Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
  • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• This invention relates to screening for agents active on peroxisome proliferator activated receptor (PPA- R) and thyroid hormone receptor (TR) related molecules.
• PPA- R peroxisome proliferator activated receptor
• TR thyroid hormone receptor
• This invention also relates to the cloning and sequencing of a new human peroxisome proliferator activated receptor named hNUClB.
• Peroxisomes are subcellular organelles found in animals and plants. Peroxisomes contain enzymes for cholesterol and lipid metabolism and respiration.
• peroxisome proliferators induce the proliferation of peroxisomes.
• Peroxisome proliferators include unsaturated fatty acids, hypolipidemic drugs (Reddy, J. K. , and Azarnoff, D. L.
• Hypolipidemic drugs such as clofibrates have been found to lower triglycerides and cholesterol levels in plasma and to be beneficial in the prevention of ischemic heart disease in individuals with elevated levels of cholesterol (Havel,
• mPPAR mouse peroxisome proliferator activated receptor
• cDNA mouse liver complementary DNA
• rPPAR rat peroxisome proliferator activated receptor
• XPPAR ⁇ Three members of the Xenopus nuclear hormone receptor superfamily (i.e., XPPAR ⁇ , XPPAR ⁇ and XPPAR ⁇ ) have also been found to be structurally and functionally related to the mPPAR (Dreyer et al. , Cell 68:879-887, 1992) .
• Peroxisome proliferator activated receptor is a member of the steroid receptor family. PPAR is divided into several subfamilies based upon their primary sequence homology. Fang et al., Biochem. Biophy. Res. Com. 196:671- 677, 1993 discussed XPPAR ⁇ , XPPAR/3, XPPAR ⁇ , mPPAR and hNUCl.
• This invention relates to the cloning, sequencing and expression of a human peroxisome proliferator activated receptor subtype, hNUClB.
• hNUClB differs in amino acid sequence from hNUCl by one amino acid (i.e., alanine at position 292) .
• hNUClB protein represses hPPAR ⁇ (hPPAR ⁇ , referred to as hPPARl in U.S. Application Serial No. 08/143,215, is a subtype of PPAR) and TR protein activity, and that relief from such repression is therapeutically useful.
• the present invention features methods for identifying therapeutic agents that alleviate the repressive effects of NUC protein on PPAR ⁇ protein and TR protein activity and for using these agents to treat diseases and pathological conditions affected by the level of NUC protein activity, such as, but not limited to, hyperlipidemia, hypercholesteremia and hyper ⁇ lipoproteinemia. These methods make it possible to screen large collections of natural, semisynthetic, or synthetic compounds for therapeutic agents that relieve the repression of PPAR ⁇ or TR activity by NUC protein.
• PPAR ⁇ protein is meant a PPAR subtype protein that is substantially homologous (i.e., no less than 90% homologous in amino acid sequence) to hPPAR ⁇ protein, including, but not limited to, hPPAR ⁇ .
• NUC protein is meant a PPAR subtype protein or a PPAR related protein that represses the transcription activation activity of PPAR ⁇ protein and/or TR protein, including, but not limited to, hNUClB protein, hNUCl protein and proteins with homologous sequences to hNUClB or hNUCl protein.
• This invention is also directed to compounds, compositions, and methods for modulating processes affected by NUC protein activity and useful for treating a patient exhibiting a pathological condition caused, induced or aggravated by the level of NUC protein activity. More particularly, the invention relates to compounds and pharmaceutical compositions that relieve the repression of PPAR ⁇ protein and TR protein activity by a NUC protein.
• the present invention features a method for identifying therapeutic agents for treatment of a pathological condition affected by the level of NUC protein activity, comprising the step of screening for an inhibitor of NUC protein activity.
• One method comprises identifying therapeutic agents which, when added to a system containing NUC protein and PPAR ⁇ protein, relieve the repression of PPAR ⁇ protein activity by NUC protein.
• this system further contains a reporter gene responsive to PPAR ⁇ protein activation, the reduction or relief of the repression of PPAR ⁇ by NUC protein is measured by the expression level of the reporter gene.
• reporter gene is meant a gene encoding a product that is easily detected and assayed by techniques known to those skilled in the art.
• a reporter gene in this invention is driven by a promoter that is responsive to PPAR ⁇ protein or TR protein, including, but not limited to, the native promoter of a gene such as acylcoenzyme A oxidase, enoyl-CoA hydratase/3-hydrosyacyl-CoA dehydrogenase bifunctional enzyme or 3-ketoacyl thiolase.
• the screening assay is conducted in a cell.
• NUC gene NUC gene
• PPAR ⁇ gene and the reporter gene are encoded in vectors and introduced into the cell by transfection.
• the reporter gene has a peroxisome proliferator responsive element (PPRE) and can be activated by PPAR ⁇ protein.
• PPRE peroxisome proliferator responsive element
• the screening assay is conducted in an extract of a cell by in vitro transcription.
• a PPAR activator is added to the screening assay.
• PPAR activator is meant a chemical agent that is capable of activating the transcription activation activity of PPAR ⁇ protein, such as, but not limited to, CFA (clofibric acid), ETYA (5, 8, 11, 14-eicosatetraynoic acid) or WY-14, 643 ( [4-chloro-6- (2,3-xylidino) -2- pyrimidinylthio] acetic acid) .
• CFA clofibric acid
• ETYA 5, 8, 11, 14-eicosatetraynoic acid
• WY-14, 643 [4-chloro-6- (2,3-xylidino) -2- pyrimidinylthio] acetic acid
• Another method comprises identifying therapeutic agents which, when added to a system containing NUC protein and TR protein, relieve the repression of TR protein activity by NUC protein.
• this system further contains a reporter gene responsive to TR protein activation, the repression or relief of the repression of TR by NUC is measured by the expression level of the reporter gene.
• the screening assay is conducted in a cell.
• NUC gene, TR gene and the reporter gene are encoded in vectors and introduced into the cell by transfection.
• the reporter gene has a thyroid hormone responsive element (TRE) and can be activated by TR protein.
• TREs include, but are not limited to, TREp (palindromic TRE) and DR4 (direct repeat with a 4 nucleotide spacing 5' -AGGTCACAGGAGGTCA-3' ) .
• the screening assay is conducted in an extract of a cell by in vitro transcription.
• TR activator is added to the screening assay to activate TR.
• TR activator is meant a chemical agent that is capable of activating the transcription activation activity of TR protein, such as, but not limited to, LT3 (3, 3' , 5-triiodo-L-thyronine) , LT4 (L-thyroxine) or Triac (3,3' , 5-triiodothyroacetic acid) .
• a third method comprises identifying agents which, when added to a system containing a NUC protein and a nucleic acid (such as an oligonucleotide) including a PPRE, reduce the binding of NUC protein to the nucleic acid.
• the level of binding can be detected in a gel retardation assay or other assays known to those skilled in the art.
• a fourth method comprises identifying agents which, when added to a system containing a NUC protein and a PPAR ⁇ protein, reduce the formation of NUC-PPAR ⁇ complexes.
• NUC protein is labeled and the formation of NUC-PPAR ⁇ complexes is measured by the amount of labeled NUC protein precipitated by PPAR ⁇ specific antibody.
• a fifth method comprises identifying agents which, when added to a system containing a NUC protein and a TR protein, reduce the formation of NUC-TR complexes.
• NUC protein is labeled and the formation of NUC-TR complexes is measured by the amount of labeled NUC protein precipitated by TR specific antibody.
• this invention features a method for treatment of a pathological condition affected by the level of NUC protein activity by providing an agent that represses or reduces the NUC protein activity.
• the pathological conditions treated by this method include, but are not limited to, hyperlipidemia, hypercholesteremia and hyperlipoproteinemia.
• This invention also relates to novel or unique therapeutic agents discovered by the above methods, i.e., agents that are not known per se or agents that are not already known for use related to treatment of a pathological condition affected by the level of NUC protein activity.
• Applicant is particularly interested in the identification of agents of low molecular weight (less than 10,000 daltons, preferably less than 5,000, and most preferably less than 1,000) which can be readily formulated as useful therapeutic agents.
• Such agents can then be screened to ensure that they are specific to tissues with pathological conditions induced or aggravated by NUC protein with little or no effect on healthy tissues such that the agents can be used in a therapeutic or prophylactic manner. If such agents have some effect on healthy tissues they may still be useful in therapeutic treatment, particularly in those diseases which are life threatening.
• the therapeutic agents discovered by the above assays can then be screened for tissue specificity and toxicity with methods known to those skilled in the art. They can be put in pharmaceutically acceptable formulations, and used for treatment of diseases and pathological conditions induced or aggravated by NUC protein activity.
• a NUC inhibitor can be used to study the mechanism of NUC inhibition of PPAR ⁇ and TR protein activity. Applying techniques known to those skilled in the art, such as those described in J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2 Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, a NUC inhibitor can also be used to study the structural changes in NUC protein when the inhibitor binds to the NUC protein, and how the binding affects the ability of the NUC protein to interact with other proteins and to bind to DNA.
• Figure 1 is a graph showing normalized luciferase activity.
• HepG2 cells were transfected with (A) vector pCMVhPPAR ⁇ , or pBKCMV (see “Vector construction” in the detailed description of the invention) and treated with CFA.
• Cells were transfected with pCMVhNUClB (see “Vector construction” in the detailed description of the invention) or vector and treated with CFA (B) or ETYA (C) and luciferase and /3-galactosidase assays performed as described in materials and methods.
• Figure 2 is a graph showing normalized response of reporter gene to increasing dose of hNUClB.
• HepG2 cells were transfected with 0.1 ⁇ g pCMVhPPAR ⁇ and increasing amounts of pCMVhNUClB (indicated in ⁇ gs) .
• CFA was added to a final concentration of 1 mM.
• Control cells received an equal volume of ethanol (vehicle) .
• Figure 3 is a graph showing normalized response of various receptors to hNUClB.
• HepG2 cells were transfected with (A) the ER expression vector HEO (l ⁇ g) and pLPwtCAT (see “Vector construction” in the detailed description of the invention) (1.5 ⁇ g) reporter (B) pRShRAR ⁇ (see “Vector construction” in the detailed description of the invention) and MTV-TREp2-LUC (C) pRShRAR ⁇ and CRBPII-tk-LUC (D) pRShTR ⁇ (see “Vector construction” in the detailed description of the invention) and MTV-TREp2-LUC. 0.1 ⁇ g of expression plasmid and 0.5 ⁇ g luciferase reporter were used (B-D) .
• pCMVhNUClB was co-transfected where indicated, l ⁇ g (A) and
• Figure 4 is a graph showing normalized response of hPPAR ⁇ to hNUClB with increasing ratio of hPPAR ⁇ to hNUClB.
• HepG2 cells were transfected with 0.05 ⁇ g of pCMVhNUClB and different amounts of pCMVhPPAR ⁇ plasmid
• CFA (indicated in ⁇ gs) .
• CFA was added to a final concentration of 1 mM.
• Figure 5 is a radiograph of a gel retardation assay. DNA binding assays were performed with extracts from
• hypolipidemic drugs like gemfibrozil that have significant cardio-protective effect are mediated via the PPARs.
• PPAR ⁇ and hNUClB two human PPAR subtypes, i.e., PPAR ⁇ and hNUClB.
• hNUClB is not activated by PPAR activators such as clofibric acid.
• hNUClB is a specific repressor of the transcriptional activation effected by PPAR ⁇ and thyroid hormone receptor.
• NUC protein The repressive action of NUC protein on PPAR ⁇ and TR receptors may limit the clinical efficacy of PPAR ⁇ and TR activators (e.g., fibrates, synthroid) . Agents that relieve this repression will increase activity of PPAR ⁇ and TR and increase the efficacy of existing drugs, or render these drugs unnecessary.
• a subtype of NUC protein, hNUCl has been shown to be present in the human heart, brain, and liver tissues where PPARs and TRs are active. Therefore, the screening methods of this invention and agents identified thereby may have widespread therapeutic significance. We have demonstrated co-operative binding of hNUClB and RXR ⁇ to a PPAR response element, PPRE.
• a candidate agent can be screened by either A) indirect evaluation of derepression of a PPAR ⁇ or TR responsive gene, B) direct evaluation of NUC protein binding to a PPAR ⁇ or TR responsive element, or C) direct evaluation of complex formation between NUC protein and PPAR ⁇ protein or TR protein.
• E TYA , jS - e s t r a d i o l , ATRA , LT 3 (3,3' , 5-triiodo-L-thyronine) and CFA were purchased from Sigma, and WY-14,643 from Chemsyn Science Laboratories, Lenexa, Kansas, USA. Stock solutions of these compounds were made in ethanol, methanol or dimethyl sulfoxide.
• hPPAR ⁇ cDNA was cloned into the NotI site of pBKCMV (Stratagene) to give pCMVhPPAR ⁇ .
• the hNUClB cDNA was directionally cloned into the Sall-SacII site of pBKCMV to give pCMVhNUClB.
• the reporter plasmid pPPREA3-tk-luc was generated by inserting three copies of the synthetic oligonucleotide (5'-CCCGAACGTGACCTTTGTCCTGGTCC-3' ) containing the "A" site of the Acyl-CoA oxidase gene regulatory sequence (Osumi, T., Wen, J. and Hashimoto, T. (1991) Biochem. Biophys. Res. Commun. 175, 866-871) into the Xhol site 5' of the tk promoter in the previously described pBLtk-luciferase vector (Giguere, V., Hollenberg, S. M. , Rosenfeld, M. G. , Evans, R. M.
• the human TR ⁇ l cDNA (Nakai, A., Sakurai, A., Bell, G.I., and DeGroot, L.J. (1988) Molec. Endoc. 2, 1087-1092) was liberated from pME21 by digestion with EcoRl and blunt ended by digestion with mung bean nuclease.
• pRS plasmid (Giguere, V., Hollenberg, S. M. , Rosenfeld, M. G. , Evans, R. M. (1986) Cell 46, 645-652) was digested with BamHl, dephosphorylated and repaired with Klenow enzyme. The TR ⁇ l cDNA was then joined to the vector by blunt end ligation.
• the ER expression plasmid HEO has been described in (Kumar, V. and Chambon, P. (1988) Cell 55, 145-156) .
• the estrogen inducible brain creatine kinase promoter was cloned into pUCPLCAT to give pLPwtCAT.
• HepG2 cells were grown in Dulbecco's modified
• DMEM Eagle's medium
• Hyclone fetal bovine serum
• 2 mM L-glutamine 2 mM L-glutamine
• 55 ⁇ g/ml gentamicin BioWhittaker
• Cells were plated at 2 x 10 5 cells per well for HepG2 in 12 well cell culture dishes (Costar) . The media was replaced with fresh media 20 hours later. After 4 hours, DNA was added by the calcium phosphate coprecipitation technique (Berger, T. S.,
• hNUClB plasmid plasmid dosage was kept constant by the addition of appropriate amounts of the empty expression vector pBKCMV. Total amount of DNA was kept at 20 ⁇ g by the addition of pGEM DNA (Promega) . After 14 hours the cells were washed with IX PBS and fresh media added (DMEM with 10% charcoal stripped fetal bovine serum (Hyclone) plus the above supplements) . Ligands or PPAR activators were added to the final concentrations indicated. Control cells were treated with vehicle.
• COS cells were transfected with 5 ⁇ g of pCMVhNUClB or pRShRXR ⁇ (Ptashne, M. (1988) Nature 335, 683-689) per 100 mm dish for 48 hours.
• Whole cell extracts were made by four cycles of freeze-thawing in 0.4 M KCl containing buffer followed by centrifugation. Gel retardations were performed by incubating 5 ⁇ g of cell extract in buffer containing 10 mM Hepes (7.8), 50 mM KCl, 1 mM DTT, 2.5 mM MgCl 2 , 0.5mg/ml dldC and 20% glycerol at 4°C for 5 minutes. About 100,000 cpm of 32 P-end-labeled probe was then added and incubated at 25°C for another 5 minutes.
• Protein-DNA complexes were resolved by electrophoresis on 5% polyacrylamide gels in 0.5X TBE.
• the PPRE sequence from the acyl-coenzymeA oxidase (AOX) gene used as probe is
• Example 1 Cloning of hPPAR ⁇ A human homologue of rat PPAR ⁇ was isolated from a human liver 5' -stretch lgtlO cDNA library (Clontech) . The library was screened at medium stringency (40% formamide, 5X SSC at 37°C) , with a rPPAR nick translated DNA fragment specific to the A/B and DNA binding domain (from the EcoRl to the Bglll site, nucleotides 450-909) (Gottmaschine, M. ,
• hPPAR ⁇ by CFA The activation profile of hPPAR ⁇ by CFA is shown in Fig. 1A.
• This receptor is also activated by other known activators of PPARs, e.g., WY-14,643 and ETYA in HepG2 and CV-1 cells.
• Example 2 Cloning of hNUClB hNUClB was isolated from a human kidney cDNA library by screening with a probe specific to the rat PPAR DNA binding domain (from the PvuII to the Bglll site, nucleotides 618-909, reference (Gott Anlagen, M. , Widmar, E., Li, Q., and Gustafsson, J. A. (1992) Proc. Natl. Acad. Sci. USA. 89, 4653-4657)) using procedures as described above in Example 1. A recombinant clone was isolated, subcloned into pGEM-5Zf and sequenced.
• hNUClB is a member of the PPAR family. hNUClB has 61% homology to hPPAR ⁇ and the two cysteine residues in the "D" box are separated by three amino acids (E, R and S, positions 112-114 of the amino acid sequence) . This is a characteristic of PPARs (Dreyer, C, Krey, G. , Hansjorg, K., Givel, F., Helftenbein, G., and Wahli, W. (1992) Cell 68, 879-887) . All the other nuclear receptors have five amino acids in the same region.
• the hNUClB protein unlike hPPAR ⁇ , is not transcriptionally activated in HepG2 or CV-1 cells by CFA, ETYA or WY-14,643 (Fig. IB, C) .
• the slight activation seen in the absence of transfected receptor is probably due to the endogenous PPARs in the cell line utilized. This has also been observed by Schmidt, A., N. Endo, S. J. Rutledge, R. Vogel, D. Shinar, and G. A. Rodan. (1992) Mol. Endocrinol. 6, 1634-1641 with hNUCl and certain fatty acids. This data suggests the absence of a PPAR activator inducible transactivation function in hNUClB.
• hNUClB did decrease the response from the endogenous PPARs. This suggested that hNUClB may act as a repressor of hPPAR function.
• Figure 4 shows that increasing ratio of hPPAR ⁇ to hNUClB overcame the repression by hNUClB.
• FIG. 2 shows a strong dose dependent repression of hPPAR ⁇ activity by hNUClB in the presence of CFA. Repression was 85% with 0.1 ⁇ g of cotransfected hNUClB plasmid. Repression by hNUCl was also observed on the rat PPAR (Gott Anlagen, M. , Widmar, E., Li, Q., and Gustafsson, J. A. (1992) Proc. Natl. Acad. Sci. USA. 89, 4653-4657) and on hPPAR ⁇ in the presence of ETYA and WY-14,643.
• hNUClB is a specific repressor of hPPAR ⁇
• hNUClB has minimal effect on activation of ER and RAR ⁇ by their respective ligands.
• hNUClB does not repress RXR ⁇ in the absence of CFA and only 25% repression was detected in its presence.
• hNUClB is not a general transcription repressor, but a dominant negative repressor of hPPAR ⁇ and hTR. Repression occurred in the absence of clofibric acid, but was enhanced in its presence.
• PPAR ⁇ and hNUClB or TR and hNUClB expressing plasmids will be contransfected into CV-1 (a monkey kidney cell line) or HepG2 (a human liver cell line) cells along with a reporter containing PPAR or TR binding elements (such as
• PPREs PPREs, or TREs
• a PPAR activator e.g., clofibiric acid, WY-14,643
• a TR activator e.g., LT3
• Clofibric acid or LT3 normally activate their respective receptors and will therefore give a strong signal. In the presence of hNUClB the signal will be very weak because of repression of these receptors by hNUClB.
• Example 5 Screening for hNUClB Inhibitors with Immuno- precipitation assay hNUClB may simply dimerize with PPAR ⁇ or TR to form an inactive heterodimer.
• To screen for agents that relieve the repression PPAR ⁇ and TR activity by hNUClB we will mix labeled hNUClB with unlabeled TR or PPAR ⁇ . TR or PPAR ⁇ specific antibodies will then be used to immunoprecipitate the hNUClB-TR or hNUClB-PPAR ⁇ complexes respectively. Test compounds will be added to this mix and only those that disrupt the formation of these hetero- dimers will be selected. These compounds will then be further tested by the methods described above to see if they relieve repression of PPAR and TR by hNUClB.
• Methods are provided for determining whether an agent active in any of the methods listed above has little or no effect on healthy cells. Such agents are then formulated in a pharmaceutically acceptable buffer or in buffers useful for standard animal tests.
• pharmaceutically acceptable buffer any buffer which can be used in a pharmaceutical composition prepared for storage and subsequent administration, which comprise a pharmaceutically effective amount of an agent as described herein in a pharmaceutically acceptable carrier or diluent.
• Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceu ⁇ tical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) .
• Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
• sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. Id. at 1449.
• antioxidants and suspending agents may be used. Id.
• Method 1 Putative NUC inhibitors are assessed for toxicity to cultured human cells. This assessment is based on the ability of living cells to reduce 2, 3, -bis [2-methoxy-4- nitro-5-sulphonylphenyl] -5- [ (phenylamino) carbonyl] -2H- tetrazolium hydroxide] otherwise referred to as XTT (Paull et al., J. Heterocyl. Chem. 25:763-767 (1987) ; Weislow et al., (1989), J. Natl. Cane. Inst. 81:577) .
• Viable mammalian cells are capable of reductive cleavage of an N-N bond in the tetrazole ring of XTT to form XTT formazan. Dead cells or cells with impaired energy metabolism are incapable of this cleavage reaction. The extent of the cleavage is directly proportional to the number of living cells tested.
• Cells from a human cell line such as HeLa cells are seeded at 10 3 per well in 0.1 ml of cell culture medium (Dulbecco's modified minimal essential medium supplemented with 10% fetal calf serum) in the wells of a 96 well microtiter plate. Cells are allowed to adhere to the plate by culture at 37 * C in an atmosphere of 95% air, 5% co 2 .
• cell culture medium Dulbecco's modified minimal essential medium supplemented with 10% fetal calf serum
• test substances are added in duplicate to wells at concentrations that represent eight half-decade log dilutions.
• solvent used to dissolve the test substance is added in duplicate to other wells.
• the culture of the cells is continued for a period of time, typically 24 hours.
• HeLa cells are grown in 96 well plates in Dulbecco's minimal essential medium supplemented with 10% fetal calf serum and 50 ⁇ g/ml penicillin and streptomycin. Cells are initially seeded at 10 3 cells/well, 0.1 ml/well. Cells are grown for 48 hrs without exposure to the NUC inhibitor, then medium is removed and varying dilutions of the NUC inhibitor prepared in complete medium are added to each well, with control wells receiving no NUC inhibitor.
• Medium is changed every 24 hrs and replaced with fresh medium containing the same concentration of the NUC inhibitors. Medium is then removed and replaced with complete medium without NUC inhibitor.
• the particular agent that affects hNUCl activity and the pathological condition of interest can be administered to a patient either by themselves, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient (s) .
• a therapeutically effective amount of a agent or agents such as these is administered.
• therapeutically effective amount is meant an amount that relieves (to some extent) one or more symptoms of the disease or condition in the patient. Additionally, by “therapeutically effective amount” is meant an amount that returns to normal, either partially or completely, physiological or biochemical parameters associated with or causative of a disease or condition.
• Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effec ⁇ tive in 50% of the population) .
• Each candidate compound is tested for its efficacy in relieving the repression of PPAR ⁇ and TR by hNUCl in cell lines, in animal models, and in controlled clinical studies using methods known to those skilled in the art and approved by the Food and Drug Administration, such as, but not limited to, those promulgated in the Federal Register 47 (no. 56) : 12558- 12564, March 23, 1982.
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
• Compounds which exhibit large therapeutic indices are preferred.
• the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
• the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
• the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
• the therapeutically effective dose can be estimated initially from cell culture assays.
• a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal disrup ⁇ tion of the protein complex, or a half-maximal inhibition of the cellular level and/or activity of a complex compo ⁇ nent) .
• IC 50 as determined in cell culture
• levels in plasma may be measured, for example, by HPLC.
• the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g. Fingl et al. , in The Pharmacological Basis of Therapeutics, 1975, Ch. 1 p. 1) . It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dys ⁇ functions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity) .
• the magnitude of an administrated dose in the management of the cardiovascular disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
• Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal admini ⁇ stration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intra- thecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few.
• the agents of the invention may be formulated in aqueous solutions, preferably in physio ⁇ logically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
• penetrants appropriate to the barrier to be permeated are used in the formula ⁇ tion.
• penetrants are generally known in the art.
• Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention.
• the compositions of the present invention in particular, those formulated as solutions, may be adminis ⁇ tered parenterally, such as by intravenous injection.
• the compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration.
• Such carriers enable the compounds of the invention to be formulated as tab ⁇ lets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
• Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvi- ronment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.
• compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
• these pharmaceutical compositions may contain suitable pharma- ceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceuti ⁇ cally.
• suitable pharma- ceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceuti ⁇ cally.
• the preparations formulated for oral administra- tion may be in the form of tablets, dragees, capsules, or solutions.
• compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspen- sions of the active compounds may be prepared as appropri ⁇ ate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
• Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
• the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
• compositions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
• suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxy- methylcellulose, and/or polyvinylpyrrolidone (PVP) .
• disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• Dragee cores are provided with suitable coatings.
• suitable coatings may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combina ⁇ tions of active compound doses.
• compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, option- ally, stabilizers.
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added.
• Ala Ala lie lie Leu Cys Gly Asp Arg Pro Gly Leu Met Asn Val Pro 355 360 365

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• 1995
  • 1995-06-29 EP EP95925440A patent/EP0769146A2/de not_active Withdrawn
  • 1995-06-29 CA CA002193685A patent/CA2193685A1/en not_active Abandoned
  • 1995-06-29 AU AU29571/95A patent/AU2957195A/en not_active Abandoned
  • 1995-06-29 JP JP8503950A patent/JPH10502454A/ja active Pending
  • 1995-06-29 WO PCT/US1995/008328 patent/WO1996001430A2/en not_active Application Discontinuation

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