl group, an isothiazolyl group, a 1,2,3-oxadiazolyl group, a triazolyl group, a tetrazolyl group, a thiadiazolyl group, a pyranyl group, a pyridy] group, a pyridazinyl group, a pyrimidinyl group, a pyrazinyl group, an azepinyl group, an azociny] group or an azoninyl group; or a group wherein the unsaturated heterocyclic groups described above are partially or completely reduced, such as a morpholinyl group, a thiomorpholinyl group, a pyrrolidinyl group, a pyrrolinyl group, a imidazolidinyl group, a jmidazolinyl group, a pyrazolidinyl group, a pyrazolinyl group, a piperidy] group, a piperazinyl group, a perhydroazepinyl group, a perhydroazocinyl group or a perhydroazoninyl group; preferably it is a 5- to 7-membered heterocyclic group containing one or more nitrogen atom and optionally containing an oxygen atom and/or a sulfur atom, which is, for example, an unsaturated heterocyclic group such as a pyrrolyl group, an azepinyl group, a pyrazolyl group, an imidazolyl group, an oxazolyl group, an isoxazolyl group, a thiazolyl group, an isothiazolyl group, a 1,2,3-oxadiazolyl group, a triazolyl group, a tetrazolyl group, a thiadiazolyl group, a pyridyl group, a pyridazinyl group, a pyrimidinyl group or a pyrazinyl group; or a group wherein this unsaturated heterocyclic group is partially or completely reduced, such as a morpholinyl group, a thiomorpholinyl group, a pyrrolidinyl group, a pyrrolinyl group, a imidazolidinyl group, a imidazolinyl group, a pyrazolidinyl group, a pyrazolinyl group, a piperidyl group or a piperazinyl group; and more preferably it is an isoxazolyl group or a pyrazolyl group for R7, R8, R9 and R10 together with the carbon atoms to which they are attached and an isoxazolyl group for R25, R26, R27 and R28 together with the carbon atoms to which they are attached.
The alkyl group having from 1 to 6 carbon atoms in the definitions of R1, R3, R4, R5, R6, R7, R8, R9, R10, Rn, R12, R13, R14, R]S, R16, R17, R18, R19, R20, R21, R23, R24, R25, R26, R27, R28, R29, R30 and substituent group α is a straight or branched chain alkyl group having from 1 to 6 carbon atoms and may be, for example, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, an s-butyl
group, a t-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a t-pentyl group, a 1-methylbutyl group, a hexyl group, a 1-methylpentyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 1-ethylbutyl group or a 2-ethylbutyl group; preferably it is an alkyl group having from 1 to 4 carbon atoms such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, an s-butyl group or a t-butyl group; more preferably it is a methyl group, an ethyl group, a propyl group or an isopropyl group; and most preferably it is a methyl group.
The alkenyl group having from 2 to 6 carbon atoms in the definition of R1 and R17 is a straight or branched chain alkenyl group having from 2 to 6 carbon atoms and may be, for example, vinyl, 2-proρenyl, 1-methyl-2-propenyl, 2-methyl-2-proρenyl, 2~ethyl-2- propenyl, 2-bulenyl, 1-methyl-2-butenyl, 2~rnethyl-2~butenyl, 1-ethyl-2-butenyl, 3- butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 1-ethyl-3-butenyl, 2-ρentenyl, 1- methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-pentenyl, 1-methyl-3-pentenyl, 2-methyl-3- pentenyl, 4-pentenyl, 1-methyl-4-ρentenyl, 2-methyl-4-pentenyl, 2-hexenyl, 3-hexenyl, 4- hexenyl and 5-hexenyl groups. Alkenyl groups having from 2 to 4 carbon atoms are preferred, and alkenyl groups having 2 or 3 carbon atoms are most preferred.
The alkynyl group having from 2 to 6 carbon atoms in the definition of R1 and R17 is a straight or branched chain alkynyl group having from 2 to 6 carbon atoms and may be, for example, ethynyl, 2-propynyl, 1-methyl-2-propynyl, 2-butynyl, 1-methyl-2-butynyl, 1-ethyl-2-butynyl, 3-butynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-ethyl-3-butynyI, 2-pentynyl, 1-melhy1-2-pentynyl, 3-pentynyl, 1-methyl-3-pentynyl, 2-methy.-3-pen.yny], 4-pentynyl, 1-methyl-4-pentynyl, 2-methy]-4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl groups. Alkynyl groups having from 2 to 4 carbon atoms are preferred, and alkynyl groups having 2 or 3 carbon atoms are most preferred.
The alkanoyloxy group having from 1 to 7 carbon atoms in the definitions of R2 and R22 is a carbonyloxy group (-COO-) the carbon atom of which is substituted with a hydrogen atom or an alkyl group having from 1 to 6 carbon atoms as described above and may be, for example, a formyloxy group, an acetyloxy group, a propionyloxy group, a butyryloxy group, an isobutyryloxy group, a pentanoyloxy group or a hexanoyloxy group; it is preferably an alkanoyloxy group having from 2 to 5 carbon atoms such as an
acetyloxy group, a propionyloxy group, a butyryloxy group or an isobutyryloxy group; and more preferably it is an acetyloxy group.
The alkoxy group having from 1 to 6 carbon atoms in the definitions of R2, R7, R8, R9, R10, R11, R22 and substituent group α is a hydroxy group in which the hydrogen atom is substituted with an alkyl group having from 1 to 6 carbon atoms as described above and may be, for example, a methoxy group, an ethoxy group, a n-ρroρoxy group, an isopropoxy group, a n-butoxy group, an isobutoxy group, an s-butoxy group, a tert- butoxy group, an n-pentyloxy group, an isopentyloxy group, a 2-rnethylbutoxy group, a neopentyloxy group, an n-hexyloxy group, a 4-methylpentyloxy group, a 3- methylpentyloxy group, a 2-methylpentyloxy group, a 3,3-dimethylbutoxy group, a 2,2- dimethylbutoxy group, a 1,1-dimethylbutoxy group, a 1,2-dimethylbutoxy group, a 1,3- dimethylbutoxy group or a 2, 3 -dimethyl butoxy group; it is preferably an alkoxy group having from 1 to 4 carbon atoms such as a methoxy group, an ethoxy group, an n- propoxy group or an n-butoxy group; and more preferably it is a methoxy group.
The alkylthio group having from 1 to 6 carbon atoms in the definition of substituent group α is a mercapto group substituted with an alkyl group having from 1 to 6 carbon atoms as described above and may be, for example, a methylthio group, an ethylthio group, an n-propylthio group, an isopropylthio group, an n-butylthio group, an isobutylthio group, an s-butylthio group, a tert-butylthio group, an n-pentylthio group, an isopentylthio group, a 2-methylbutylthio group, a neopentylthio group, a 1- ethylpropylthio group, an n-hexylthio group, an isohexylthio group, a 4-methylpentylthio group, a 3-methylρentylthio group, a 2-methylρentylthio group, a 1-methylpentylthio group, a 3,3-dimethylbutylthio group, a 2,2-dimethylbutylthio group, a 1,1- dimethylbutylthio group, a 1,2-dimethylbutylthio group, a 1,3-dirnethylbutylthio group, a 2,3~dimethylbutylthio group or a 2-ethylbutylthio group; it is preferably an alkylthio group having from 1 to 4 carbon atoms such as a methylthio group, an ethylthio group, an n-propylthio group or an n~butylthio group; and more preferably it is a methylthio group.
The alkylsulfinyl group having from 1 to 6 carbon atoms in the definition of substituent group a is a sulfinyl group (-SO-) which is substituted with an alkyl group having from 1 to 6 carbon atoms as described above and may be, for example, a methanesulfinyl group, an ethanesulfinyl group, an n-propanesulfinyl group, an
isopropanesulfinyl group, an n-butanesulfinyl group, an isobutanesulfinyl group, an s- butanesulfinyl group, a tert-butanesulfinyl group, an n-pentanesulfinyl group, an isopentanesulfinyl group, a 2-methylbutanesulfinyl group, a neopentanesulfinyl group, an n-hexanesulfinyl group, a 4-methylpentanesulfinyl group, a 3-methylpentanesulfinyl group, a 2-methylρentanesulfinyl group, a 3,3-dimethylbutanesulfinyl group, a 2,2- dimethylbutanesulfmyl group, a 1,1-dimethylbutanesulfinyl group, a 1,2- dimethylbutanesulfinyl group, a 1,3-dimethylbutanesuIfinyl group or a 2,3- dimethylbutanesulfinyl group; preferably it is an alkylsulfinyl group having from 1 to 4 carbon atoms such as a methanesulfinyl group, an ethanesulfinyl group, an n- propanesulfinyl group, an isopropanesulfinyl group or an n-butanesulfinyl group; and more preferably it is a methanesulfinyl group.
The alkylsulfonyl group having from 1 to 6 carbon atoms in the definition of substituent group α is a sulfonyl group (-SO2-) substituted with an alkyl group having from 1 to 6 carbon atoms as described above and may be, for example, a methanesulfonyl group, an ethanesulfonyl group, an n-propanesulfonyl group, an isopropanesulfonyl group, an n-butanesulfonyl group, an isobutanesulfonyl group, an s-butanesulfonyl group, a tert-butanesulfonyl group, an n-pentanesulfonyl group, an isopentanesulfonyl group, a 2-methylbutanesulfonyI group, a neopentanesulfonyl group, an n-hexanesulfonyl group, a 4-methylpentanesulfonyl group, a 3-methylpentanesuIfonyl group, a 2- methylpentanesulfonyl group, a 3,3-dimethylbutanesulfonyl group, a 2,2- dimethylbutanesulfonyl group, a 1,1-dimethylbutanesulfonyl group, a 1,2- dimethylbutanesulfonyl group, a 1,3-dimethylbutanesulfonyl group or a 2,3- dimethylbutanesulfonyl group; preferably it is an alkylsulfonyl group having from 1 to 4 carbon atoms such as a methanesulfonyl group, an ethanesulfonyl group, an n- propanesulfonyl group or an n-butanesulfonyl group; and more preferably it is a methanesulfonyl group.
Where the compound of formula (I) or (TV) of the present invention or a pharmacologically acceptable ester thereof has a basic group, the compound can be converted to a salt by reacting it with an acid, and in the case where the compound of formula (I) or (IV) of the present invention or a pharmacologically acceptable ester thereof has an acidic group, the compound can be converted to a salt by reacting it with a
base. The compounds of the present invention encompass such salts. Where said salts are to be used for a therapeutic use, they must be pharmacologically acceptable.
Preferred examples of the salts formed with a basic group present in the compound of formula (I) or (IV) of the present invention include inorganic acid salts such as hydrohalogenated acid salts (e.g. hydrochlorides, hydrobromides and hydroiodides), nitrates, perchlorates, sulfates and phosphates; organic acid salts such as lower alkanesulfonates in which the lower alkyl moiety thereof is an alkyl group having from 1 to 6 carbon atoms as defined above (e.g. methanesulfonates, trifluoromethanesulfonates and ethanesulfonates), aryl sulfonates in which the aryl rnoiety thereof is an aryl group having from 6 to 14 carbon atoms (e.g. benzenesulfonate or p-toluenesulfonate), acetates, malates, fumarates, succinates, citrates, ascorbates, tartrates, oxalates and maleates; and amino acid salts such as glycine salts, lysine salts, arginine salts, ornithine salts, glutamates and aspartates. Hydrohalogenated acid salts are particularly preferred.
Preferred example of the salts formed with an acidic group present in the compound of formula (I) or (IV) of the present invention include metal salts such as alkali metal salts (e.g. sodium salts, potassium salts and lithium salts), alkali earth metal salts (e.g. calcium salts and magnesium salts), aluminium salts and iron salts; amine salts such as inorganic amine salts (e.g. ammonium salts) and organic amine salts (e.g. t- octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycineallcyl ester salts, ethylenedi amine salts, N-methylglucamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'- dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzylphenethyl amine salts, piperazine salts, tetramethylammonium salts and tris(hydroxymethyl)aminomethane salts; and amino acid salts such as glycine salts, lysine salts, arginine salts, ornithine salts, glutamates and aspartates. Alkali metal salts are particularly preferred.
The compounds of formulae (I) and (IV) of the present invention and pharmacologically acceptable salts and esters thereof of the present invention can sometimes take up water upon exposure to the atmosphere or when recrystallized to absorb water or to form a hydrate and such hydrates are also included within the scope of the present invention. Additionally, certain other solvents may be taken up by the
compounds of the present invention to produce solvates, which also form a part of the present invention.
The compounds of formulae (I) and (IV) of the present invention sometimes contain one or more asymmetric centres, and can therefore form optical isomers (including diastereoisomers). For the compounds of the present invention, each of said isomers and mixture of said isomers are depicted by a single formula, i.e. the formula (I) and (IV) respectively. Accordingly, the present invention covers both the individual isomers and mixtures thereof in any proportion, including racemic mixtures.
The present invention encompasses esters of the compounds of formulae (I) and (IV). These esters are compounds of formulae (I) and (IV) in which a hydroxyl group or a carboxy group of said compound of formula (I) or (IV) is modified by the addition of a protecting group using conventional techniques well-known in the art (see, for example, "Protective Groups in Organic Synthesis, Second Edition, Theodora W. Greene and Peter G.M. Wuts, 1991, John Wiley & Sons, Inc.).
There is no particular restriction on the nature of this protecting group, provided that, where the ester is intended for therapeutic purposes, it must be pharmacologically acceptable, i.e. the protecting group must be capable of being removed by a metabolic process (e.g. hydrolysis) on administration of said compound to the body of a live mammal to give a compound of formula (I) or (TV) or a salt thereof. In other words, the pharmacologically acceptable esters are pro-drugs of the compounds of formula (I) or (IV) of the present invention.
Whether an ester of a compound of formula (I) or (IV) of the present invention is pharmacologically acceptable can be easily determined. The compound under investigation is intravenously administered to an experimental animal such as a rat or mouse and the body fluids of the animal are thereafter studied. If a compound of formula (I) or (IV) or a pharmacologically acceptable salt thereof can be detected in the body fluids, the compound under investigation is judged to be a pharmacologically acceptable ester.
The compounds of formula (I) or (IV) of the present invention can be converted to an ester, examples of which include a compound of formula (I) or (IV) in which a hydroxyl group present therein is esterified. The ester residue must be capable of being
removed by a metabolic process (e.g. hydrolysis) in vivo in order for the esterified compound to be one which is pharmacologically acceptable. Preferred examples of such a protecting group include the following: (i) 1-(acyloxy)lower alkyl groups, examples of which include 1-(aliρhatic acyloxy)lower alkyl groups which comprise an alkyl group having from 1 to 6 carbon atoms as defined above which is substituted with an alkylcarbonyloxy group having from 1 to 6 carbon atoms, examples of which include formyloxymethyl, acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl, valeryloxymethyl, isovaleryloxymethyl, hexanoyloxymethyl, 1-formyloxyethyl, 1- acetoxyethyl, 1-propionyloxyethyl, 1-butyryloxyethyl, 1-ρivaloyloxyethyl, 1- valeryloxyethyl, 1-isovaleryloxyethyl, 1-hexanoyloxyethyl, 1-formyloxypropyl, 1- acetoxypropyl, 1-propionyloxypropyl, 1-butyryloxypropyl, 1-pivaloyloxyproρyl, 1- valeryloxypropyl, 1-isovaleryloxypropyl, 1-hexanoyloxy-propyl, 1-acetoxybutyl, 1- propionyloxybutyl, 1-butyryloxybutyl, 1-pivaloyloxybutyl, 1-acetoxypentyl, 1- propionyloxypentyl, 1-butyryloxypentyl, 1-pivaloyloxypentyl and 1-pivaloyloxyhexyl groups, 1-(cycloalkylcarbonyloxy)lower alkyl groups which comprise an alkyl group having from 1 to 6 carbon atoms as defined above which is substituted with a cycloalkylcarbonyloxy group in which a carbonyloxy group is substituted with a cycloalkyl group having from 1 to 6 carbon atoms, examples of which include cyclopentylcarbonyloxymethyl, cyclohexylcarbonyloxy-methyl, 1- cyclopentylcarbonyloxyethy], 1-cyclohexylcarbonyloxyethyl, 1-cyclopentylcarbonyloxypropyl, 1-cyclohexylcarbonyloxypropyl, 1-cyclopenty1- carbonyloxybutyl and 1-cyclohexylcarbonyloxybutyl groups, and 1-(aromatic acyloxy)lower alkyl groups which comprise an alkyl group having from 1 to 6 carbon atoms as defined above which is substituted with an aryl carbonyloxy group which comprises an oxygen atom which is substituted with an arylcarbonyl group, examples of which include benzoyloxymethyl groups; (ii) substituted carbonyloxyalkyl groups, examples of which include
(lower alkoxycarbonyloxy)alkyl groups which comprise an alkyl group having from 1 to 6 carbon atoms as defined above or a cycloalkyl group having from 1 to 6
carbon atoms which is substituted with a lower alkoxycarbonyloxy group which comprises a carbonyloxy group substituted with an alkoxy group having from 1 to 6 carbon atoms as defined above or a cycloalkoxy group having from 1 to 6 carbon atoms, examples of which include methoxycarbonyloxymethyl, ethoxycarbonyloxymethyl, propoxy-carbonyloxymethyl. isopropoxycarbonyloxymethyl, butoxycarbonyloxymethyl, isobutoxycarbonyloxymethyl, pentyloxycarbonyloxymethyl, hexyloxycarbonyloxy- methyl, cyclohexyloxycarbonyloxymethyl, cyclohexyloxycarbonyloxy(cyclohexyl)- methyl, 1-(methoxycarbonyloxy)ethyl, 1-(ethoxycarbonyloxy)ethyl, 1-(proρoxy- carbonyloxy)ethyl, 1-(isopropoxycarbonyloxy)ethyl, 1-(butoxycarbonyloxy)ethyl, 1-(isobutoxycarbonyloxy)ethyl, 1-(t-butoxycarbonyloxy)ethyl, 1-(pentyloxy- carbonyloxy)ethyl, 1-(hexyloxycarbonyloxy)ethy3, 1-(cyclopentyloxycarbonyloxy)-ethy3, l~(cycloρentyloxycarbonyloxy)proρyl, 1-(cyclohexyloxycarbonyloxy)proρyl, 1-(cycloρentyloxycarbonyloxy)butyl, 1-(cyclohexyloxycarbonyloxy)butyl, 1-(cyclohexyloxycarbonyloxy)ethyl, 1-(ethoxycarbonyloxy)propyl, 1-(methoxy~ cai'bonyloxy)propyl, 1-(ethoxycarbonyloxy)proρyl, 1-(propoxycarbonyloxy)propyl, 1-(isopropoxycarbonyloxy)proρyl, 1-(butoxycarbonyloxy)propyl, 1-(isobutoxy- carbon yloxy)propyl, 1 -(pentyloxycarbonyloxy)propyl, 1 -(hexyloxycarbonyloxy)-propyl, 1-(methoxycarbonyloxy)butyl, 1-(ethoxycarbonyloxy)butyl, 1-(propoxy- carbonyloxy)butyl, 1-(isopropoxycarbonyloxy)butyl, 1-(butoxycarbonyloxy)butyl, 1-(isobutoxycarbonyloxy)butyl, 1-(methoxycarbonyloxy)peπtyl, 1-(ethoxy- carbonyloxy)pentyl, 1-(methoxycarbonyloxy)hexyl and 1-(ethoxycarbonyloxy)hexyl groups, and oxodioxolenylmethyl groups, which comprise a methyl group which is substituted with an oxodioxolenyl group which itself may optionally be substituted with a group selected from the group consisting of alkyl groups having from 1 to 6 carbon atoms as defined above and aryl groups having from 6 to 14 carbon atoms as defined above which may optionally be substituted with at least one alkyl group having from 1 to 6 carbon atoms as defined above, alkoxy group having from 1 to 6 carbon atoms as defined above or a halogen atom, examples of which include (5~pheny1-2-oxo-1,3-dioxolen-4- yl)methyl, [5-(4-methylρhenyl)-2-oxo-1,3-dioxolen-4-yl]methyl, [5-(4-rnethoxyphenyl)- 2-oxo-1,3-dioxolen-4-yl]methyl, [5-(4-fluorophenyl)-2-oxo-1,3-dioxolen-4-yl]methyl, [5- (4-chlorophenyl)-2-oxo-1,3-dioxolen-4-yl]methyl, (2-oxo-1,3-dioxolen-4-yl)-methyl, (5-
methyl-2-oxo-1,3-dioxolen-4-yl)methyl, (5-ethyl-2-oxo-1,3-dioxolen-4-yl)methyl, (5- propy1-2-oxo-1,3-dioxolen-4-yl)methyl, (5-isopropy1-2-oxo-1,3-dioxolen-4-yl)methyl and (5-buty1-2-oxo-1,3-dioxolen-4-yl)methyl groups;
(iii) phthalidyl groups which comprise a phthalidyl group which may optionally be substituted with a substituent selected from the group consisting of alkyl groups having from 1 to 6 carbon atoms as defined above and alkoxy groups having from 1 to 6 carbon atoms as defined above, examples of which include phthalidyl, dimethylphthalidyl and dimethoxyphthalidyl groups;
(iv) aliphatic acyl groups, examples of which include alkylcarbonyl groups having from 1 to 25 carbon atoms, examples of which include formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovalery], octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyϊoctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl, 14-methyl- pentadecanoyl, 13,13-dimethyltetradecanoyl, heptadecanoyl, 15-methylhexadecanoyl, octadecanoyl, 1-methylheρtadecanoyl, nonadecanoyl, eicosanoyl and heneicosanoyl groups, ester forming residues of a saturated or unsaturated C2-CiO aliphatic di-carboxylic acids such as a fumarate, a maleate, oxalate, malonate or succinate, halogenated alkylcarbonyl groups having from 1 to 25 carbons in which the alkyl moiety thereof is substituted by at least one halogen atom, examples of which include chloroacetyl, dichloroacetyl, trichloroacetyl and trifluoroacetyl groups, lower alkoxyalkylcarbonyl groups which comprise an alkylcarbonyl group having from 1 to 25 carbon atoms in which the alkyl moiety thereof is substituted with at least one C1-C6 alkoxy group as defined above, examples of said lower alkoxyalkylcarbonyl groups including methoxyacetyl groups, and unsaturated alkylcarbonyl groups having from 1 to 25 carbon atoms, examples of which include acryloyl, propioloyl, methacryloyl, crotonoyl, isocrotonoyl and (E)-2- methyl-2-butenoyl groups; of these, alkylcarbonyl groups having from 1 to 6 carbon atoms are preferred; (v) aromatic acyl groups, examples of which include
arylcarbonyl groups which comprise a carbonyl group which is substituted with an aryl group having from 6 to 14 carbon atoms as defined above, examples of which include benzoyl, ct-naphthoyl and β-naphthoyl groups, halogenated arylcarbonyl groups which comprise an arylcarbonyl group as defined above which is substituted with at least one halogen atom, examples of which include 2-bromobenzoyl, 4-chlorobenzoyl and 2,4,6-trifluorobenzoyl groups, lower alkylated arylcarbonyl groups which comprise an arylcarbonyl group as defined above which is substituted with at least one alkyl group having from 1 to 6 carbon atoms as defined above, examples of which include 2,4,6-trimethyl-benzoyl and 4-toluoyl groups, lower alkoxylated arylcarbonyl groups which comprise an arylcarbonyl group as defined above which is substituted with at least one alkoxy group having from 1 to 6 carbon atoms as defined above, examples of which include 4-anisoyl groups, nitrated arylcarbonyl groups which comprise an arylcarbonyl group as defined above which is substituted with at least one nitro group, examples of which include 4~ nitrobenzoyl and 2-nitrobenzoyl groups, lower alkoxycarbonylated arylcarbonyl groups which comprise an arylcarbonyl group as defined above which is substituted with a carbonyl group which is itself substituted with an alkoxy group having from 1 to 6 carbon atoms as defined above, examples of which include 2-(methoxycarbonyl)benzoyl groups, and arylated arylcarbonyl groups which comprise an arylcarbonyl group as defined above which is substituted with at least one aryl group having from 6 to 14 carbon atoms as defined above, examples of which include 4-phenylbenzoyl groups; (vi) half-ester salt residues of succinic acid; (vii) phosphate ester salt residues ;
(vϋi) ester-forming residues of an amino acid such as glutamate and aspartate; (ix) carbamoyl groups which may optionally be substituted with 1 or 2 alkyl groups having from 1 to 6 carbon atoms as defined above; and
(x) 1-(acyloxy)alkoxycarbonyl groups which comprise a lower alkoxycarbonyl group as defined above in which the lower alkoxy moiety is substituted with an aliphatic acyloxy group as defined above or an aromatic acyloxy group as defined above, examples of
which include pivaloyloxymethyloxycarbonyl groups.
Of the above protecting groups which are capable of being removed by a metabolic process (e.g. hydrolysis) in vivo which are used to synthesise a compound of formula (I) or (IV) in which a hydroxyl residue therein is modified, the C1-C25 alkylcarbonyl groups and substituted carbonyloxyalkyl groups are preferred.
Preferred compounds of formula (I) are compounds of formula (Ia) and pharmacologically acceptable salts and esters thereof:
wherein R
1, R
2, R
3, R
4, R
5, R
e, R
7, R
8, R
9 and R
10 are as defined and exemplified above. Of these compounds of formula (Ia) and pharmacologically acceptable salts and esters thereof, preferred are those wherein:
(i) R1 is an alkyl group having from 1 to 4 carbon atoms, an alkenyl group having from 2 to 4 carbon atoms or an alkynyl group having from 2 to 4 carbon atoms; (ii) R1 is a methyl group or an ethynyl group;
(iii) R2 is hydroxyl, an alkanoyloxy group having from 2 to 5 carbon atoms, a group of formula (II) wherein n is 0, 1 or 2, and R! 1 is an alkyl group having from 1 to 4 carbon atoms, a hydroxyl group, an alkoxy group having from 1 to 4 carbon atoms or a group of formula -N(R14J2 wherein each group R!4 is the same or different and is hydrogen or an alkyl group having from 1 to 4 carbon atoms or a group of formula (III) wherein m is 0, 1 or 2, and each of R12 and R13 is the same or different and is hydrogen or an alkyl group having from 1 to 4 carbon atoms;
(iv) R2 is hydroxyl, an alkanoyloxy group having 2 or 3 carbon atoms, a group of formula (EE) wherein n is 0 and R11 is a methyl group, an ethyl group, a hydroxyl group, a methoxy group, an ethoxy group, an amino group, a methylamino group or a dimethylamino group, or a group of formula (III) wherein m is 0 or 1, and each of R12 and R13 is the same or different and is hydrogen, a methyl group or an ethyl group;
(v) R2 is hydroxyl;
(vi) R3 is hydrogen or an alkyl group having from 1 to 4 carbon atoms;
(vii) R3 is a methyl group;
(viii) R4 is hydrogen or an alkyl group having from 1 to 4 carbon atoms;
(ix) R4 is a methyl group;
(x) each of R7 and RB is a hydrogen atom and R9 and R10 together with the carbon atom to which they are attached represent a carbonyl group, or
R7, R8, R9 and R10 together with the carbon atoms to which they are attached represent a 5- to 7-membered heterocyclyl group, said 5- to 7-membered heterocyclyl group optionally being substituted with from 1 to 3 substituents (said substituents are the same or different and are selected from substituent group α1 defined below), and substituent group α1 represents a group consisting of a halogen atom, a hydroxy] group, an amino group, an alkyl group having from 1 to 4 carbon atoms, an alkoxy group having from 1 to 4 carbon atoms, a phenyl group and a group of formula -N(R16a)2 wherein each group Ri6a is the same or different and is hydrogen or an alkyl group having from 1 to 4 carbon atoms;
(xi) R7, R8, R9 and R10 together with the carbon atoms to which they are attached represent an isoxazolyl group; and
(xia) each of R5 and R6 is a hydrogen atom or R5 and R6 together represent a single bond.
In each group of (i) to (ii), (iii) to (v), (vi) to (vii), (viii) to (ix) and (x) to (xi) compounds of formula (Ia) and pharmacologically acceptable salts and esters thereof having substituents falling within the larger numbered group are more preferred.
The compounds of formula (Ia) and pharmacologically acceptable salts and esters thereof which are given by an optional combination of R1 selected from (i) to (ii), R2 selected from (iii) to (v), R3 selected from (vi) to (vii), R4 selected from (viii) to (ix), R7,
Rs, R9 and R10 selected from (x) to (xi) and R5 and R6 selected from (xia) are also preferred.
Compounds of formula (Ia) and pharmacologically acceptable salts and esters thereof having the following combinations are particularly preferred:
(a) R1 = (i), R2 = (iii), R3 = (vi), R4 = (viii), R5 and R6 = (xia), R7, R8, R9 and R10 = (x);
(b) R1 - (ii), R2 = (iv), R3 = (vii), R4= (ix), R5 and R6 = (xia), R7, R8, R9 and R10 = (x); and
(c) R! = (ii), R2 = (v), R3 = (vϋ), R4 = (ix), R5 and R6 = (xia), R7, R8, R9 and R!0 = (xi).
The most preferred compounds of formula (Ia) and pharmacologically acceptable salts and esters thereof are ethisterone, danazol and stanozolol and pharmacologically acceptable salts and esters thereof:
Ethisterone Danazol
Stanozolol
Ethisterone, danazol and stanozolol are known synthetic steroid hormones having antiandrogen activity. Ethisterone (17α-hydroxypregn-4-en-20-yn-3-one) is a progestogen that has been used in the past to treat menstrual disorders and as a component of combined oral contraceptives. Danazol (17α-pregna-2,4-dien-2G-yno[2,3- d]-isoxazo1-17β-ol) is a derivative of ethisterone that is a weak androgen that binds to numerous steroid hormone receptors and blocks the synthesis of estradiol, progesterone, testosterone and glucocorticoids; it is known for use as an oral agent employed in the treatment of endometriosis. Stanozolol (17~methyl-5α -androstano[3,2-c]pyrazol-17β~ol) is a synthetic testosterone analogue.
Preferred compounds of formula (TV) are compounds of formula (IVa) and pharmacologically acceptable salts and esters thereof:
wherein R
17, R
18, R
19, R
20, R
21, R
22, R
23, R
24, R
25, R
26, R
27, R
28 and R
29 are as defined and exemplified above.
Of these compounds of formula (IVa) and pharmacologically acceptable salts and esters thereof, preferred are those wherein:
(xii) each of R and R is the same or different and is hydrogen or an alkyl group having from 1 to 4 carbon atoms; (xiii) each of RI S and R19 is a methyl group;
(xiv) each of R20, R21 and R24 is the same or different and is a hydrogen atom or an alkyl group having from 1 to 4 carbon atoms or R21 is a hydrogen atom and R20 and R24 together represent a single bond;
(xv) each of R20, R21 and R24 is a hydrogen atom or R2' is a hydrogen atom and R20 and R24 together represent a single bond;
(xvi) R17 is hydrogen or an alkyl group having from 1 to 4 carbon atoms;
(xvii) R17 is hydrogen;
(xviii) R22 is hydroxyl, an alkanoyloxy group having from 2 to 5 carbon atoms, a group of formula (II) wherein n is 0, 1 or 2, and Rn is an alky] group having from 1 to 4 carbon atoms, a hydroxyl group, an alkoxy group having from 1 to 4 carbon atoms or a group of formula -N(R14)2 wherein each group R14 is the same or different and is hydrogen or an alkyl group having from 1 to 4 carbon atoms or a group of formula (III) wherein m is 0, 1 or 2, and each of R12 and R13 is the same or different and is hydrogen or an alkyl group having from 1 to 4 carbon atoms;
(xix) R22 is hydroxyl, an alkanoyloxy group having 2 or 3 carbon atoms, a group of formula (IE) wherein n is 0 and R11 is a methyl group, an ethyl group, a hydroxyl group, a methoxy group, an ethoxy group, an amino group, a methylamino group or a dimethylamino group, or a group of formula (TU) wherein m is 0 or 1, and each of R12 and R13 is the same or different and is hydrogen, a methyl group or an ethyl group;
(xx) R17 and R22 together represent an oxo group;
(xxi) each of R23 and R29 represents a hydrogen atom or R23 and R39 together represent an epoxy linkage or a double bond;
(xxii) R2S is hydrogen or an alkyl group having from 1 to 4 carbon atoms, R26 is a cyano group and R27 and R2B together with the carbon atom to which they are attached represent a carbonyl group, or
R25, R26, R27 and R2E together with the carbon atoms to which they are attached represent a 5- to 7-membered heterocyclyl group, said 5- to 7-membered heterocyclyl group optionally being substituted with from 1 to 3 substituents (said substituents are the same or different and are selected from substkuent group α1 defined above); and (xxiii) R25 is hydrogen, R26 is a cyano group and R27 and R28 together with the carbon atom to which they are attached represent a carbonyl group, or
R25, R26, R27 and R28 together with the carbon atoms to which they are attached represent an isoxazolyl group.
In each group of (xii) to (xiii), (xiv) to (xv), (xvi) to (xvii), (xviii) to (xix) and (xxii) to (xxiii), compounds of formula (IVa) and pharmacologically acceptable salts and esters thereof having substituents falling within the larger numbered group are more preferred.
The compounds of formula (IVa) and pharmacologically acceptable salts and esters thereof which are given by an optional combination of R1 E and R19 selected from (xii) to (xiii), R20, R21 and R24 selected from (xiv) to (xv), R17 selected from (xvi), (xvii) and (xx), R22 selected from (xviii) to (xx), R23 and R29 selected from (xxi) and R25, R26, R27 and R2S selected from (xxii) to (xxiii) are also preferred.
Compounds of formula (IVa) and pharmacologically acceptable salts and esters thereof having the following combinations are particularly preferred:
(d) R18 and R!9 = (xii), R20, R21 and R24 = (xiv), R17 = (xvi) and R22 = (xviii), R23 and R29 = (xxi), and R25, R26, R27 and R28 - (xxii);
(e) R18 and R19 - (xiii), R20, R21 and R24 = (xv), R17 = (xvii) and R22= (xix), R23 and R29 = (xxi), and R25, R26, R27 and R28 = (xxiii);
(f) R18 and R19 = (xii), R20, R21 and R24 = (xiv), R17 and R22 together = (xx), R23 and R29 = (xxi), and R25, R26, R27 and R28 = (xxii); and
(g) R18 and R19 = (xiii), R20, R21 and R24 = (xv), R17 and R22 together = (xx), R23 and R29 = (xxi), and R25, R26, R27 and R28 = (xxiii).
The most preferred compounds of formula (IVa) and pharmacologically acceptable salts and esters thereof are trilostane, trilostane IE, trliostane HI, keto-trilostane and pharmacologically acceptable salts and esters thereof:
Trilostane Trilostane II
Kelo-Triloslane Trilostane III
Trilostane (2α-cyano-4α,5α-epoxyandrostari-17β-o1-3-one) and derivatives thereof are synthetic steroid hormones having activity in lowering the blood concentrations of glucocorticoids such as Cortisol. Trilostane is known as an oral medication for the treatment of Cushing's Syndrome and advanced breast cancer and is described in UK Patent Nos. 1,123,770, 2,130,588 and 2,345,851, US Patent No. 3,296,295 and WO-A- 02/080930, the contents of which are incorporated herein by reference thereto.
The topical formulations of the present invention comprise a pharmaceutically acceptable carrier or diluent and an effective amount of a steroid selected from the group consisting of ethisterone and derivatives thereof and trilostane and derivatives thereof. These topical formulations can be in any suitable form known to the person skilled in this field and can, for example, take the form of an ethanol solution, cleansing foam, cleansing cream, skin gel, skin lotion, shampoo gel, cream shampoo or the like.
Topical formulations are prepared by adding an exemplified compound to a base well known to those skilled in the art; for example, suspending agents (examples include gum arabic, tragacanth, methyl cellulose, sodium carboxymethylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, sodium alginate and bentonite), emulsifying agents
(examples include triethanolarnine, sodium lauryl sulfate, sorbitan sesquioleate, polysorbate 80 and stearic acid polyoxyl 40), moistening agents (examples include sorbitol, ethylene glycol, propylene glycol, butylene glycol and glycerin), preservatives (examples include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and butyl paraoxybenzoate) or solvents (examples include water; alcohols such as ethanol, isopropyl alcohol, propylene glycol, cetanol and isostearyl alcohol; hydrocarbons such as natural fats and oils, waxes and liquid paraffin; aliphatic acids such as stearic acid, isostearic acid, oleic acid and linoleic acid; and esters such as isopropyl myristate) or a mixture thereof.
The amount of the ethisterone or derivative thereof or trilostane or derivative thereof locally administered will vary depending on the condition, age or the like of the patient. It is desirably administered at a concentration of 0.01 mg/ml formulation (preferably 0.1 mg/ml formulation, most preferably 1 mg/ml formulation) as a lower limit and 100 mg/ml formulation (preferably 25 mg/ml formulation, most preferably 10 mg/ml formulation) as an upper limit and administered in a single dose or in several divided doses a day.
The Best Mode for Carrying Out the Invention
Additional objects, advantages and novel features of the present invention will become apparent to those skilled in the art by consideration of the following non-limiting examples. Reference is made to accompanying Figures 1 to 5 in which:
Figure 1 shows the fluorescence measured after treatment of HUVEC cells with danazol and trilostane IH as a measure of their ability to prevent endothelial cell invasion;
Figure 2 shows the OD levels measured after incubation of HUVEC cells with trilostane IEI as a measure of its ability to prevent initial proliferation of endothelial cells;
Figure 3 shows photographs of HlJVEC cells taken after incubation with trilostane HI as a measure of its ability to prevent tube formation of endothelial cells;
Figure 4 shows the OD levels measured after incubation of HUVEC cells with
danazol as a measure of its ability to prevent initial proliferation of endothelial cells; and
Figure 5 shows photographs of HUVEC cells taken after incubation with danazol as a measure of its ability to prevent tube formation of endothelial cells.
Example 1 Trilostane IH and Danzol Effect on HUVEC Angiogenesis: Invasion Chamber
Purpose:
To examine the effect of trilostane III and danazol on fetal calf serum induced endothelial cells invasion through matrigel treated inserts.
Materials:
• Passage 5 Human umbilical vein endothelial cells 7016 (HUVEC), Cambrex
• EGM-2 medium, Cambrex: supplemented to include 0.1% or 5% fetal calf serum
• 10 mM LY294002 and LY303511 in DMSO, CalBiochem
• 50 mM trilostane El in ethanol, Bowman Research, Newport, South Wales, UK (prepared as, for example, described in GB 1,123,770)
• 50 mM danazol in ethanol, Sigma
• 4 mM Calcein AM in DMSO, Sigma
• Hepes buffered saline solution (HBSS), Cambrex
• BD Biocoat Mangel Invasion Chamber, BD Biosciences
• microplate fluorescence reader
Protocol: in short
L Trypsinized HUVEC cells from flasks grown in Cambrex EGM-2 media to 70-80% confluence were washed two times with 37 °C EGM-2 with 0.1% FCS.
2. Cell suspensions containing 30,000 cells and compounds in EGM-2 0.1% FCS were added to the upper chamber of inserts.
3. EGM-2 containing 5% FCS was added to the bottom chamber and then incubated for 24 hours at 37 °C and 5% CO2.
4. Non-invading cells were removed from the upper chamber with cotton swab and the inserts were washed two times with 37 ° C HBSS.
5. Inserts were then placed in wells containing 10 μM Calcein AM in HBSS.
6. Following four hours at 37 °C and 5% CO2, fluorescence was measured at 485 nm excitation and 595 nm emission.
Results and Observations:
Results presented as mean fluorescent units (FU) in triplicate (n=3) with background fluorescence subtracted. HUVEC 7016 cells were used for this experiment exhibiting 95% viability by trypan blue exclusion at time of seeding. To determine background invasion, nil inserts were included in triplicate that had EGM-2 with 0.1% FCS added to the bottom chamber. Without a chemotactic signal, these inserts will give a background invasion to compare the FCS and FCS plus compound wells. The results are as shown in Table 1 below and depicted graphically in Figure 1.
Table 1
Mean std SEM p values
Sample FU FU FU vs 5% FCS
Nil 9034 1688.2 974.687 0.024186
5% FCS 12039.7 764.86 441.591
50 uM Danazol 8867.67 743.84 429.454 0.003373
25 uM Danazol 10067 552.21 318.818 0.01116
50 uM Trilostane 9101.33 540.28 311.928 0.002781
25 uM Trilostane 11212.3 2748.7 1586.94 0.320938
5O uM LY 303511 12795.3 609.45 351.867 0.125899
5O uM LY 294002 9454.33 1877.9 1084.18 0.045893
Discussion and Conclusion:
Treatment with 25 μM danazol lead to a significant decrease in endothelial cell invasion into the lower chamber. At 50 μM danazol, the invasion was reduced to
background levels similar to wells without fetal calf serum added to initiate invasion. Trilostane IE may have been less effective then danazol, but the high dose did drop the levels back to background. LY294002, a known inhibitor of invasion, was included as a control and exhibited similar inhibition as 50 μM danazol and trilostane. LY303511 is an inactive variant of the LY294002 and had no effect on invasion as expected, In conclusion, it would seem that danazol and trilostane IE may have an effect on endothelial cell invasion in certain individuals or at the appropriate time in the cell cycle.
Example 2 Trilostane III Effect on HUVEC Cell Proliferation
Purpose:
To determine the effect trilostane III has on HUVEC cell proliferation.
Materials:
Passage 2 Human umbilical vein endothelial cells (HUVEC), Cambrex
EGM-2 medium supplemented to include 0.1% and 5% fetal calf serum, Cambrex • 50 mM trilostane III in ethanol, Bowman Research, Newport, South Wales, UK (prepared as, for example, described in GB 1,123,770)
Hepes buffered saline solution (HBSS), Cambrex
Celltiter 96 Aqueous One reagent, Promega
Falcon 96 well tissue culture plates
Microplate fluorescence reader
Protocol:
1. HUVEC cells were plated in 96 well plates at 5,000 cells/cm2 and incubated for 24 hours at 37 °C and 5% CO2 in EGM-2 media.
2. Medium was aspirated and the cells were then washed two time with 37 °C HBSS.
3. EGM-2 containing 5% FCS with and without the compound (0.01 μM - 200 μM trilostane IU) was added to the wells and incubated for 24, 48, or 72 hours.
4. Cells were again washed to times with warm HBSS and then Celltiter reagent in EGM-2 with 0.1% FCS was added.
5. After 4 hours in culture the OD of each well was determine at 470 nm.
6. Repeat steps 4 and 5 for each time point.
Results and Observations:
Results are presented as mean OD of samples performed in triplicate (n=3) with mean blank OD subtracted. HUVEC 8750 cells were used for this experiment with 98% viability by trypan blue exclusion at time of seeding, Extra fetal calf serum (2% increased to 5%) was added to keep trilostane III in solution. The raw data are shown in Table 2 below and depicted graphically in Figure 2.
Table 2
Trilostane III
Sample 24 hour 48 hour 72 hour
Nil 0.414 0.629333 1.1135
0.1 uM 0.387333 0.656 0.918667
I uM 0.311 0.434667 0.387667 10 uM 0.246667 0.203 0.072
100 uM 0 .278667 0.214667 0.108
Trilostane III 24 hour results pvalue vs
Sample OD l OD 2 OD 3 std Nil
Nil 0.345 0.457 0.44 0.060357
0.1 uM 0.362 0.368 0.432 0.038799 0.277404
I uM 0.284 0.328 0.321 0.023643 0.025631
10 uM 0.18 0.267 0.293 0.059181 0.013283
100 uM 0.235 0.334 0.267 0.050521 0.020409
Trilostane III 48 hour results pvalue vs
Sample OD 1 OD 2 OD 3 std Nil
Nil 0.521 0.666 0.701 0.095438
0.1 uM 0.575 0.698 0.695 0.070164 0.358232
I uM 0.358 0.489 0.457 0.068296 0.022665
10 uM 0.159 0,208 0.242 0.041725 0.001045
100 uM 0.17 0.23 0.244 0.039311 0.001121
Trilostane III 72 hour results pvalue vs
Sample OD 1 OD 2 OD 3 std Nil
Nil 1.075 1.152 0.054447
0.1 uM 0.733 0.962 1.061 0.168239 0.113558
1 uM 0.387 0.437 0.339 0.049003 0.000285
10 uM 0.031 0.095 0.09 0.035595 5.8E-05
100 uM 0.092 0.119 0.113 0.014177 3.09E-05
Discussion and Conclusion:
Trilostane III proved effective at inhibiting endothelial cell proliferation. Concentrations as low as 1 μM (at 24 hours), exhibited statistically relevant decreases in cell proliferation. Increasing the dose to 100 μM, inhibited cultures by 33, 67, and 90 % at 24, 48, and 72 hours respectively. The initial seeding of 1,500 cells per well is not detectable by the celltiter assay. Cells must expand to detectable levels so 100% inhibition can be expected and would not infer cell toxicity. Viable cells were visible in all wells examined under the inverted microscope, even at the highest doses after 72 hours in culture. These results indicate that trilostane DI may be an effective an ti angiogenic compound, by interfering with the initial proliferation of endothelial cells.
Example 3 Trilostane III Effect on HUVEC Angiogenesis: Tube formation
Purpose:
To examine the effect of trilostane IH on the formation of tube-like structures by HUVEC cells in an extracellular matrix gel.
Materials:
• Passage 3 Human umbilical vein endothelial cells (HUVEC), Cambrex
• EGM-2 medium, Cambrex: supplemented to include 0.1% or 5% fetal calf serum
• 10 mM LY294002 and LY303511 in DMSO, CalBiochem
• 50 mM trilostane HI in ethanol, Bowman Research, Newport, South Wales, UK (prepared as, for example, described in GB 1,123,770)
• BD Biocoat Angiogenesis system: tube formation assay, BD Biosciences « Microscope with camera
Protocol: in short
1. Trypsinized HUVEC cells from flasks grown in Cambrex EGM-2 media to 70-80% confluence were washed two times with 37 °C EGM-2 with 0.1% FCS.
2. Cell suspensions containing 10,000 cells and compounds in both EGM-2 0.1% and 5% FCS were added per well then incubated for 18 hours at 37 °C and 5% CO2.
3. Following incubation, tube formations were photographed under microscope.
Results and Observations:
Passage 3 HUVEC 8750 cells were used for this experiment at 98 % viability by trypan blue exclusion at time of seeding. Few cells were obtained for this experiment but did not seem to interfere with the development of tubes. Pictures of representative wells for the compound and controls are shown in Figure 3.
Discussion and Conclusion:
The final step of angiogenesis is the formation of new vascular structures. HUVEC cells when grown in gels consisting of extracelluler matrix proteins, will exhibit a "latticework" of vacuoles that mimic the inner lumen of the capillary. Addition of fetal calf serum, or other angiogenic substances, will enhance the length and definition of these structures. Dosing with 50 μM trilostane HE led to a decrease in branching, vacuole formation, and increase in satellite cells. 50 μM LY294002, a PΪ3 kinase inhibitor known to interfere with tube formation, completely inhibited the development seen in the nil wells while the inactive form exhibited comparable tube formations to untreated cells. The inclusion of 5% fetal calf serum to the wells increased tube definition and vacuole formation. 50 μM trilostane UL and 50 μm LY294002 treatment greatly reduced tube formation in the presence of fetal calf serum. The untreated cells are far more susceptible to the effects of the control compounds and trilostane HE. In conclusion, trilostane HI appears to prohibit tube formation of HUVEC cells.
Example 4 Danazol Effect on HUVEC Cell Proliferation
Protocol:
Cryopreserved ampoules of passage 2 HUVEC cells were thawed and plated in 96 well tissue culture plates at 5,000 cells/cm2. A 50 mM stock solution of danazol was prepared in ethanol and the FCS in the medium was increased to 5% to keep danazol in solution. The cells were treated with medium containing final concentrations of danazol ranging from 0.1 to 100 μM in triplicates. 24, 48, and 72 hour incubations were performed and cell proliferation was determined utilizing Celltiter 96 AQUCOus One Solution Cell Proliferation assay from Promega (Madison, WI). In short, medium was aspirated from each well and the cells were washed with 200 μl Hepes buffered saline (HBSS) from Cambrex warmed to 37 °C. 100 μl diluted celltiter solution (15 μl stock + 85 μl EGM-2 containing 0.1% FCS) were added to each well and incubated for an additional 4 hours. Optical density was determined by microplate reader using a 530 nm
filter after blank subtraction and data presented as OD + standard deviation. The final concentration of ethanol in the wells was less then 0.2% and had no effect on cell proliferation or viability.
Results, Observations and Discussion:
Culturing primary HUVEC endothelial cells in the presence of danazol decreased the OD obtained from the Promega celltiter proliferation assay in a time and dose dependent fashion (Fig. 4). The celltiter assay is based on the reduction of the assay solution by dehydrogenase enzymes to a formazan dye that directly correlates to cell number. Danazol treatment at 24 hours seemed to be effective only at very high doses. Significant decreases (p value < 0.05) in assay OD were seen at 10 μM or greater concentrations of danazol. The OD detected in the nil wells was 0.414 + 0.06 and treatment with 10 μM danazol decreased the OD to 0.288 + 0.037 while 100 μM to 0.162 + 0.017, equating to percent inhibitons of 30% and 65% respectively. At 48 hours, the inhibition observed was significant even at physiological levels or approximately 1 μM. The nil reading obtained after 48 hours in culture increased to 0.629 + 0.095 and was reduced to 0.378 ± 0.037 by 1 μM, 0.241 ± 0.012 by 10 μM, and 0.19 μM ± 0.033 by 100 μM (or percent inhibitons of 40%, 61%, and 70% respectively). After 72 hours, all danazol treatments tested exhibited significant reduction in HUVEC proliferation. The OD obtained in nil wells was 1.113 ± 0.054 and after 0.1 μM treatment fell to 0.798 ± 0.037, 1 μM to 0.484 + 0.022, 10 μM to 0.229 ± 0.016, and 100 μM to 0.156 ± 0.018 (28%, 57%, 80%, and 86% respectively). Examination of the OD obtained from all 100 μM danazol doses was consistent at all time points indicating a complete arrest of cell proliferation at this concentration. In summary, danazol exhibited strong inhibition of endothelial cell proliferation.
Example 5 Danazol Effect on HUVEC Angiogenesis: Tube Formation
Protocol:
To investigate the formation of capillary-1ike structures by HUVEC cells, the Angiogenesis System: Endothelial Cell Tube Formation Assay was purchased from BD Biosciences and used according to the manufacturers protocol. In brief, 100,000 HUVEC cells were seeded onto rehydrated matrigel plugs in 96 well tissue culture plates in the presence of 5% FCS to induce tube formation. Danazol was added to final concentrations of 1 μM, 10 μM, or 100 μM and LY294002 was added at 100 μM. After 18 hours the wells were photographed using a Kodak DCS Pro SLR/N digital camera (Rochester, NY) mounted on an inverted microscope. Ethanol treated wells were included to determine if the vehicle had any effects on cell differentiation.
Results, Observations and Discussion:
HUVEC cells grown with danazol exhibited fewer organized structures with thin and less defined interconnections than controls (see Figure 5, in which A = control, B = I μM danazol, C = 10 μM danazol, D = 50 μM danazol, and E = 50 μM LY294002). Treatment with 50 μM danazol led to isolated colonies of HUVEC located in the plug with very few, thin connections or vessel lumen spaces. The effect of danazol was very similar to the positive control compound LY294002. To ensure that the vehicle used had no effect, wells were treated with ethanol at concentrations corresponding to the highest dose of danazol used and no effect on tube formation was observed (data not shown). This data would indicate that danazol is an effective inhibitor of tube formation.