EP0671005A1 - Rxr homodimer formation and bridged bicyclic aromatic compounds and their use in modulating gene expression - Google Patents
Rxr homodimer formation and bridged bicyclic aromatic compounds and their use in modulating gene expressionInfo
- Publication number
- EP0671005A1 EP0671005A1 EP94904805A EP94904805A EP0671005A1 EP 0671005 A1 EP0671005 A1 EP 0671005A1 EP 94904805 A EP94904805 A EP 94904805A EP 94904805 A EP94904805 A EP 94904805A EP 0671005 A1 EP0671005 A1 EP 0671005A1
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- European Patent Office
- Prior art keywords
- compound
- retinoid
- receptor
- homodimer
- structural formula
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- C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
- C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
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- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
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Definitions
- This invention relates generally to the regulation of gene expression by retinoid receptors, and more particularly relates to the retinoid x receptor and novel bridged bicyclic aromatic compounds that are useful in modulating gene expression by retinoic acid receptors, retinoid X receptors, vitamin D receptors and thyroid receptors.
- retinoids The vitamin A metabolite all-trans-retinoic acid (RA) and its natural and synthetic derivatives (retinoids) exert a broad range of biological effects 1,2 .
- retinoids are important therapeutics in the treatment of skin diseases and cancers 3'6 . Understanding how the multitude of retinoid actions can be mediated at the molecular level has been greatly enhanced by the cloning and characterization of specific nuclear receptors, the retinoic acid receptors (RARs) 7'12 and the retinoid X receptors (RXRs) 13'17 .
- RARs and RXRs are part of the steroid/thyroid hormone receptor superfamily 18,19 .
- Both types of receptors are encoded by three distinct genes, a, ⁇ , and y, from which, in the case of RARs, multiple isoforms can be generated 20'22 .
- RARs are specific to vertebrates, the RXRs have been well conserved from Drosophila to man 17,23 .
- RA stereoisomer 9-cis-RA binds with high affinity to RXRs 23,24 suggested a retinoid response pathway distinct from that of all-trans-RA.
- RARs require interaction with auxiliary receptors for effective DNA binding and function and that RXRs are such auxiliary receptors 15,16,26"29 .
- RXRs appear to function effectively only as heterodimeric RAR/RXR complexes, or in combination with comparable auxiliary proteins that still need to be identified.
- RXRs were shown to require RARs, thyroid hormone receptors (TRs), or Vitamin D 3 receptors (VDRs) for effective DNA binding 15,16,26'29 .
- RXRs appeared to be able to function predominantly if not exclusively as auxiliary receptors, thereby playing a crucial role in generating a high degree of diversity and specificity of transcriptional controls and mediating the highly pleiotropic effects of different hormones by increasing DNA affinity and specificity for at least 3 different classes of ligand- activated receptors.
- the present invention provides that RXRs form homodimers.
- the invention provides that these homodimers effectively bind to,specific response elements in the absence of auxiliary receptors and their DNA binding specificity is distinct from that of the RXR containing heterodimers.
- the invention demonstrates a novel mechanism for retinoid action by which a ligand induced-homodimer mediates a distinct retinoid response pathway. Additionally, ligands are provided which selectively activate RXR homodimer formation.
- the present invention also provides a new class of retinoids in the form of bridged bicyclic aromatic compounds as will be described in detail herein. These new compounds are useful for regulating and/or eliciting selective gene expression by receptors in the retinoic acid family, i.e., RARs, RXRs, vitamin D receptors (VDRs), and thyroid hormone receptors (THRs). While not wishing to be bound by theory, it is postulated that the presently disclosed and claimed compounds are effective in modulating gene expression by virtue of their capability of interacting with a receptor protein that binds to the aforementioned receptors.
- the compounds are also believed to be useful in modulating gene expression by inducing formation of RXR homodimers in addition to RAR-RXR, VDR-RXR and THR- RXR heterodimers.
- the novel compounds are thus useful for controlling cellular processes that are regulated by thyroid hormone, vitamin D, and retinoids such as 9-cis-retinoic acid, the natural ligand for RXR.
- retinoids such as 9-cis-retinoic acid, the natural ligand for RXR.
- acne, leukemia, psoriasis, and skin aging all of which are regulated by retinoic acid, may be treated using the compounds of the invention, as may bone calcification, regulated by vitamin D, and energy levels, regulated by thyroid hormone.
- the present compounds are believed to provide for substantially reduced side effects and teratogenicity.
- the invention provides a method of screening a substance for the ability to affect the formation of a retinoid X receptor homodimer comprising combining the substance and a solution containing retinoid X receptors and determining the presence of homodimer formation. Also provided is a method of screening a substance for an effect on a retinoid X receptor homodimer's ability to bind DNA comprising combining the substance with the homodimer and determining the effect of the compound on the homodimer's ability to bind DNA.
- a method of inhibiting an activity of a retinoid X receptor heterodimer comprising increasing the formation of a retinoid X receptor homodimer, thereby preventing the retinoid X receptor from forming a heterodimer and preventing the resulting heterodimer activity is also provided.
- a method of inhibiting an activity of a retinoid X receptor homodimer is also provided.
- a method of determining an increased probability of a pathology associated with retinoid X receptor homodimer formation and treating such pathology are further provided.
- a method of screening a response element for binding with a retinoid X receptor homodimer is provided.
- the invention provides methods of activating retinoid X receptor homodimer formation.
- the invention relates to novel compounds having the structural formula (I)
- R 1 is selected from the group consisting of lower alkyl and adamantyl
- R 2 is -0-R 6 or -S-R 6 where R 6 is lower alkyl; or where R 1 is ortho to R 2 , R 1 and R 2 may be linked together to form a 5- or 6-membered cycloalkylene ring, either unsubstituted or substituted with 1 to 4 lower alkyl groups, and optionally containing 1 or 2 heterocyclic atoms selected from the group consisting of 0, S and NR where R is hydrogen or lower alkyl, preferably adjacent to the aromatic ring;
- R 3 is selected from the group consisting of carbonyl
- X 1 and X 2 are independently selected from the group consisting of 0, S and methylene, wherein at least one of X 1 and X 2 is 0 or S, or wherein one of X 1 and X 2 is NR, and the other is methylene, is 2 or 3,
- R 6 , R 7 , R 8 and R 9 are independently hydrogen or lower alkyl, with the proviso that when n is 0, R 6 and R 7 are not both hydrogen and R 8 and R 9 are not both hydrogen, or R 8 and R 9 may be linked together to form a cycloalkylene ring containing 3 to 6 carbon atoms, and * represents the point of attachment of the R 3 substituent to the remainder of the molecule;
- R* is selected from the group consisting of
- R 10 is hydrogen or methyl
- 1 is 0 or 1
- ** represents the point of attachment of the R* substituent to the remainder of the molecule
- the invention also encompasses pharmaceutically acceptable esters, amides and salts of such compounds, as will be explained in detail, infra.
- the invention relates to pharmaceutical compositions containing the aforementioned compounds and to methods of using the compounds to modulate selective gene expression by a receptor in the retinoic acid family of receptors.
- Figure 1 shows 9-cis-retinoic acid induces RXR homodimer binding on TREpal .
- Lane 1 represents the nonspecific binding of unprogram ed reticulocyte lysate. Open triangles indicate the nonspecific complex observed with unprogrammed reticulocyte lysate. Solid triangles indicate the specific TR ⁇ -RXR heterodimer binding. Arrows indicate specific RXR ⁇ homodimer binding. The RXR ⁇ /RAR ⁇ heterodimer migrates at the same position as the RXR ⁇ homodimer. For comparison, the effect of 9-cis-RA on RAR ⁇ binding is shown.
- F-RXR Flag-RXR ⁇
- Flag eight-amino-acid epitope
- Lane 1 represents the nonspecific binding of unprogrammed reticulocyte lysate (open triangles). Arrows indicate the specific F-RXR ⁇ homodimer and RAR-RXR heterodimer binding. Diamonds indicate the anti-Flag antibody up-shifted F-RXR homodimer.
- Figure 2 shows the characterization of 9-cis-RA induced
- FIG. 1 shows 9-cis-RA induces RXR homodimer binding on
- RXR-specific response elements (a) Nuclear receptor binding elements used in this study. These oligonucleotides were synthesized with appropriate restriction sites at both ends as indicated by the small letters. Sequences that are closely related to the AGG/TTCA motif are indicated by arrows.
- Figure 4 shows response element specific binding of RXR homodimer.
- the effect of 9-cis-RA on RXR binding on RA specific response elements (a), T 3 specifip response elements (b), or estrogen specific response element (c) was analyzed by gel retardation assays as described in Figure la.
- the binding of RXR/RAR heterodimer (a), RXR/TR heterodimer (b) or estrogen receptor (c) is shown.
- Open triangles indicate the nonspecific binding of unprogrammed reticulocyte lysate. Solid triangle indicates the
- RAR/RXR heterodimer complex (a), TR/RXR heterodimer complex (b) or ER complexes (c).
- FIG. 5 shows RXR homodimerization occurs in solution.
- 35 S-labeled in vitro synthesized RXR ⁇ proteins were incubated with partially purified bacterially expressed Flag-RXR (F-RXR) (+) or similarly prepared glutathione transferase control protein (-) either in the presence or absence of response elements or chemical cross- linker DSP as indicated. After incubation, either anti-Flag antibody (F) or nonspecific preimmune serum (NI) was added. 10 "7 M 9-cis-RA was maintained during working process. The immune complexes were washed in the presence of 10 *7 M-cis-RA. boiled in SDS sample buffer and separated on a 10% SDS-PAGE. The 35 S-labeled in vitro synthesized RXR ⁇ protein is shown in the right panel.
- F-RXR Flag-RXR
- NI nonspecific preimmune serum
- FIG. 6 shows transcriptional activation of RXR and RAR ⁇ : RXR heterodimers by 9-cis-RA on natural response elements.
- CV-1 cells were cotransfected with 100 ng of the reporter plasmids (a) TREpal-tk- CAT (b) 0RARE-tk-CAT (c) ApoAI-RARE-tk-CAT and (d) CRBPI-RARE-tk-CAT and 5 ng of empty pECE expression vector, pECE-RXR ⁇ , pECE RAR ⁇ or combination of both as indicated.
- Transfected cells were treated with no hormone (open bars), 10 "7 M RA (shadowed bars) or 10" 7 M 9-cis-RA (dark shadowed bars). The results of a representative experiment performed in duplicate are shown.
- Figure 7 shows RXR -dependent transactivation of reporter constructs (a) TREpal-tk-CAT (10) or (b) CRBPII-tk-CAT (10) by
- alkyl refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t- butyl, octyl, decyl, tetradecyl , hexadecyl, eicosyl, tetracosyl and the like.
- Preferred alkyl groups herein contain 1 to 12 carbon atoms.
- the term “lower alkyl” intends an alkyl group of one to six carbon atoms, preferably one to four carbon atoms.
- cycloalkyl intends a cyclic alkyl group of three to eight, preferably five or six, carbon atoms.
- alkoxy 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 one to six, more preferably one to four, carbon atoms.
- alkylene refers to a di- functional saturated branched or unbranched hydrocarbon chain containing from 1 to 24 carbon atoms, and includes, for example, methylene (-CH 2 -), ethylene (-CH 2 -CH 2 -), propylene (-CH 2 -CH 2 -CH 2 -), 2- methylpropylene [-CH 2 -CH(CH 3 )-CH 2 -], hexylene [-(CH 2 ) 6 -] and the li e.
- “Lower alkylene” refers to an alkylene group of 1 to 6, more preferably 1 to 4, carbon atoms.
- cycloalkylene as used herein refers to a cyclic alkylene group, typically a 5- or 6-membered ring.
- an 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.
- 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 bicyclic 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.
- 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 bicyclic 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 receptor, i.e., a receptor in the retinoic acid family.
- retinoic acid family of receptors also termed “retinoid receptors,” is intended to encompass retinoic acid receptors, retinoid X receptors, vitamin D receptors and thyroid hormone receptors.
- retinoic acid receptors may also be loosely referred to herein as "retinoic acid receptors,” i.e., such that the term includes retinoid X receptors, vitamin D receptors and thyroid hormone receptors in addition to retinoic acid receptors themselves.
- “Bridged, bicyclic aromatic compounds” intends all compounds encompassed by the structure of formula (I). These compounds may also be termed “retinoids,” with that term further intended to include such species as retinoic acid and 9-cis retinoic acid.
- the invention provides a method of screening a substance for the ability to affect the formation of an RXR homodimer comprising combining the substance and a solution containing RXRs and determining the presence of a homodimer formation.
- the presence of homodimer formation can, for example, be determined by detecting the activation of transcription by the RXR homodimer or by coprecipitation.
- the affect can be the induction of homodimer formation, for example, an activity similar to that activated by 9-cis-RA or an activity which selectively activates homodimer formation over heterodimer formation.
- selectively activates is meant a compound which activates homodimer formation but does not significantly activate heterodimers.
- “selectively activates” includes compounds which activate heterodimer formation but do not significantly activate homodimer formation.
- “Significant activation” includes activation that is sufficient to cause a harmful pharmological effect on the subject. The affect can also be the inhibition of homodimer formation. Examples of inhibition include a substance which competes for 9-cis-RA binding to the receptor but itself does not activate or induce dimerization or which binds 9-cis-RA to block its activity.
- Such screening of substances is routinely carried out given the subject discovery of homodimer formation. In particular assays set forth below can generally be used for screening by merely substituting the substance of interest for 9-cis-RA.
- a good starting point for screening such "substances” is the activity of 9-cis-RA described herein.
- the substituents on 9-cis-RA can be varied to make 9-cis-RA analogs and screened in the method to determine any increase or decrease in homodimer formation.
- any substance can be screened in this assay to determine any affect on homodimer formation.
- Such compounds can then be used to promote homodimer formation and gene transcription in a cell.
- a cell as used herein includes cells found either in vitro or in vivo.
- the compounds can be administered to a human subject to effect RXR homodimer formation and promote transcription of a gene activated by an RXR homodimer.
- Such compounds are set forth in the Examples.
- the data set forth herein utilizes RXR ⁇ .
- each protein should form homodimers and have the activity described for RXR ⁇ homodimers.
- homodimers can form between different RXRs.
- homodimers can form between RXR ⁇ and RXR ⁇ or between RXR ⁇ and RXRp or between RXR ⁇ and RXRy. The activity of these homodimers can be confirmed using the methods set forth herein.
- the invention also provides a method of screening a substance for an effect on an RXR homodimer's ability to bind DNA comprising combining the substance with the homodimer and determining the effect of the compound on the homodimer's ability to bind DNA. For example, compounds which might bind the homodimer or bind the DNA response element recognized by an RXR homodimer can be screened in this method.
- the invention further provides a method of inhibiting an activity of an RXR-containing heterodimer comprising increasing the formation of an RXR homodimer, thereby preventing the RXR from forming a heterodimer and preventing the resulting heterodimer activity.
- the activity can be any activity but is generally the activation or repression of transcription.
- the activity can be blocked, for example, by utilizing RXRs to form homodimers which otherwise would be available to form heterodimers. Since the number of heterodimers are decreased, the activity of the heterodimers is decreased.
- the RXR heterodimer is comprised of thyroid hormone receptor and RXR. The activity of the RXR/TR heterodimer was decreased. Other heterodimers can be tested using standard methods given the teaching set forth herein.
- the invention also provides a method of inhibiting an activity of an RXR receptor homodimer comprising preventing the formation of the RXR homodimer.
- Such inhibition can be obtained, for example, by inhibiting 9-cis-RA or the transcription or activation by 9-cis-RA.
- the activity inhibited is generally the activation or repression of transcription.
- the invention also provides a method of inhibiting an activity of an RXR homodimer comprising preventing the binding of the RXR homodimer to its response element. For example, the activity of a receptor which competes for the same response element can be promoted. In general, the activity which is inhibited is the activation or repression of transcription.
- the invention still further provides a method of determining an increased probability of a pathology associated with RXR homodimer formation comprising detecting a modulation of RXR homodimer formation in the subject when compared to a normal subject.
- the modulation can be an increase or a decrease in homodimer formation. Such a modulation can result, for example, from a mutated RXR.
- the decrease can be detected by an assay for the homodimers in a sample or by detecting mutations known to decrease homodimer formation.
- the invention provides a method of treating a pathology associated with RXR homodimer formation in a subject comprising modulating homodimer formation in the subject.
- the modulation can be an increase or a decrease depending on the pathology. Such an increase can be accomplished, for example, utilizing compounds which promote RXR transcription.
- the pathology can be associated with the skin, e.g., acne and psoriasis.
- the pathology can be a cancer.
- the exact amount of such compounds 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 activity promoting amount. However, an appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
- the invention also provides a purified RXR homodimer.
- purified is meant free of at least some of the cellular components associated with RXR homodimers in a natural environment.
- the invention further provides a method of screening a response element for binding with a RXR homodimer comprising combining the response element with the RXR homodimer and detecting the presence of binding.
- the presence of binding can be determined by a number of standard methods.
- binding is detected by the transcriptional activation of a marker which is operably linked to the response element.
- operably linked is meant the marker can be transcribed in the presence of the transcriptional activator.
- the invention grows out of our study of the effects of the natural vitamin A derivative 9-cis-RA on retinoid receptor DNA binding and transcriptional activation.
- the 9- cis analog dramatically enhances RXR ⁇ binding at 10 "9 to 10" 8 M concentrations to several RXR-specific RAREs but not to natural TREs or the ERE.
- the effect is specific to RXR since 9-cis-RA did not induce binding of RAR ⁇ , ⁇ or y to response elements (Fig. 1, Fig. 3).
- RXR ⁇ homodimers exert response element specificity distinct from heterodimers.
- the rCRBPI response element did not interact with RXR homodimers, while the CRBPII response element, the only natural RARE identified so far that contains perfect repeats, was a strong binder of 9-cis-RA induced RXR ⁇ homodimers. It has been shown previously that this response element is well activated by RXR ⁇ 24,30 . Although this response element is also bound effectively by the RXR ⁇ -RARff heterodimer (Fig. 3) 27,29 , the heterodimer appears to have a repressor function 30 .
- RXRs exist mainly as monomer in solution 16 and require high concentrations or the presence of RARs, TRs or VDR to display effective DNA binding activity »- 15 - 16 - 26 - 30 .
- the observation of the enhanced cooperative RXR DNA binding activity in the presence of 9-cis-RA demonstrates that 9-cis-RA induced he formation of RXR homodimers which have an increased affinity for DNA.
- binding of 9-cis-RA to RXR caninduce a conformational change, which allows homodimerization to occur. It is interesting that although 9-cis-RA and RA can bind to RAR, 24,25 they do not induce RAR homodimer formation.
- RXR ⁇ homodimer formation can occur in solution in the absence of DNA.
- 9-cis-RA becomes available to cells, the equilibrium between monomeric and dimeric receptors is changed and an additional species, the RXR homodimer can be formed, allowing for novel response pathways.
- the concept of ligand-induced homodimer binding as observed by in vitro gel shift assay has not been previously observed for nuclear receptors with the exception of a mutated estrogen receptor (ER-val-400) 44,45 .
- RXRs The data presented in this application clearly demonstrate the central role of the RXRs, having dual functions that allow them to act as auxiliary receptors for three classes of hormone receptors, the RARs, TRs and VDRs through heterodimerization.
- 9-cis-RA can have therapeutic properties distinct from that of all-trans-RA.
- novel compounds provided herein are those defined by structural formula (I) above.
- Preferred compounds within this generic structure include
- R" is selected from the group consisting of 0, S, (CH 3 ) 2 C and CH 2 , and R 12 is hydrogen or methyl. Particularly preferred compounds within this group are as shown in structural formula (II).
- R 3 when R 3 has the general structure
- the invention also encompasses pharmaceutically acceptable nontoxic ester, amide and salt derivatives of those compounds of formula (I) containing a carboxylic acid moiety.
- Pharmaceutically acceptable salts are prepared by treating the free acid with an appropriate amount of 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 compounds of structural formula (I) to base used are chosen to provide the ratio desired for any particular salts.
- the ammonium salts of the free acid starting material a particular preferred embodiment herein—the starting material can be treated with approximately one equivalent of pharmaceutically acceptable base to yield a neutral salt.
- 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—as illustrated in the Examples below-- and accordingly may serve as prodrugs. Generally, these derivatives will be lower alkyl esters such as acetate, propionate, and the like.
- Amide derivatives -(C0)NH 2 , -(CO)NHR and -(C0)NR 2 , where R is lower alkyl, may be prepared by reaction of the carboxylic acid-containing compound with ammonia or a substituted amine (as illustrated in Example VII below). Synthetic Methods:
- the compounds of the invention may be readily synthesized using techniques generally known to synthetic organic chemists. Suitable experimental methods for making and derivatizing bridged bicyclic aromatic compounds are described, for example, in the Maignan et al. patents summarized above, the disclosures of which patents are hereby incorporated by reference. Methods for making specific and preferred compounds of the present invention are described in detail in Examples III - XVII below.
- the compounds of the invention defined by structural formula (I), including the pharmacologically acceptable esters, amides or salts thereof, are useful to elicit and/or regulate selective gene expression by receptors in the retinoic acid family and to control cell differentiation processes regulated by retinoids, vitamin D and/or thyroid hormone.
- the compounds of the invention are thus useful for treating acne, leukemia, psoriasis, skin aging, bone calcification and energy levels, as well as other indications related to cellular processes regulated by retinoic acid, vitamin D, thyroid hormone and 9-cis-retinoic acid.
- the compounds of the invention may be conveniently formu ⁇ lated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable carrier.
- a pharmaceutically acceptable carrier e.g., Remington's Pharmaceutical Sciences, latest edition, by E.W. Martin (Mack Publ. Co., Easton PA) 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.
- the compounds may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, topically, transdermally, or the like, although oral or topical administration is typically preferred.
- parenterally e.g., intravenously
- intramuscular injection e.g., intraperitoneal injection
- topically e.g., transdermally
- transdermally e.g., transdermally
- oral or topical administration is typically 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 judgment 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 2 ⁇ g/kg/day to 2 g/kg/day.
- 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 administration 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.
- 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 excipi- ent, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
- an excipi- ent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like
- 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.
- wetting or emulsifying agents such as sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine o
- 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.
- Parenteral administration if used, is generally charac ⁇ terized 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 parenteral 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, wiich is incorporated by reference herein.
- RXRs have been shown to bind RA response elements (RAREs) when used at high concentrations, 28,30,31 more recent investigations revealed that RXR exists mainly as monomers in solution 10 and that effective DNA interaction requires heterodimer formation with RARs or TRs or VDR 15,16,26"29 . Binding of the heterodimers to a variety of response elements was found to be ligand independent 32 .
- the newly discovered natural RA isomer, 9-cis-RA. has been reported to be an effective activator of RXRs in Drosophila Schneider cells that are known to contain neither RAR nor TRs 17,24 .
- Flag-RXR ⁇ was constructed as described previously 33 by ligation of a double-stranded oligonucleotide containing an ATG codon and a DNA sequence encoding Flag (Arg-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) [SEQ ID N0:1] to the N-terminus of RXR ⁇ . The fusion product was then cloned into pBluescript.
- RXR containing heterodimers have a highly specific interaction with various natural response elements in that TR-RXR heterodimers only bind strongly to TREs but not to RAREs, whereas the opposite is true for RAR-RXR heterodimers 15,32 .
- TR-RXR heterodimers only bind strongly to TREs but not to RAREs, whereas the opposite is true for RAR-RXR heterodimers 15,32 .
- Fig. 1 Gel retardation assays using in vitro synthesized receptor protein are described in the Fig. 1 legend. The following oligonucleotides and their complements were used as probes in Figs. 3 and 4.
- ApoAI-RARE a direct repeat response element with 2 bp spacer 31 , gatcAGGGCAGGGGTCAAGGGTTCAGTgatc [SEQ ID N0:2]
- CRBPII-RARE a direct repeat RXR-specific response element with 1 bp spacer 30 , gatcCAGGTCACAGGTCACAGGTCACAGTTCAAgatc [SEQ ID N0:3]
- BRARE a direct repeat of RA response element present in RAR ⁇ promoter 36,37 , gatctGTAGGGTTCACCGAAAGTTCACTCagatc [SEQ ID N0:4]
- CRBPI-RARE a direct repeat RA specific response element present in rat CRBPI promoter 38 , gatccAGGTCAA
- TREpal The sequence of TREpal [SEQ ID NO:11] is shown for comparison.
- ApoAI-RARE a direct repeat response element that contains a 2 bp spacer
- RXR-specific 31 The sequence of TREpal [SEQ ID NO:11] was shown for comparison.
- ApoAI-RARE a direct repeat response element that contains a 2 bp spacer
- RXR-specific 31 resultsed in a strong RXR complex in the presence of 9-cis-RA and to a lesser degree with RA (10 "6 M).
- the RXR-RAR heterodimer also bound effectively to this response element. Since the heterodimer complex migrated at the same position as RXR homodimers, the effect of 9-cis- RA on RXR-RAR heterodimers cannot be clearly determined.
- RAR homodimer binding was not induced by 9-cis-RA (Fig. 3b).
- RXR homodimer band was considerably weaker than the RAR-RXR heterodimer band at the protein concentrations used.
- another natural RARE derived from the rat CRBPI promoter 38 that like the ApoAI-RARE contains a 2 bp spacer, did not show any binding of RXR in the presence of 9-cis-RA (Fig. 4a), indicating that the actual sequence of the repeated core motif is critical for RXR homodimer binding.
- the DR-5- RARE, a perfect repeat element derived from the ⁇ -RARE 39 did not exhibit interaction with RXR in the presence of 9-cis-RA. while it interacted strongly with RXR when RARE was present (Fig. 4a).
- Flag-RXRtf was cloned in frame in the expression vector pGex 2T (Pharmacia) and was expressed in bacteria using the procedure provided by the manufacturer. Protein was partially purified on a prepacked glutathione sepharose 4B column (Pharmacia) and tested for its function by gel retardation assays and western blotting using anti-Flag antibody. Immunocoprecipitation assay was performed essentially as described 26 .
- the immune complexes were washed extensively with cold NET-N buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM DTT, 0.5% NP-40) containing 10' 7 M 9-cis-RA. boiled in SDS sample buffer and resolved by SDS-polyacrylamide gel electrophoresis. The gel was fixed, dried and visualized by autoradiography.
- the ApoAI element containing reporter was also very effectively activated by RXR in the presence of 9-cis-RA (Fig. 6c) while RA did not induce above the level obtained in the absence of RXR ⁇ . Similar to the TREpal, maximal activation was seen when both receptors RXR ⁇ and RAR ⁇ were cotransfected. Under these conditions RA also led to a strong activation. In contrast, the CRBPI element, where we did not observe DNA binding by RXR in the presence of 9-cis- RA, also was not activated by RXR and 9-cis-RA in the transient transfection studies (Fig. 6d) while RAR ⁇ alone led to significant activation that was mostly 9-cis-RA dependent. The heterodimer
- RAR ⁇ RXR ⁇ allowed maximal activation in the presence of 9-cis-RA. Not unexpectedly, no induction by RXR ⁇ and 9-cis-RA was observed on the MHC-TRE, the ME-TRE or the ERE. These in vivo analyses showed a very significant correlation to the results obtained with the in vitro DNA binding studies, in that strong activation by RXR ⁇ in the presence of 9-cis-RA is only observed on the response elements that strongly interact with the 9-cis-RA induced RXR ⁇ homodimer.
- CAT reported gene that is activated by a thyroid hormone receptor/RXR heterodimer in the presence of thyroid hormone (T 3 ) can be inhibited by adding 9-cis-RA. This type of inhibition is most easily measured by using a transfection assay.
- TREpal-tk-reporter gene in a transient transfection assay essentially as described 51 to evaluate compounds for induction of RXR activity. Briefly, CV-1 cells or Hep G2 cells were grown in DME medium supplemented with 10% fetal calf serum. Cells were plated at 1.0 x 10 s per well in a 24-well plate 16-24 h before transfection. In general, 100 ng of reporter plasmid, 150 ng of ⁇ -galactosidase expression vector (pCHHO, Pharmacia), and variable amounts of receptor expression vector were mixed with carrier DNA (pBluescript) to 1,000 ng of total DNA per well.
- carrier DNA pBluescript
- Chloramphenicol actyl transferase (CAT) activity was normalized for transfection efficiency by the corresponding ⁇ -galactosidase activity as previously described 52 .
- the TREpal represents a response element that is activated by both RAR/RXR heterodimers and RXR homodimers.
- the RXR expression vector is cotransfected with the TREpal-tk-reporter gene into CV-1 cells, all-trans-RA does not efficiently activate the reporter, whereas 9-cis-RA does. Evaluation of a series of retinoids indicated, that several showed activity with RXR.
- the activity rankings for this series of retinoids were the same for both the TREpal and CRBPII reporter genes.
- the ketal SR11237 was the most active, followed by the isopropylidenyl retinoid SR11217, the hemithioketal SR11235 and the thioketal SR11234.
- the dithiane SR11203 and dioxane SR11236 had the lowest activity.
- In vitro translated receptor receptor protein (1 to 5 ml depending on the translation efficiency) was incubated with the 32 P-labeled oligonucleotides in a 20-ml reaction mixture containing 10 mM Hepes buffer, pH 7.9, 50 mM KCI , 1 mM DTT, 2.5 mM MgCl, 10% glycerol, and 1 mg of poly(dl-dC) at 25°C for 20 minutes.
- Retinoids SR11217, and SR11237 induced RXR homodimer binding to the response element in a concentration-dependent manner.
- Retinoid 11203 which behaved as a weak activator in the transient transfection assays, also induced only weak RXR binding.
- SR11231 which did not activate the RXR homodimer was also not able to induce RXR homodimer binding. Similar results were obtained with the CRBPII-RARE and the ApoAI-RARE. We have thus defined here a class of synthetic retinoids that activate RXR ⁇ by inducing homodimer formation and binding to DNA.
- RXR-active compounds like 9-cis-RA. would also activate RAR/RXR heterodimers or whether they would be truly RXR selective.
- CBPI rat cytoplasmic retin I
- the four different reporter constructs were cotransfected with RAR ⁇ , RAR ⁇ , RXR ⁇ , or with RXR ⁇ and RAR ⁇ together 51 .
- the retinoids were analyzed at a concentration of 5 x 10 "7 M (a dose shown to yield almost full induction (Fig. 7)).
- CV-1 cells were cotransfected with 100 ng reporter plasmid a) CRBPI-tk-CAT, b) BRARE-tk-CAT, c) CRBPII-tk-CAT, and d) apoAI-tk-CAT. Retinoids were applied at 5 x 10" 7 M. Results of a representative experiment are shown.
- the RXR-specific retinoids behaved strikingly different from 9-cis-RA (or RA) in that they only activated RXR homodimers but not RAR/RXR heterodimers.
- 9-cis-RA both SR11217 and SR11237 were strong activators of the CRBPII-RARE (i.e. the RARE that is significantly activated only by the RXR homodimer).
- 9-cis-RA did not induce the CRBPI-RARE that is activated only by the RAR/RXR heterodimer.
- SR11217 and SR11237 behaved very similarly to 9-cis-RA on the CRBPII-RARE, they showed no response on the CRBPI-RARE, where 9-cis-RA is the optimal activator.
- the BRARE was slightly activated by SR11217 and SR11237, consistent with the relatively low affinity of RXR homodimers for this response element.
- the apoAI-RARE was most effectively activated by RAR/RXR heterodimers in the presence of 9-cis-RA.
- retinoids SR11217 and SR11237 were also seen in various other cell lines, including Hep G2 cells, where a particular high response was seen.
- RAR ⁇ and RAR ⁇ when cotransfected alone, were not activated significantly by any of the synthetic retinoids on any of the response elements tested. Similar negative results were obtained for RARy.
- RAR ⁇ and ⁇ are assumed to form heterodimers with endogenous RXR-like proteins in CV-1 cells, thus these heterodimers are also unresponsive to the synthetic retinoids.
- keto-ester 3 80 mg, 0.228
- ethylene glycol 1 L
- l,2-bis(trimethylsilyloxy)ethane 2 mL
- a catalytic amount of JJ-TSOH a catalytic amount of JJ-TSOH.
- the reaction mixture was heated at reflux for 4 h and then cooled to room temperature.
- the solution was poured into saturated aqueous NaHC0 3 and extracted with 40% ethyl acetate/hexane.
- the combined organic layers were dried over anhydrous MgS0 4 , filtered, and concentrated to afford a solid.
- the acid 9 was dissolved in dichloromethane (4 mL) under argon, and ammonia gas was condensed into the solution, which was stirred for 5 min at -33°C. The solution was warmed to room temperature for 20 min to evaporate the ammonia and concentrated to afford the ammonium salt .10 as a white powder (47 mg, 93%): m.p. 259-261°C. The structure of the product was also confirmed using IR, l NMR and mass spectroscopy. Exampl e VI
- keto-ester 3 (88 mg, 0.251) in 2 ml of benzene was added 2-mercaptoethanol (1 mL), and a catalytic amount of 2-Ts0H.
- the reaction mixture was heated at reflux overnight and then cooled to room temperature.
- the solution was poured into saturated aqueous NaHC0 3 and extracted with 40% ethyl acetate/hexane.
- the combined organic layers were dried over anhydrous MgS0 4 , filtered, and concentrated to afford a solid. Flash chromatography (50% CH 2 Cl 2 /hexane) yielded the desired ketal H as a white solid (0.09 g, 87%): m.p. 122-124oC; R f 0.24 (50% CH 2 Cl 2 /hexane) .
- the structure of the product was also confirmed using IR, W NMR and mass spectroscopy.
- keto-ester 3 150 mg, 0.428 mmol
- 1,3-propanediol 1.5 mL
- p_-Ts0H a catalytic amount of p_-Ts0H.
- the reaction mixture was heated at reflux overnight and then cooled to room temperature.
- the solution was poured into saturated aqueous NaHC0 3 and extracted with 40% ethyl acetate/hexane.
- the combined organic layers were dried over anhydrous MgS0 4 , filtered, and concentrated to afford a solid.
- Flash chromatography (50% CH 2 Cl 2 /hexane) yielded the desired ketal 13 as a white solid (0.164 g, 94%): m.p. 157-159oC; R f 0.24 (5% ethyl acetate/hexane).
- the structure of the product was also confirmed using IR, l H NMR and mass spectroscopy.
- Ammonia gas was condensed into the flask and the mixture was stirred for 5 min at -33oC.
- the solution was warmed to room temperature for 20 min to evaporate ammonia and concentrated to afford the ammonium salt 15 as a white powder (0.238 g, 97%): m.p. 228-230oC.
- the structure of the product was also confirmed using IR, Hi NMR and mass spectroscopy.
- keto-ester 42 (0.12 g, 0.33) in 2 mL of benzene was added ethylene gylcol (0.8 mL), l,2-bis(trimethylsilyloxy)ethane (2 mL) and a catalytic amount of ⁇ -Ts0H.
- the reaction mixture was heated at reflux for 2 days and then cooled to room temperature.
- the solution was poured into saturated aqueous NaHC0 3 and extracted with 40% ethyl acetate/hexane.
- the combined organic layers were dried over anhydrous MgS0 4 , filtered, and concentrated to afford a solid.
- the reaction mixture was diluted with 20% ethyl acetate/hexane and filtered through a plug of silica gel with 20% ethyl acetate/hexane. The solution was concentrated to afford a yellow gum. Chromatography (38% diehloromethane/hexane) yielded the mixture of 45 as a pale-yellow gum (51 mg, 50%): R f 0.43, 0.47 (40% CH 2 Cl 2 /hexane) .
- the ester 45b was hydrolyzed as above to give 25 mg of pale-yellow solid. Recrystallization from ethyl acetate afforded the acid 47 as a pale-yellow powder (21 mg, 20% overall yield): m.p.
- the olefin 48 (0.0105 g, 0.0314 mmol) was hydrogenated over 5% palladium on charcoal (1 mg) in 0.5 mL of ethanol at room temperature and atmospheric pressure. After one equivalent (0.7 mL) of hydrogen was taken up, the catalyst was removed by filtration through a small Celite pad. The solvent was removed in vacuo to give the crude acid as a white solid (0.019 g) . Recrystallization from benzene-hexane afforded the desired acid 49 as a white crystalline solid (0.0078 g, 74%): m.p. I86-I880C. The structure of the product was also confirmed using IR, Hi NMR and mass spectroscopy.
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
Abstract
Description
Claims
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US07/982,305 US5466861A (en) | 1992-11-25 | 1992-11-25 | Bridged bicyclic aromatic compounds and their use in modulating gene expression of retinoid receptors |
US982305 | 1992-11-25 | ||
US07/982,174 US5552271A (en) | 1992-06-16 | 1992-11-25 | RXR homodimer formation |
US982174 | 1992-11-25 | ||
PCT/US1993/011492 WO1994012880A2 (en) | 1992-11-25 | 1993-11-24 | Rxr homodimer formation and bridged bicyclic aromatic compounds and their use in modulating gene expression |
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AU (1) | AU700706B2 (en) |
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ATE307795T1 (en) * | 1992-04-22 | 2005-11-15 | Ligand Pharm Inc | COMPOUNDS WITH RETINOID X RECEPTOR SELECTIVITY |
US5780676A (en) * | 1992-04-22 | 1998-07-14 | Ligand Pharmaceuticals Incorporated | Compounds having selective activity for Retinoid X Receptors, and means for modulation of processes mediated by Retinoid X Receptors |
US5962731A (en) * | 1992-04-22 | 1999-10-05 | Ligand Pharmaceuticals Incorporated | Compounds having selective activity for retinoid X receptors, and means for modulation of processes mediated by retinoid X receptors |
JPH08506323A (en) * | 1992-11-25 | 1996-07-09 | ラ ホヤ キャンサー リサーチ ファウンデーション | RXR homodimer formation and bridged bicyclic aromatic compounds and their use in regulated gene expression |
US7115728B1 (en) | 1995-01-30 | 2006-10-03 | Ligand Pharmaceutical Incorporated | Human peroxisome proliferator activated receptor γ |
FR2733684B1 (en) * | 1995-05-03 | 1997-05-30 | Cird Galderma | USE OF RETINOIDS IN A COSMETIC COMPOSITION OR FOR THE MANUFACTURE OF A PHARMACEUTICAL COMPOSITION |
FR2735370B1 (en) * | 1995-06-19 | 1997-07-18 | Cird Galderma | METHOD FOR IDENTIFYING AGONIST COMPOUNDS OF RXRS RECEPTORS |
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AU8588798A (en) | 1997-07-25 | 1999-02-16 | Institut Pasteur | Human peroxisome proliferator activated receptor gamma (ppargamma) gene re gulatory sequences and uses therefor |
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JPH08506323A (en) | 1996-07-09 |
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