WO1998046076A1

WO1998046076A1 - Retinoid related molecules for the inhibition of endothelin-1 overproduction in disease

Info

Publication number: WO1998046076A1
Application number: PCT/US1998/007125
Authority: WO
Inventors: Magnus Pfahl; Ju-Yu Hsu
Original assignee: Sidney Kimmel Cancer Center
Priority date: 1997-04-11
Filing date: 1998-04-10
Publication date: 1998-10-22
Also published as: AU6895198A; BR9808866A; JP2001522350A; EP0973390A1; EP0973390A4

Abstract

A method of inhibiting endothelin-1 in a subject comprising administering to the subject an inhibiting amount of a suitable retinoid or retinoid related molecule and a method of treating pain and diseases associated with the presence of increased levels of endothelin-1in subjects comprising administering an endothelin-1 inhibiting amount of a suitable retinoid or retinoid related molecule, are provided.

Description

RETINOID RELATED MOLECULES FOR THE INHIBITION OF ENDOTHELIN-1 OVERPRODUCTION IN DISEASE

ACKNOWLEDGMENTS

This invention was made with government support under Grant CA55681 awarded by the National Cancer Institute of the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to retinoid molecules that are potent inhibitors of endothelin-1 messenger RNA transcription and which thus can inhibit the production of endothelin-1. The invention has application to the treatment of various diseases associated with the presence and production of increased levels of endothelin- 1 , and the treatment of pain due to increased levels of endothelin-1.

BACKGROUND OF THE INVENTION

Endothelin-1 (ET-1) is a polypeptide with very potent vasoconstrictor activity (38). However, it is also known that elevated plasma ET-1 levels are found in a variety of diseases (39-50) including systemic sclerosis, Kawasaki disease, cardiogenic shock, acute myocardial infarction, advanced atherosclerosis, advanced prostate cancer, localized prostate cancer, hemangioendothelioma, pulmonary hypertension, sepsis, congestive heart failure and hepatorenal syndrome.

It has been discovered that ET-1 not only functions as a vasoconstrictor but that it also has other functions, serving, for instance, as a growth factor alone or in combination with other hormones and peptides, as in prostate cancer (51). In addition, ET-1 is related to the sorafotoxins, which are painful and lethal moieties in the venom of certain snakes of the genus Atractapsis (52,53). Similar to venom in a snake bite, recombinant ET-1 has been reported to produce intense pain in humans after the localized injection of high concentrations (54). Thus, for instance, in prostate cancer, a high localized concentration of ET-1 produced by the cancer cells is likely to be a major contributor to the high degree of pain associated with the late stage of the disease. Molecules that prevent or inhibit the production of ET-1 can therefore be expected to be useful for the treatment of all disease where ET-1 is overproduced and also in situations where ET-1 production or overproduction induces pain.

Retinoids, which regulate cell differentiation by modulating gene expression and are thus able to reverse the preneoplastic transformation of cells, have excellent potential as therapeutic agents for the treatment and prophylaxis of cancer (58,59). Retinoids, particularly retinoic acid (RA) analogs, have been used in the treatment of leukemia, mycosis fungoides, basal cell carcinoma, psoriasis and other hyperproliferative diseases of the skin (60). An increasing volume of data (1-5) suggests that retinoids (vitamin A derivatives and their synthetic analogs) are promising new therapeutics against proliferative diseases.

However, the systemic side effects of these compounds and their teratogenicity limit their utility. Side effects include, for example, bone remodeling, palmoplantar peeling, dermatitis, alopecia, hepatotoxicity and systemic toxicity (61,62).

The mechanism by which retinoids function has therefore attracted great interest, and many retinoids have been synthesized (6), with the goal of separating beneficial activities from undesirable ones. The lack of a detailed understanding of the mechanism of retinoid action, however, has previously inhibited progress in the development of retinoids for clinical usage. More recent advances made in the molecular analysis of their response pathways and the observation that receptor and pathway selective compounds can be defined (7-10) has greatly enhanced the possibility that retinoids with narrow biological activities - and thus reduced adverse effects - can be designed.

A class of retinoid compounds has been identified that surprisingly inhibits the expression of ET-1. This class of retinoids comprises numerous small molecules that are structurally related to all-trans retinoic acid (tRA) and 9-cis retinoic acid (9-cisRA). The retinoids are useful in the treatment of diseases or pain associated with the overproduction of ET- 1. Unlike other retinoids, the retinoid molecules of this invention have reduced side effects, which correlates with their reduced ability or inability to induce differentiation in F9 teratocarcinoma cells (ATCC accession No. CRL 1720) and S19 pluripotent teratocarcinoma cells (ATCC accession No. CRL 1825).

SUMMARY OF THE INVENTION In one embodiment, the invention provides a method of inhibiting ET-1 in a subject comprising administering to the subject an ET-1 inhibiting amount of a suitable retinoid.

In another embodiment, the invention provides a method of treating a disease associated with the presence of increased levels of ET-1 in a subject comprising administering to the subject an ET-1 inhibiting amount of a suitable retinoid.

In a further embodiment, the invention provides a method of treating pain associated with the presence of increased levels of ET-1 in a subject comprising administering to the subject an

ET-1 inhibiting amount of a suitable retinoid.

Additional advantages of the invention will be set forth in part in the description which follows and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION

The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention.

Before the present compounds, compositions and methods are disclosed and described, it is to be understood that this invention is not limited to specific synthetic methods, specific pharmaceutical carriers, or to particular pharmaceutical formulations or administration regimens, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an aromatic compound" includes mixtures of aromatic compounds, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

The term "alkyl" as used herein refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, w-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. Preferred alkyl groups herein contain from 1 to 12 carbon atoms. The term "lower alkyl" intends an alkyl group of from one to six carbon atoms, preferably from one to four carbon atoms, even more preferably one to two carbon atoms. The term "cycloalkyl" intends a cyclic alkyl group of from three to eight, preferably five or six carbon atoms.

The term "alkoxy" as used herein intends an alkyl group bound through a single, terminal ether linkage; that is, an "alkoxy" group may be defined as -OR where R is alkyl as defined above. A "lower alkoxy" group intends an alkoxy group containing from one to six, more preferably from one to four, carbon atoms.

The term "alkylene" as used herein refers to a difunctional saturated branched or unbranched hydrocarbon chain containing from 1 to 24 carbon atoms, and includes, for example, methylene (-CH₂-), ethylene (-CH₂-CH₂-), propylene (-CH₂-CH₂-CH₂-), 2-methylpropylene [-CH₂-CH(CH₃)-CH₂-], hexylene [-(CH₂)₆-] and the like. "Lower alkylene" refers to an alkylene group of from 1 to 6, more preferably from 1 to 4, carbon atoms. The term "cycloalkylene" as used herein refers to a cyclic alkylene group, typically a 5- or 6-membered ring. The term "alkene" as used herein intends a mono-unsaturated or di-unsaturated hydrocarbon group of 2 to 24 carbon atoms. Preferred groups within this class contain 2 to 12 carbon atoms. Asymmetric structures such as (AB)C-C(CD) are intended to include both the E and Z isomers. This may be presumed in structural formulae herein wherein an asymmetric alkene is present, or it may be explicitly indicated by the bond symbol ==.

The term "alkyne" as used herein intends a mono-unsaturated or di-unsaturated hydrocarbon group of 2 to 24 carbon atoms, having the generic formula CnH_2n-₂, and a structural formula containing a triple bond. Preferred groups within this class contain 2 to 12 carbon atoms.

The term "aryl" includes any compound derived from an aromatic hydrocarbon and includes, for example, benzene and naphthalene, and heteroaryls such as pyridine, furan, and pyrrole.

The term "heteroatom" as used herein refers to sulfur, oxygen, or nitrogen, and preferably sulfur or oxygen. The prefix "hetero-" as used herein refers to a compound having at least one heteroatom present in the structural unit, in substitution for a carbon atom. For example, heteroaryl refers to an aryl group having a heteroatom in the aryl ring.

The term "Me" is an abbreviation for methyl. "Ac" is an abbreviation for acetyl. "Ad" is an abbreviation for adamantyl.

A "radical having two bonding sites" refers to a moiety, bound to two other moieties through covalent bonding, but viewed in isolation as if the bonds between moieties had been broken and two unpaired electrons were available at different points on the radical for bonding with another suitable moiety or moieties. A "radical having two bonding sites" thus includes, for example, a divalent radical. A "backbone," when used to refer to a "radical having two bonding sites," means the shortest continuous chain of carbon atoms between the two bonding sites for the radical, wherein the carbon atoms in such chain may be substituted by heteroatoms or aryl moieties.

"Bonding by aryl or heteroaryl fusion" means bonding that occurs along one side each of two aryl or heteroaryl moieties. Bonding by aryl fusion would include, therefore, a naphthyl bond.

Diseases that are associated with the presence of increased or elevated levels of ET-1 include any diseases caused by the presence of increased or elevated levels of ET-1, as well as any diseases which cause an overproduction of ET-1. As used herein, "increased or elevated levels" of ET-1 means levels of ET-1 in a subject, as measured by protocols well known in the art, which are greater than normal levels of ET-1 for the subject, i.e., normal levels of ET-1 are those which are present in a subject in the absence of a disease or pain-associated condition. For example, normal plasma levels of ET-1 in human subjects are described in Nelson et al. (51).

Also as used herein, "overproduction of or "overproduced" ET-1 means the production of ET-1 in a subject in an amount greater than that which is normally produced in the subject in the absence of a disease or pain-associated condition.

"Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, the phrase "optionally substituted lower alkyl" means that the lower alkyl group may or may not be substituted and that the description includes both unsubstituted lower alkyl and lower alkyl where there is substitution.

By the term "effective amount" of a compound as provided herein is meant a nontoxic but sufficient amount of the compound to provide the desired regulation of gene expression or other activity. As will be pointed out below, the exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration and the like. Thus, it is not possible to specify an exact "effective amount." However, an appropriate effective amount may be determined by one of ordinary skill in the art using only routine experimentation.

By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

"Eliciting," "modulating" or "regulating" selective gene expression is intended to mean that a compound is capable of acting as an activator or an antagonist of gene expression by a particular mechanism, e.g., a ligand binding a receptor in the retinoic acid family.

To achieve the advantages, and in accordance with the purpose of the invention, as embodied and broadly described herein, the invention comprises in a first embodiment a method of inhibiting endothelin-1 in a subject comprising administering to the subject an ET-1 inhibiting amount of a suitable retinoid. The suitable retinoids of this invention function by binding via the retinoic acid receptors RAR , β or γ or RXRoc, β or γ. This class of suitable retinoidal molecules comprises potent inhibitors of ET-1 messenger RNA transcription, which effectively inhibit the production of ET-1. These molecules do not, however, possess the typical activities well known for classical retinoids, especially the ability to induce differentiation in various cell lines. In particular, the suitable retinoids of this invention are not able to induce differentiation in F9 teratocarcinoma cells and/or PI 9 pluripotent teratocarcinoma cells at a concentration of 10^" ⁶ M or less. In addition, the suitable retinoids of this invention do not exhibit typical retinoid toxicities in vivo and are therefore well tolerated in mammals and humans.

Suitable retinoids can be screened for the inhibition of ET-1 messenger RNA (mRNA) transcription by using a reverse transcription polymerase chain reaction (RT-PCR) assay. RNA is isolated from cells grown in culture in the absence or presence of the retinoid molecule to be tested. The cells can be any cell type in which one desires to examine ET-1 expression, including but not limited to vascular cells, myocardial cells and prostate cancer cells, as well as any cell type in which ET-1 is produced. RNA isolation is done following procedures well known in the art, or by using commercially available kits. The isolated RNA is subsequently incubated with reverse transcriptase, a DNA polymerase, suitable buffer and upstream and downstream primers specific for the ET-1 gene sequence. A reagent kit from, for example,

Promega, which contains all these ingredients, can be used. As a control, the same reaction can be carried out with primers specific for a housekeeping gene, such as GAPDH, which is not regulated by the activity of the retinoids. The RT-PCR reaction is carried out in a thermal cycler by procedures well known in the art. Subsequently, the amplified DNA samples are loaded onto a 2% agarose gel and are analyzed for inhibition of ET-1 mRNA transcription.

Retinoids which inhibit ET-1 mRNA transcription can also be assayed to determine the degree of induction of cell differentiation, in order to identify specific retinoids suitable for the invention. As previously noted, suitable retinoids are not able to induce differentiation in F9 teratocarcinoma cells and/or P 19 pluripotent teratocarcinoma cells at a concentration of 10⁶ M or less. This cell differentiation-inducing activity of the retinoids of the present invention is conveniently assayed in F9 teratocarcinoma cells and/or PI 9 pluripotent teratocarcinoma cells, in which differentiation is not induced or is only minimally induced by the retinoids of the present invention, as compared to the differentiation-inducing activity of all-trans retinoic acid. The differentiation-inducing activity can be measured by inspecting cells, after exposure to the compounds (at a concentration of 10^"6 M or less), for morphological changes (55). Alternatively, the differentiation-inducing activity can be assayed by measuring the inhibition of F9 and/or P19 cell proliferation upon exposure to the retinoids of this invention, on the premise that induction of differentiation in these cells is associated with a strong inhibition of cell proliferation. To measure cell proliferation, a standard colorimetric assay kit (MTT or MTS) from Promega or other suppliers can be used.

Suitable retinoids of the instant invention are sometimes referred to hereinafter as the inventive compounds. The inventive compounds are potent inhibitors of ET-1 mRNA transcription in at least one or more particular cell types. The inhibition of ET-1 transcription by these retinoids results in reduced levels of the ET-1 peptide, which has a beneficial effect on diseases where ET-1 is overproduced, or in alleviating pain due to elevated levels of ET-1. The inventive compounds can include certain known structural entities previously described as AP-1 selective retinoids (55) and also molecules previously reported as RXR selective compounds (56, 57), provided these compounds have ET-1 inhibiting activity. In addition, suitable retinoids include molecules whose selectivity has not been previously reported, which are discussed herein.

Particularly suitable retinoid compounds include compounds of the following structures A(i) - A(ii):

wherein: (a) Rj , is a compound of the following structures B(i) - B(iv):

(b) R_[ is a radical having two bonding sites, wherein bonding can occur at each of the two bonding sites, independently, by a single covalent bond or by aryl or heteroaryl fusion, (c) R₂, R₃, R₄, R₅, and R₁₀ are independently the same or different lower alkyl, (d) R^ is hydrogen or methyl, (e) R₇, R₈, and Rg are hydrogen, hydroxy, lower alkoxy, alkyl, or adamantyl, and (f) X and Y are, independently, a heteroatom. The symbol rm- , as used herein and throughout the application and claims, represents a single or double bond. The squiggly line (

), as used above and throughout the application and claims, represents a bonding site.

Suitable retinoid compounds also include the salt, amide, and ester of the foregoing compounds. In a particular embodiment of the invention, in formula B(i) for R_π the COOH is at the para position. In another particular embodiment, in formula B(iii) for R_{j h} the COOH is at the 2 position. R, is preferably a hydrocarbon or heterohydrocarbon having from one to five, preferably from one to three, carbon atoms, heteroatoms and/or aryl moieties in its backbone, with the proviso that there is no more than one aryl moiety in the backbone.

R_] is also preferably: (a) a heteroatom, methyl, methyl hydroxy, carbonyl, imine, heteroaryl, or a divalent methyl substituted with lower alkyl or methyl aryl through a double or single bond; (b) a compound of formula C(i):

wherein R¹ and R² are methyl or heteroatoms, optionally linked together to form a 3, 5, or 6 membered cycloaliphatic or heterocycloaliphatic ring; or (c) a compound of formula C(ii):

^ ^R3-_R ^ ^C(il)

wherein R³ is a heteroatom, methyl, methyl hydroxy, carbonyl, imine, heteroaryl, a divalent methyl substituted with lower alkyl or methyl aryl through a double or single bond; and R⁴ is a heteroatom, carbonyl, methoxycarbonyl, ethene, methoxy, acetylene, aryl, or carbonyl amine.

In yet another embodiment of the invention, R_j is a moiety selected from formulas C(iii) through C(xxiv):

_jT¹ C(viii)

o

O

^

C(xxii) C(xxiii)

R₁₂ and R₁₃ preferably are each, independently, lower alkyl, and more preferably, independently methyl, and X and Y are each, independently, a heteroatom.

The moieties represented by C(iii) through C(xxiv) are radicals having two bonding sites, wherein bonding can occur at each of the two bonding sites, independently, by a single covalent bond or by aryl or heteroaryl fusion. The bonding sites are represented by the squiggly line and, in those structures that have only one squiggly line, a second bonding site is along one side of the terminal aryl group. In structure C(xxii), for example, there is one bonding site represented by a squiggly line and an aryl group through which bonding occurs through aryl fusion.

Particularly suitable retinoids can also include compounds of the following structures D(i) through D(xxxx):

D(xviii)

D(xχiii)

D(xxxii)

D(xxxiii)

D(xxxiv)

D(xxxvii)

D(xxxix)

In the above structures D, the bond symbols — and = represent a bond terminated by a lower alkyl group, preferably methyl.

The invention also encompasses pharmaceutically acceptable nontoxic ester, amide, and salt derivatives of the inventive compounds containing a carboxylic acid moiety.

Pharmaceutically acceptable salts are prepared by treating the free acid with an appropriate amount of a pharmaceutically acceptable base. Representative pharmaceutically acceptable bases are ammonium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, and the like. The reaction is conducted in water, alone or in combination with an inert, water-miscible organic solvent, at a temperature of from about 0°C to about 100°C, preferably at room temperature. The molar ratio of retinoid to base used is chosen to provide the ratio desired for any particular salts. For preparing, for example, the ammonium salts of the free acid starting material, the starting material can be treated with approximately one equivalent of pharmaceutically acceptable base to yield a neutral salt. When calcium salts are prepared, approximately one-half a molar equivalent of base is used to yield a neutral salt, while for aluminum salts, approximately one-third a molar equivalent of base will be used.

Ester derivatives are typically prepared as precursors to the acid form of the compounds and accordingly may serve as prodrugs. Generally, these derivatives will be lower alkyl esters such as methyl, ethyl, and the like. Amide derivatives -(CO)NH₂, -(CO)NHR and -(CO)NR₂, where R is lower alkyl, may be prepared by reaction of reactive derivatives of the carboxylic acid-containing compound with ammonia or a substituted amine.

The present invention also provides for pharmaceutical compositions comprising the inventive compound and a pharmaceutically acceptable carrier. Preferred is a pharmaceutical composition for inhibiting ET-1 mRNA transcription comprising a therapeutic amount of the inventive compound and a pharmaceutically acceptable carrier.

Synthetic Methods:

The compounds of the invention may be readily synthesized using techniques generally known to synthetic organic chemists. General suitable experimental methods for making and derivatizing the compounds are described (see, for example, refs 63-72, the disclosures of which are herein incorporated by reference). Utility and Administration:

The compounds of the invention, including the pharmacologically acceptable esters, amides or salts thereof, are useful in many contemplated applications. A principal use for the compounds and methods of the present invention is in the prevention and treatment of various diseases, including the pain associated with various diseases. Such diseases include, for example, systemic sclerosis, Kawasaki disease, cardiogenic shock, acute myocardial infarction, advanced atherosclerosis, advanced prostate cancer, localized prostate cancer, hemangioendothelioma, pulmonary hypertension, sepsis, congestive heart failure and hepatorenal syndrome. Another use for the retinoids of this invention is in the treatment of pain associated with the presence of increased levels of ET-1.

The compounds and methods of the present invention also permit the identification of therapeutic compounds which can be administered to inhibit ET-1 expression. Specifically, the present invention provides a method for screening a retinoid for ET- 1 production inhibiting activity comprising contacting the retinoid with cells producing ET-1 and assaying the cells for ET-1 production, a decrease in ET-1 production as compared to ET-1 producing cells not contacted with the retinoid indicating a retinoid having ET-1 production inhibiting activity. ET- 1 production can be assayed by measuring the amount of ET-1 secreted into the cell culture supernatant according to protocols standard in the art, as well as by assaying the amount of ET-1 mRNA transcribed by RT-PCR as described herein.

The present invention further provides a method for identifying a retinoid as suitable for treatment of a subject having a disease or pain-associated condition caused by overproduction of ET-1 comprising contacting the retinoid with cells producing ET-1 and assaying the cells for ET-

1 production and contacting the retinoid with cells which differentiate in the presence of retinoids (e.g., F9 teratocarcinoma cells and S19 teratocarcinoma pluripotent cells) and assaying the cells for differentiation, a decrease in ET-1 production as compared to ET-1 producing cells not contacted with the retinoid and the inability to induce differentiation in cells which differentiate in the presence of retinoids indicating a retinoid suitable for treatment of a subject having a disease or pain-associated condition caused by overproduction of ET-1. As described above, ET-1 production can be assayed by measuring the amount of ET-1 secreted into the cell culture supernatant according to protocols standard in the art, as well as by assaying the amount of ET-1 mRNA transcribed by RT-PCR as described herein. The inability of the retinoid to induce differentiation in cells which differentiate in the presence of retinoids can be determined according to the differentiation and/or proliferation assays described herein.

A variety of diseases and pain-associated conditions may thereby be treated with the retinoids identified by this method to be inventive compounds, especially those associated with the presence or expression of increased levels of ET-1. Inhibition of ET-1 mRNA transcription will result in reduced levels of the ET-1 peptide, which has a beneficial effect on the diseases or pain situations mentioned above where ET-1 is overproduced.

There are several particular embodiments of the invention related to particular diseases that should be mentioned. For example, when treating prostate cancer, one preferably uses a retinoid selected from structures D(i) - D(xxii) and D(xxxv). Even more preferably, one uses a retinoid selected from structures D(i) - D(v), D(vii), D(xiv), D(xix) - D(xxi), and D(xxv). Still even more preferably, one uses the retinoid of structure D(xxxvii).

When treating ovarian cancer the retinoid is preferably selected from structures D(i) - D(xxi) and D(xxxv), and even more preferably selected from structures D(i) - D(v), D(vii), D(xiv), D(xix) - D(xxi), and D(xxv). When treating breast cancer the retinoid is preferably selected from structures D(i) - D(xiv), D(xvi) - D(xxi), and D(xxxv), even more preferably selected from D(i) - D(v), D(vii), D(xiv), D(xix) - D(xxi), and D(xxv), and still even more preferably, structure D(xxxvii).

As previously noted, the method of the invention can also be used to treat heart related conditions such as cardiogenic shock, acute myocardial infarction, and congestive heart failure. The invention has particular application to the treatment of patients that have experienced a myocardial infarction, in order to inhibit chronic heart disease and/or myocardial hypertrophy. When treating heart disease the retinoid is preferably selected from structures D(xxiv) - D(xxxiv) and D(xxxvi) - D(xxxx). Most preferably the retinoid is structure D(xxxvii). The invention can also be characterized by excluding certain diseases from those which are known to be associated with the presence of increased levels of endothelin-1. For example, in one embodiment the invention provides a method of treating disease, other than cancer and heart disease, associated with the presence of increased levels of endothelin-1 by administering a suitable retinoid. In a preferred embodiment when using the method of the invention to treat a disease other than cancer or heart disease, the retinoid is preferably selected from structures D(i) - D(xxii) and D(xxxv). In a separate embodiment, when using the invention to treat a disease other than cancer of heart disease, the retinoid is preferably selected from structures D(xxiv) - D(xxxiv) and D(xxxvi) - D(xxxx).

In another particularly suitable embodiment the retinoid has structure A(ii) from above, wherein R₇ is adamantyl, R_g is alkoxy (preferably methoxy), R_*> is hydrogen, R, is structure C(ii), wherein R³ is carbonyl and R⁴ is a heteroatom, preferably sulfur, and Rj j is strucutre B(i), wherein the COOH is at the para position, and wherein the two ----- bonds are, independently, single and/or double bonds. Such embodiment is particularly suitable for treating heart disease, prostate cancer, ovarian cancer, and and/or breast cancer.

The retinoids of the present invention can be administered to any subject which produces ET-1. The subject can be a mammal and is most preferably a human. These and other uses of the present invention will be apparent to one skilled in the art.

The compounds of the invention may be conveniently formulated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable carrier. See, e.g., Remington's Pharmaceutical Sciences (74), which discloses typical carriers and conventional methods of preparing pharmaceutical compositions that may be used in conjunction with the preparation of formulations of the inventive compounds and which is incorporated by reference herein.

The compounds may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, topically, transdermally, or the like, although oral admimstration is preferred. The amount of active compound administered will, of course, be dependent on the subject being treated, the subject's weight, the manner of administration and the judgement of the prescribing physician. Generally, however, dosage will approximate that which is typical for the administration of retinoic acid and will preferably be in the range of about 0.5 μg/kg/day to 60 mg/kg/day for humans, and even more preferably in the range of about 2.0 g/kg/day to 30 mg/kg/day for humans. The exact dosage will be determined by the clinician administering the retinoid and will be optimized on the basis of bioavailability and the presence or absence of toxicity symptoms and other side effects for a given subject. Such toxicity symptoms and side effects can include, but are not limited to, nausea, adverse effects on bone growth; teratogenicity, headache, skin rashes and the like.

The retinoid can be administered therapeutically to the subject until symptoms of the disease or pain-associated condition subside or diminish or the retinoid can be administered prophylactically for an extended and indefinite period of time, for example, to prevent chronic heart disease. Various clinical parameters that can be monitored by the clinician in determining the efficacy of the retinoid in treating the disease or pain-associated condition can include, but are not limited to, decrease in symptomology (e.g., reduction in pain sensation; increased motor function or mobility; improved overall well being; change in weight or growth rate); decrease in plasma or serum ET-1 levels; decrease in levels of a specific tumor marker (e.g. prostatic serum antigen); reduction in tumor size, etc. These parameters are monitored and measured according to protocols well known in the art.

Suitable models for testing the efficacy of the retinoids of the present invention in treating or preventing disease or pain-associated conditions caused by increased levels of ET-1 include animal models for tumor growth and mortality caused by malignancy as well as animal models for acute and chronic heart disease. For example, the rat model of Sakai et al. (74), in which acute myocardial infarction is induced, can be used to study the effects of the inventive compounds on myocardial vascular tissue in these animals. As another example, an animal model for cancer can be used in the present invention. For example, an animal model in which tumor growth has been established can be assayed for reduction in tumor size upon administration of the retinoids of this invention or malignant cells can be introduced into an animal to which the retinoids have been administered and assays for survival or establishment of the malignant cells in the animal can be conducted.

In addition, the retinoids of the present invention can be administered to human subjects with cancer and other disease or pain-associated conditions caused by elevated levels of ET- 1. For example, the inventive compounds can be administered to human patients diagnosed with prostate cancer (51). The efficacy of the retinoids in these patients can be monitored by assaying such clinical parameters as prostatic serum antigen levels; ET-1 serum or plasma levels; tumor size reduction, weight loss or gain, etc. Such a patient would also be an appropriate subject for evaluating the pain reducing effects of administering the retinoids of the present invention, on the basis that prostate cancer patients, particularly in the later stages of the cancer, experience a significant degree of bone pain, which generally cannot be alleviated by conventional pain- relieving therapies. Thus, the efficacy of the inventive compounds in reducing pain in prostate cancer patients can also be determined according to standard methods known in the art for evaluating pain sensation.

Depending on the intended mode of administration, the pharmaceutical compositions may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, lotions, creams, gels, or the like, preferably in unit dosage form suitable for single admimstration of a precise dosage. The compositions will include, as noted above, an effective amount of the selected drug in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.

For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc., an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example see Remington 's Pharmaceutical Sciences (73) .

For oral administration, fine powders or granules may contain diluting, dispersing, and/or surface active agents, and may be presented in water or in a syrup, in capsules or sachets in the dry state, or in a nonaqueous solution or suspension wherein suspending agents may be included, in tablets wherein binders and lubricants may be included, or in a suspension in water or a syrup. Where desirable or necessary, flavoring, preserving, suspending, thickening, or emulsifying agents may be included. Tablets and granules are preferred oral administration forms, and these may be coated.

Parental admimstration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parental administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Patent No. 3,710,795, which is incorporated by reference herein.

For topical administration, liquids, suspension, lotions, creams, gels or the like may be used as long as the active compound can be delivered to the surface of the skin.

Throughout this application, various publications are listed or referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Although the present invention has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

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Claims

What is claimed is:

1. A method of inhibiting endothelin-1 in a subject comprising administering to the subject an endothelin-1 inhibiting amount of a suitable retinoid.

2. The method of Claim 1, wherein the suitable retinoid is a compound selected from the group of structures consisting of A(i) through A(ii) below:

wherein: a. R, j is a compound selected from the group of structures consisting of B(i) through B(iv) below:

b. Rj is a radical having two bonding sites, wherein bonding can occur at each of the two bonding sites, independently, by a single covalent bond or by aryl or heteroaryl fusion, c. R₂, R₃, R₄, R₅, and R₁₀ are independently the same or different lower alkyl, d. R_₅ is hydrogen or methyl, e. R₇, R₈, and Rg are hydrogen, hydroxy, lower alkoxy, alkyl, or adamantyl, and f. X and Y are, independently, a heteroatom,

or a salt, amide, or ester thereof.

3. The method of Claim 2 wherein Rj j is formula B(i) and, in formula B(i) for R-. _j , the COOH is at the para position.

4. The method of Claim 2, wherein R-. is a hydrocarbon or heterohydrocarbon having from one to five carbon atoms, heteroatoms and/or aryl moieties in its backbone, with the proviso that there is no more than one aryl moiety in the backbone.

5. The method of Claim 2, wherein R-. is:

(a) a heteroatom, methyl, methyl hydroxy, carbonyl, imine, heteroaryl, a divalent methyl substituted with lower alkyl or methyl aryl through a double or single bond;

(b) a compound of structure C(i),

wherein R¹ and R² are methyl or heteroatoms, optionally linked together to form a 3, 5, or 6 membered cycloaliphatic or heterocycloaliphatic ring; or or (c) a compound of structure C(ii),

R³ ^

^ C(ii)

^{^}R^4'

wherein R³ is a heteroatom, methyl, methyl hydroxy, carbonyl, imine, heteroaryl, or a divalent methyl substituted with lower alkyl or methyl aryl through a double or single bond; and R⁴ is a heteroatom, carbonyl, methoxycarbonyl, ethene, methoxy, acetylene, aryl, or carbonyl amine.

6. The method of Claim 2, wherein R is a moiety selected from structures C(iii) through C(xxiv):

l2

^R12

I C(ix) C(xi)

C(xxiii)

and R₁₂ and R₁₃ are each, independently, lower alkyl, and X and Y are each, independently, a heteroatom.

7. The method of Claim 1, wherein the suitable retinoid is a compound selected from D(i) through D(xxxx):

D(xviii)

D(xxiii)

OH

D(xxxii)

D(xx╧çiii)

D(xxxiv)

D(xxxvi)

D(xxxvii)

D(xxxviii)

D(xxxix)

whererin, the bond symbol _ or ΓÇö is terminated by lower alkyl.

8. The method of Claim 1 , wherein the subject is a mammal.

9. The method of Claim 8, wherein the mammal is a human.

10 A method of treating a disease associated with the presence of increased levels of endothelin-1 in a subject comprising administering to the subject an endothelin-1 inhibiting amount of a suitable retinoid.

11. The method of Claim 10, wherein the disease is systemic sclerosis, Kawasaki disease, cardiogenic shock, acute myocardial infarction, advanced atherosclerosis, advanced prostate cancer, localized prostate cancer, hemangioendothelioma, pulmonary hypertension, sepsis, congestive heart failure and/or hepatorenal syndrome.

12. The method of Claim 10, wherein the suitable retinoid is a compound selected from the group of structures consisting of A(i) through A(ii) below:

R] ! is a compound selected from the group of structures consisting of B(i) through B(iv) below:

b. R] is a radical having two bonding sites, wherein bonding can occur at each of the two bonding sites, independently, by a single covalent bond or by aryl or heteroaryl fusion, c. R₂, R , R , R₅, and R_]0 are independently the same or different lower alkyl, d. R₅ is hydrogen or methyl, e. R₇, R₈, and R^ are hydrogen, hydroxy, lower alkoxy, alkyl, or adamantyl, and f. X and Y are, independently, a heteroatom,

or a salt, amide, or ester thereof.

13. The method of Claim 12, wherein R_u is formula B(i), and in formula B(i) for R_n, the COOH is in the para position.

14. The method of Claim 12, wherein Rj is a hydrocarbon or heterohydrocarbon having from one to five carbon atoms, heteroatoms and/or aryl moieties in its backbone, with the proviso that there is no more than one aryl moiety in the backbone.

15. The method of Claim 12, wherein R, is:

(b) a compound of structure C(i),

R3 ^^" c^ _R4' C(ii)

16. The method of Claim 12, wherein Rj is a moiety selected from structures C(iii) through C(xxiv):

O OH

C(iii) C(iv) O

^ C(v) ^* \ -r^

R 12 ┬░ 12 ^R13 _jj^ C(vi) A C(vii) _C ^╬║ C(viii)

-r^ -^ X

O

C(xviii)

O

^ '

C(xxiii)

and R₁₂ and R₁₃ are each, inpependently, lower alkyl, and X and Y are each, independently, a heteroatom.

17. The method of Claim 10, wherein the suitable retinoid is a compound selected from structures D(i) through D(xxxx):

D(xviii)

D(xxiii)

D(x╧ç╧çii)

D(xx╧çiii)

D(xxxiv)

D(xxx╧çvi)

D(xxxvii)

D(xxxviii)

D(xxxix)

wherein the bond symbol - and/or = are terminated by lower alkyl.

18. The method of Claim 10, wherein the subject is a mammal.

19. The method of Claim 18, wherein the mammal is a human.

20. A method of treating pain associated with the presence of increased levels of endothelin-1 in a subject comprising administering to the subject an endothelin-1 inhibiting amount of a suitable retinoid.

21. The method of Claim 20, wherein the suitable retinoid is a compound selected from the group of structures consisting of A(i) through A(ii) below:

wherein:

(a) R, , is a compound of the following structures B(i) - B(iv):

b. Rj is a radical having two bonding sites, wherein bonding can occur at each of the two bonding sites, independently, by a single covalent bond or by aryl or heteroaryl fusion, c. R₂, R₃, R₄, R₅, and R₁₀ are independently the same or different lower alkyl, d. R₆ is hydrogen or methyl, e. R₇, R₈, and R₉ are hydrogen, hydroxy, lower alkoxy, alkyl, or adamantyl, and f. X and Y are, independently, a heteroatom, or a salt, amide, or ester thereof.

22. The method of Claim 21, wherein R_j , is formula B(i) and, in formula B(i) for R_╧Ç, the COOH is at the para position.

23. The method of Claim 21 wherein Rj is a hydrocarbon or heterohydrocarbon having from one to five carbon atoms, heteroatoms and/or aryl moieties in its backbone, with the proviso that there is no more than one aryl moiety in the backbone.

24. The method of Claim 21 , wherein R_[ is:

(b) a compound of structure C(i),

wherein R¹ and R² are methyl or heteroatoms, optionally linked together to form a 3, 5, or 6 membered cycloaliphatic or heterocycloaliphatic ring; or (c) a compound of structure C(ii),

^ ^R3^_R4^ ^C(")

wherein R³ is a heteroatom, methyl, methyl hydroxy, carbonyl, imine, heteroaryl, or a divalent methyl substituted with lower alkyl or methyl aryl through a double or single bond; and R⁴ is a heteroatom, carbonyl,, methoxycarbonyl, ethene, methoxy, acetylene, aryl, or carbonyl amine.

25. The method of Claim 21, wherein R, is a moiety selected from structures C(iii) through C(xxiv):

O OH

C(iii) C(iv) O _j^ X C(v) ^ΓÇó \ J^ X

R 12 ^ 12\ .^R13 _jj^ X C(vi) C(vii) C(viii) j^ x ^ X

O

O

^

C(xxiii)

26. The method of Claim 20, wherein the suitable retinoid is a compound selected from structures D(i) through D(xxxx):

D(xviii)

D(xxiii)

D(xxxii)

D(xxxiii)

D(xxxiv)

D(xxxvi)

D(xxxvii)

D(xxxviii)

D(xxxi╧ç)

wherein the bond symbol - and/or = are terminated by lower alkyl.

27. The method of Claim 20, wherein the subject is a mammal.

28. The method of Claim 27, wherein the mammal is a human.

29. The method of Claim 2 wherein R₂, R₃, R , and R₅ are all methyl.

30. The method of Claim 12 wherein R₂, R₃, R₄, and R₅ are all methyl.

31. The method of Claim 21 wherein R₂, R₃, R₄, and R₅ are all methyl.

32. The method of claim 17 for treating prostate cancer wherein the suitable retinoid is selected from structures D(i) - D(xxii) and D(xxxv).

33. The method of claim 17 for treating prostate cancer wherein the suitable retinoid is selected from structures D(i) - D(v), D(vii), D(xiv), D(xix) - D(xxi), and D(xxxv).

34. The method of claim 17 for treating prostate cancer wherein the suitable retinoid is structure D(xxxvii).

35. The method of claim 17 for treating ovarian cancer wherein the suitable retinoid is selected from structures D(i) - D(xxii) and D(xxxv).

36 The method of claim 17 for treating ovarian cancer wherein the suitable retinoid is selected from structures D(i) - D(v), D(vii), D(xiv), D(xix) - D(xxi), and D(xxxv).

37. The method of claim 17 for treating ovarian cancer wherein the suitable retinoid is structure D(xxxvii).

38. The method of claim 17 for treating breast cancer wherein the suitable retinoid is selected from structures D(i) - D(xiv), D(xvi) - D(xxii), and D(xxxv).

39. The method of claim 17 for treating breast cancer wherein the suitable retinoid is selected from structures D(i) - D(v), D(vii), D(xiv), D(xix) - D(xxi), and D(xxxv).

40. The method of claim 17 for treating breast cancer wherein the suitable retinoid is structure D(xxxvii).

41. The method of claim 17 for treating heart disease wherein the suitable retinoid is selected from structures D(xxiv) - D(xxxiv) and D(xxxvi) - D(xxxx).

42. The method of claim 17 for treating heart disease wherein the suitable retinoid is structure D(xxxvii).

43. The method of claim 17 for treating a disease, other than cancer or heart disease, associated with the presence of increased levels of endothelin-1 wherein the suitable retinoid is selected from structures D(i) - D(xxii) and D(xxxv).

44. The method of claim 17 for treating a disease, other than cancer or heart disease, associated with the presence of increased levels of endothelin-1 wherein the suitable retinoid is selected from structures D(xxiv) - D(xxxiv) and D(xxxvi) - D(xxxx).

45. The method of claim 10 for treating a disease other than cancer or heart disease.