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WO2000053174A1 - Modulation de l'expression de proteines au moyen de derives d'acide carbocyclique aryl alcenoique - Google Patents

Modulation de l'expression de proteines au moyen de derives d'acide carbocyclique aryl alcenoique Download PDF

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Publication number
WO2000053174A1
WO2000053174A1 PCT/US2000/006377 US0006377W WO0053174A1 WO 2000053174 A1 WO2000053174 A1 WO 2000053174A1 US 0006377 W US0006377 W US 0006377W WO 0053174 A1 WO0053174 A1 WO 0053174A1
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protein
optionally substituted
hsc70
compound
cftr
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WO2000053174A9 (fr
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Pamela L. Zeitlin
Saul Brusilow
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Johns Hopkins University
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Johns Hopkins University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 

Definitions

  • the present invention features compounds and methods for modulating protein expression and, more particularly, to the use of one or more of such compounds to treat a subject suffering from or susceptible to a condition facilitated by undesired protein expression.
  • Particular compounds of interest include carbocyclic aryl alkenoic acid derivatives.
  • the invention relates to methods for detecting and analyzing such derivatives for therapeutic capacity to treat such conditions.
  • Proteins help define biological systems particularly by influencing cell shape, structure and function. Proteins are generally made by steps that include transcription, translation, trafficking, and for some proteins, secretion or membrane targeting. The ability of the proteins to exist is impacted by degradative processes. See generally, Alberts, B. et al. (1989) in Molecular Biology of the Cell (2nd ed.) Garland Publishing, Inc. New York and London; and Stryer, L. (1988) in Biochemistry W.H. Freeman and Co. New York.
  • one approach has been to identify compounds that can modulate the expression of a particular protein.
  • a compound can be administered to a primate and especially a human subject to alter at least one synthetic or degradative step to treat a medical condition.
  • This strategy has been used to implement many successful therapies. See generally The Pharmacological Basis of Therapeutics (8th ed.) Gilman, A. et al. (eds.) McGraw-Hill Health Professionals Division, pp. 1264-1276, (1993).
  • CFTR cystic fibrosis transmembrane regulator
  • steps associated with protein trafficking and especially protein degradation have been disclosed. Some of these steps appear to implement various heat shock protein and/or ubiquitin-associated pathways. See e.g., Gething et al.. Nature, 355:33-45 (1992) and Chiang et al., Science. 246:382-385 (1989)
  • PBA or Buphenyl can be used to treat various medical conditions including CF.
  • This compound has been approved for the treatment of certain urea cycle disorders. There has been some discussion that this compound may also find use in the treatment of certain hemoglobinopathies, including sickle cell disease, thalassemias; as well as cancer.
  • 4-PBA can enhance fetal hemoglobin levels by transcriptional "up" regulation.
  • the effect has been reported to involve histone deacetylase. See e.g., Stamatoyannopoulos et al., Blood, 84:3198-3204 (1994); Lea et zl., Anticancer Res., 15:879-883 (1995).
  • the invention provides important carbocyclic aryl alkenoic acid derivatives that are sometimes referred to herein as "gene” or “protein” drugs.
  • gene or “protein” drugs.
  • This designation is meant to emphasize that the derivatives represent a new class of compounds that can modulate protein expression at one or more levels.
  • More particular compounds of the invention desirably modulate at least one of transcription, translation, or trafficking of a subject protein or group of such proteins.
  • trafficking is meant at least one cell pathway that has been reported to help manipulate protein (or proteins), generally in folded form, to achieve a biological objective. Examples include post-translational modification, protein degradation, secretion, and/or membrane targeting.
  • disorders treatable or preventable by the present compounds include those impacted or suspected of being impacted by incorrect (aberrant) protein folding. Such inappropriate folding (misfolding) can encompass all or part of the subject protein. More specific examples of such disorders include those afflicting or thought to inflict one or more of the nervous, hepatic, or respiratory systems.
  • lung diseases e.g., those associated with misfolding of surfactant proteins
  • lung diseases impacted by improper expression of transmembrane proteins including the cystic fibrosis transmembrane regulator (CFTR); liver diseases associated with improper protein trafficking; and brain diseases such as those associated with tangle bodies, e.g., neurofibrillary tangles.
  • CFTR cystic fibrosis transmembrane regulator
  • brain diseases such as those associated with tangle bodies, e.g., neurofibrillary tangles.
  • More specific brain disorders of interest include those manifested by genetic, infectious (e.g., a viral, bacterial or prion agent) or environmental factors. Other illustrations of such disorders or conditions are provided below.
  • Therapies of the invention are particularly effective for the treatment and/or prevention of undesired protein expression including tliose embodiments in which modulated protein expression is desired.
  • modulate or related phrase as it is used to reference protein expression is meant an increase or decrease in that expression relative to control (or baseline) expression.
  • Preferred therapies treat, prevent, delay the onset of, or reduce the severity of a targeted mammalian disease or condition.
  • Particular compounds of the present invention are derivatives of carbocyclic aryl alkenoic acid.
  • Such compounds provide a number of important advantages with respect to prior drugs, particularly derivatives of carbocyclic aryl butyric acid and especially phenyl derivatives of butyric acid, specifically 4-PBA.
  • the unsaturation associated with the present compounds can often assist solubility especially with those aqueous or semi-aqueous solvents typical of many pharmaceutical formulations.
  • many of the prior drugs do not have this unsaturation which may, with some solvents, help make those drugs more difficult to use or administer properly.
  • the therapeutic methods of the invention generally include administering to a subject, particularly a mammal such as a primate and especially a human, a therapeutically effective amount of a compound that can modulate protein expression. More particular compounds that suitably increase or decrease at least one of transcription or trafficking of the subject protein or group of proteins including degradation thereof with respect to control or baseline expression of that protein.
  • an administered compound modulates the expression of a subject protein by at least about 10%, preferably at least about 25%, in at least one of the standard in vitro assays disclosed herein.
  • Exemplary assays detect and preferably measure the protein or nucleic acid encoding same with respect to a suitable control.
  • the administered compound exhibit an IC50 of at least about 0.001 to about 10 mM in a standard in vitro assay for measuring heat shock protein 70 (hsc70).
  • more preferred compounds feature an IC 5 Q of about 500 ⁇ M or less, still more preferably an IC 50 of about 1 to about 10 ⁇ M or less in the standard in vitro hsp 70 assay.
  • IC 5 Q of about 500 ⁇ M or less
  • IC 50 of about 1 to about 10 ⁇ M or less in the standard in vitro hsp 70 assay.
  • Such compounds that inhibit hsc 70 protein expression, as determined by the assay will sometimes be referred to herein as "trafficking inhibitor compounds" or other similar term.
  • the carbocyclic aryl alkenoic acid derivatives can be selected for capacity to boost CFTR protein expression.
  • preferred compounds increase CFTR expression by at least about 10%, preferably at least about 25%, when compared to control or baseline expression of that protein.
  • the compounds will increase CFTR expression by about 50%, and more preferably about 70% to about 100% or more as determined by the assay.
  • the assay detects and preferably quantifies presence of degradative complexes that usually include the hsc70 protein.
  • selected compounds generally assist trafficking by helping CFTR avoid the complexes, thereby reducing or even eliminating significant CFTR degradation.
  • CFTR expression is assisted in this assay by the trafficking inhibitor compounds.
  • such compounds are particularly useful for treating a variety of disorders and conditions including, but not limited to, lung disorders such as CF.
  • the carbocyclic aryl alkenoic acid derivatives are selected for capacity to boost protein expression, particularly by increasing transcription of a subject protein (or group thereof)
  • the administered compound exhibit at least about a 10%, preferably at least about a 25 % increase in transcription when compared to a suitable control or baseline experiment.
  • the increase in transcription provided by the compounds will be between from about 20% to about 50%, more preferably between from about 60% to 70% as measured in a standard in vitro assay for transcription.
  • An example includes a conventional nuclease protection assay.
  • Such compounds are sometimes referred to herein as "transcription enhancing" compounds or like term. In many instances, expression of the subject protein(s) will be increased by such transcription enhancing compounds.
  • Compounds useful in the invention include carbocyclic aryl compounds substituted with a carboxy acid (-COOH); protected carboxy acid such as an ester, particularly an alkyl ester such as e.g. -COOR where R is alkyl, preferably C ⁇ - 8 alkyl; sulfonic acid (-SO 3 H); nitro; cyano; haloalkyl particularly perhaloalkyl such as trifluoromethyl and pentafluoroethyl, where such a polar functional group is spaced from the carbocyclic aryl ring, e.g.
  • linker group containing 1 to about 16 carbons, more typically 1 to about 8 or 12 carbons, still more typically a linker of about 1, 2, 3, 4, 5, or 6 carbons .
  • the polar functional group i.e. carboxy acid, ester, sulfonic acid, nitro, cyano, haloalkyl
  • the linker may contain one or more unsaturated carbons, preferably a carbon-carbon double bond, although alkynylene linkages also may be present. If a carboxy group is present, tire linker group preferably contains a carbon-carbon multiple bond, particularly an alkenylene linkage.
  • the linker also may contain a hetero atom (N, O or S) in the linker chain.
  • Typical carbocyclic aryl groups substituted with a polar functional group include e.g. phenyl, naphthyl, acenapthyl, anthracenyl, and the like, with phenyl being preferred.
  • the carbocyclic aryl group also may have ring substituents such as halo (particularly F, Cl, and Br); alkyl particularly C
  • Suitable compounds of this invention can be readily identified or confirmed by simple testing, e.g. by in vitro testing of a candidate compound relative to a control for the ability to modulate protein expression, e.g. by at lea ⁇ st 10% relative to the control.
  • preferred compounds of the invention will show better activity than 4-PBA especially at low dosages. That is, such compounds will show at least about a 10 fold increase in activity with respect to the same amount of 4-PBA in the assay, preferably about a 50 to 100 fold increase and more preferably about a 100 to about a 1000 fold increase in activity.
  • the invention further relates to methods of detecting and analyzing compounds that modulate protein expression and exhibit therapeutic capacity to treat or prevent the above-described conditions.
  • Preferred detection and analysis methods include both in vitro and in vivo assays to determine the therapeutic capacity of agents to modulate the expression of one or a group of subject proteins.
  • Preferred in vitro detection assays detect and usually quantify modulated protein expression by analyzing specific steps or pathways known or thought to impact expression.
  • such an assay will include the following steps 1) through 4):
  • particular steps of interest include transcription, translation, and protein trafficking including, but not limited to, steps or pathways associated with particular molecular complexes such as those impacting protein degradation.
  • a preferred candidate compound is a carbocyclic aryl alkenoic acid derivative.
  • the method can be adapted to detect and preferably quantify hsc70 expression, CFTR expression, or both.
  • the hsc70 and/or the CFTR can be provided to the cells as a heterologous or homologous proteins as needed.
  • the protein may be suitably provided by standard recombinant strategies involving expression or co-expression of vectors that encode the protein.
  • the foregoing general assay can effectively measure the capacity of a desired compound to modulate protein expression by detecting and preferably quantifying increases or decreases in transcription or protein trafficking.
  • References herein to a "standard in vitro assay” or other similar phrase refers to the above protocol of steps 1) through 4).
  • step 3 of the standard in vitro assay when step 3) of the standard in vitro assay is implemented to measure hsc70 protein expression, the assay will be more specifically referred to as the standard in vitro hsc70 assay.
  • the assay When that assay is adapted to measure CFTR expression, the assay will be referred as an in vitro CFTR assay.
  • the CFTR expression is suitably measured according to standard methods such as those discussed below.
  • Preferred assays of this type involve immunological detection strategies such as immunoprecipitation or related approaches.
  • step 3 of the standard in vitro assay measures transcription of a subject protein (or group of proteins)
  • the assay will often be referenced as a standard in vitro assay for measuring transcription of nucleic acid encoding the protein or similar term.
  • a preferred in vitro transcription assay is a nuclease protection assay.
  • the in vitro assays of the present invention can be conducted with nearly any population of cells that can express at least one heat shock protein and particularly hsc70 including a lysate of such cells or tissue, or a substantially purified fraction of the lysate.
  • Suitably expressing cells that may be employed in the assay include, but are not limited to, primary cells such as nasal epithelia, and certain immortalized cells having demonstrated capacity to express, as a heterologous or homologous protein, mammalian and especially human proteins e.g., hsc70 and CFTR. Preferred examples of such cells are provided below.
  • the in vitro detection assays of the invention can be adapted in accordance with intended use.
  • the standard in vitro assay generally outlined above can be modified, e.g., at step 3) above to include measurements of desired steps such as transcription or protein trafficking including measurement of hsp70 expression as degradative complexes.
  • the known or candidate compound can be employed in the assays as a sole active agent or in combination with other agents including other recognized modulators of protein expression, e.g., transcriptional inhibitors, protein trafficking inhibitors and the like. Examples of such agents include, but are not limited to, 4-PBA and hydroxyurea.
  • the in vitro assays are performed with a suitable control assay usually including or consisting of the same test conditions as in the steps above, but without adding the compound or compounds to be tested.
  • a candidate compound can be identified as exhibiting desired activity by exhibiting at least about 10% change in the specified activity relative to the control; more preferably at least about 20% change relative to the control assay; and still more preferably at least about a 30% to about a 100%, change relative to the control.
  • change is meant an increase or decrease relative to that control.
  • the invention is compatible with recognized in vivo assays to determine the therapeutic capacity of a known or candidate compound to modulate protein expression and particularly to treat a disease or condition impacted by such expression.
  • the monitored disease or condition suitably may be pre-exist in the test animal, or the cell function may be induced, e.g., genetically, chemically, or by surgical intervention.
  • Animal functions that can be suitably assayed in these methods include, but are not limited to, transcription, translation, post-translational modification, trafficking including the degradation of subject proteins, cell proliferation including metastases, cell division, apoptosis, respiratory function, cognition, membrane potential, intracellular or extracellular ion concentration, intracellular kinase activity, phosphatase activity, intracellular protein transport, endogenous or heterologous gene expression, chloride channel function and protein secretion.
  • Suitable in vivo assays can be modified in a number of ways as needed.
  • a specific carbocyclic aryl alkenoic acid derivative is administered to the animal either as a sole active agent or in combination with other active compounds (e.g., 4-PBA), including other compounds of this invention to be tested.
  • activity of the compound in a given in vivo assay is compared to a suitable control (e.g., a control animal not receiving the compound).
  • the control assay is conducted the same as the test assay but without administering the compound to the test subject.
  • a variety of test subjects can be employed, particularly mammals such as rabbits, primates, various rodents and the like including mice. Preferred test subjects are recognized models for a particular human disease or disorder.
  • the in vivo efficacy of a particular carbocyclic aryl alkenoic acid derivative can be tested in recognized human CF model ie., nasal potential differences between normal and CF patients.
  • the derivative is administered to the human subject in a therapeutically effective amount for several days.
  • Nasal tissue is removed and chloride transport detected and preferably measured in control (no ⁇ nal patient) and CF patients.
  • preferred compounds of this invention will help restore normal CFTR function in the nasal epithelial cell surface to level that is comparable to that achieved with 4-PBA.
  • a preferred assay of this type is provided below in Example 13.
  • the detection assays can be conducted in a wide variety of cells. If desired, the assays can also be conducted with tissues and organs that include such cells. Further, the assays can detect useful compounds by measuring the activity of target molecules such as nucleic acids and proteins in pathways that modulate protein expression. Thus, the present assays are readily adapted to measure activity in a variety of cell, tissue and organ settings. Significantly, use of multiple detection assays (e.g., a combination of the in vitro and/or in vivo assays) with a single compound such as a particular carbocyclic aryl alkenoic acid derivative as provided herein can extend the selectivity and sensitivity of detection as desired.
  • multiple detection assays e.g., a combination of the in vitro and/or in vivo assays
  • a single compound such as a particular carbocyclic aryl alkenoic acid derivative as provided herein can extend the selectivity and sensitivity of detection as desired.
  • in vitro assays of the invention can efficiently perform multiple analyses, thereby enhancing efficiency and probability of identifying compoimds with therapeutic capacity. This is especially useful when large numbers of compounds need to be tested.
  • libraries of candidate compounds and particularly libraries of carbocyclic aryl alkenoic acid derivatives can be made by standard synthetic methods including combinatorial-type chemistry manipulations and then tested in accord with the invention.
  • kits for performing the methods of those invention include at least one container means that includes at least one of the carbocyclic aryl compounds disclosed herein.
  • Figure 1A is a representation of a Western Blot showing hsc70 expression as a function of added 4-PBA in IB3-1 cells.
  • Figure IB is a graph showing densitometery of results from immunoblot experiments in which relative heat shock protein 70 (hsc70) levels are determined versus mM 4-PBA.
  • Figure 1C is a representation of a Western Blot showing a standard curve construction.
  • Figure ID is a graph showing densitometery of results from immunoblot experiments in which relative hsc70 levels dete ⁇ nined versus bovine hcs70.
  • Figure 2A-E are Western Blots showing amounts of Calnexin (Fig. 2A), various heat shock proteins (Figs. 2B-D) and Hdj2 ( Figure 2E) in the presence of mM 4-PBA.
  • Figures 3 A is a representation of a gel showing results of an RNase protection experiment using an hsc70 probe.
  • Figure 3B is a graph showing densitometry results of RNase protection experiments in which relative hsc70 amounts are compared to mM 4-PBA.
  • Figure 4A is a representation of a Western Blot showing results of immunoprecipitation with an anti-hsc70 antibody.
  • Figure 4B is a graph showing densitometery results of protein relative to control in the presence of mM 4-PBA.
  • Figure 4C is a graph showing densitometery results of a CFTR standard curve construction.
  • Figure 5A shows representations of Western blots analyzed for presence of hsc70 using a polyclonal serum.
  • Figure 5B is a graph showing densitometric results of hsc70 expression under various conditions and relative to a control.
  • Figure 5C is a graph showing densitometric results of chaperone under various conditions and relative to a control.
  • Figure 6A is a representation of a Western Blot showing RNase protection results for hsc70 and 18s rRNA under various conditions.
  • Figure 6B is a graph showing densitometric results of hsc70 mRNA relative to control under various conditions.
  • Figure 7A is a representation of a Western Blot showing the cystic fibrosis transmembrane regulator (CFTR) and hsc70 expression under various conditions.
  • Figure 7B is a graph showing densitometric results of hsc 70 and CFTR protein under various conditions.
  • CFTR cystic fibrosis transmembrane regulator
  • Figure 8 is a representation of a Western blot showing expression of CFTR in the presence of mM 4-phenyl- ⁇ 3-transbutenoic acid.
  • Figure 9A and 9B are representations of Western blots showing: 9 A. induction of CFTR biosynthesis and processing by a butyrate pro-drug and 9B. 4- phenyl- ⁇ 3-transbutenoic acid up-regualtion of of band C in CFTR (IB3-1 cells).
  • Figure 10A and 1 OB are representations of Western blots showing 4-phenyl- ⁇ 3-transbutenoic acid mediated up-regulation of CFTR in primary cystic fibrosis bronchial epithelial cells.
  • Figure 11 is a representation of a Western blot showing effect of 4-phenyl- ⁇ 3- transbutenoic acid on hsc70 in primary CF bronchial epithelia.
  • the phenotype is ⁇ F508/ ⁇ F5058.
  • Figure 12 is a representation of a Western blot showing effect of 4-phenyl- ⁇ 3- transbutenoic acid on hsc70 in the primary CF bronchial epithelia.
  • Figure 13 is a representation of a Western blot showing a time course of 4- phenyl- ⁇ 3-transbutenoic acid up-regulation of band C.
  • Figure 14 is a representation of a Western blot showing induction of CFTR band C and of Hsp70 chaperone protein by 4-pheylbutyrate in 1B3-1 cells.
  • Figure 15 is a representation of a Western blot showing overexpression of
  • Hsp70 by transient transfection with Hsp70 cDNA.
  • Figures 16A-D are graphs showing nasal potential difference patterns in normal subjects ( Figures 16B) and CF patients ( Figures 16A, 16C-D).
  • Administration of 4-phenylbutyrate increases presence of functional CFTR in nasal epithelia ( Figures 16C-D).
  • the present invention features therapeutic methods for treatment and prevention of conditions modulated by unacceptable protein expression.
  • the treatment methods of the invention generally include administering a therapeutically effective amount of at least one carbocyclic aryl alkenoic acid derivative to a subject, preferably a mammal such as a primate and often a human patient in need of such treatment.
  • the therapeutic methods of the invention generally comprise administration of a therapeutically effective amount of at least one and preferably one compound to the subject.
  • Treatment methods of the invention also comprise administration of an effective amount of a compound of Formula I as defined herein to the subject, particularly a mammal such as a human in need of such treatment for an indication disclosed herein.
  • Typical mammalian subjects are human patients suffering from, recovering from, or susceptible to those conditions discussed above, e.g., lung diseases afflicting normal alveolar function and including those associated with misfolding of surfactant protein protein C; lung diseases impacted by improper expression of the cystic fibrosis transmembrane regulator (CFTR) including CF; liver diseases including ⁇ l anti-trypsin disease; and brain diseases such as Alzheimer's disease and related dementia.
  • CFTR cystic fibrosis transmembrane regulator
  • liver diseases including ⁇ l anti-trypsin disease
  • brain diseases such as Alzheimer's disease and related dementia.
  • each W is the same or different linker group, such as optionally substituted alkylene preferably having 1 to about 12 chain carbons, more preferably 1 to about 8 chain carbons, still more preferably 1, 2, 3 or 4 alkylene chain carbons; optionally substituted alkenylene preferably having 2 to about 12 chain carbons, more preferably 2 to about 8 chain carbons, still more preferably 2, 3 or 4 alkenylene chain carbons; optionally substituted alkynylene preferably having 1 to about 12 chain carbons, more preferably 2 to about 8 chain carbons, still more preferably 2, 3 or 4 alkynylene chain carbons; optionally substituted heteroalkylene preferably having 1 to about 12 chain carbons, more preferably 1 to about 8 chain carbons, still more preferably 1 , 2, 3 or 4 heteroalkylene chain carbons; optionally substituted heteroalkenylene preferably having 2 to about 12 chain carbons, more preferably 2 to about 8 chain carbons, still more preferably 1, 2, 3, 4 heteroalkenylene chain carbons; or optionally substituted
  • R is a non-hydrogen substituent such as halogen, cyano, nitro, optionally substituted alkyl preferably having 1 to about 20 carbons, more preferably 1 to about 12 carbons; optionally substituted alkenyl preferably having 2 to about 20 carbon atoms, more preferably 2 to about 12 carbon atoms; optionally substituted alkynyl preferably having 2 to about 20 carbon atoms, more preferably 2 to about 12 carbon atoms; optionally substituted alkoxy preferably having from 1 to about 20 carbon atoms, more preferably 1 to about 12 carbon atoms; optionally substituted alkylthio preferably having from 1 to about 20 carbon atoms, more preferably 1 to about 12 carbon atoms; optionally substituted alkylsulfinyl preferably having from 1 to about 20 carbon atoms, more preferably 1 to about 12 carbon atoms; optionally substituted alkylsulfonyl preferably having from 1 to about 20 carbon atoms, more preferably 1 to about 12 carbon
  • Preferred compounds of Formula I have an unsaturated linker group, such as those of following Formula I A:
  • each W is the same or different optionally substituted alkenylene preferably having 2 to about 12 chain carbons, more preferably 2 to about 8 chain carbons, still more preferably 2, 3 or 4 alkenylene chain carbons; optionally substituted alkynylene preferably having 1 to about 12 chain carbons, more preferably 2 to about 8 chain carbons, still more preferably 2, 3 or 4 alkynylene chain carbons; optionally substituted heteroalkenylene preferably having 2 to about 12 chain carbons, more preferably 2 to about 8 chain carbons, still more preferably 1, 2, 3, 4 heteroalkenylene chain carbons; or optionally substituted heteroalkynynylene preferably having 2 to about 12 chain carbons, more preferably 2 to about 8 chain carbons, still more preferably 2, 3 or 4 heteroalkynylene chain carbons;
  • Y, R, m and n are the same as defined in Formula I; and pharmaceutically acceptable salts thereof.
  • Particularly preferred compounds of Formula I have an alkenylene linker group, such as those of following Formula LB:
  • each W is the same or different optionally substituted alkenylene preferably having 2 to about 10 chain carbons, more preferably 2 to about 8 chain carbons, still more preferably 2, 3, 4 or 5 alkenylene chain carbons;
  • Y, R, m and n are the same as defined in Formula I; and pharmaceutically acceptable salts thereof.
  • Specifically preferred compounds for use in methods of the invention include sterioisomers of the foregoing compounds according to Formulae I, IA and IB above. More are cis and trans isomers of 4-phenyl- ⁇ 3 -butenoic acid and 4-phenyl- ⁇ 2- butenoic acid. More specifically preferred are the following trans isomers of those compounds shown below in Table I. 4-p envi- ⁇ 3-transbutenoic acid
  • V-CH CH-CH2— COOH
  • Suitable alkyl substituent groups of compounds of the invention typically have from 1 to about 12 carbon atoms, more preferably 1 to about 8 carbon atoms, still more preferably 1, 2, 3, 4, 5, or 6 carbon atoms.
  • alkyl unless otherwise modified refers to both cyclic and noncyclic groups, although of course cyclic groups will comprise at least three carbon ring members.
  • Preferred alkenyl and alkynyl groups of compounds of the invention have one or more unsaturated linkages and typically from 2 to about 12 carbon atoms, more preferably 2 to about 8 carbon atoms, still more preferably 2, 3, 4, 5, or 6 carbon atoms.
  • alkenyl and alkynyl refer to both cyclic and noncyclic groups, although straight or branched noncyclic groups are generally more preferred.
  • Preferred alkoxy groups of compounds of the invention include groups having one or more oxygen linkages and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 , 2, 3, 4, 5 or 6 carbon atoms.
  • Preferred alkylthio groups of compounds of the invention include those groups having one or more thioether linkages and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1, 2, 3, 4, 5, or 6 carbon atoms.
  • Preferred alkylsulfmyl groups of compounds of the invention include those groups having one or more sulfoxide (SO) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1, 2, 3, 4, 5, or 6 carbon atoms.
  • Preferred alkylsulfonyl groups of compounds of the invention include those groups having one or more sulfonyl (SO 2 ) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably 1 , 2, 3, 4, 5 or 6 carbon atoms.
  • Preferred aminoalkyl groups include those groups having one or more primary, secondary and/or tertiary amine groups, and from 1 to about 12 carbon atoms, more preferably 1 to about 8 carbon atoms, still more preferably 1, 2, 3, 4, 5, or 6 carbon atoms. Secondary and tertiary amine groups are generally more preferred than primary amine moieties.
  • Suitable heteroaromatic groups of compounds of the invention contain one or more N, O or S atoms and include, e.g., coumarinyl including 8-coumarinyl, quinolinyl including 8-quinolinyl, pyridyl, pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, oxidizolyl, triazole, imidazolyl, indolyl, benzofuranyl and benzothiazole.
  • Suitable heteroalicyclic groups of compounds of the invention contain one or more N, O or S atoms and include, e.g., tetrahydrofuranyl, thienyl, tetrahydropyranyl, piperidinyl, morpholino and pyrrolindinyl groups.
  • Suitable carbocyclic aryl groups of compounds of the invention include single and multiple ring compounds, including multiple ring compounds that contain separate and/or fused aryl groups.
  • Typical carbocyclic aryl groups of compounds of the invention contain 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms.
  • carbocyclic aryl groups include phenyl; naphthyl including 1- naphthyl and 2-naphthyl; biphenyl; phenanthryl; anthracyl; and acenaphthyl.
  • Substituted carbocyclic groups are particularly suitable including substituted phenyl, such as 2-substituted phenyl, 3-substituted phenyl, 4-substituted phenyl, 2,3- substituted phenyl. 2,4-substituted phenyl, and 2,4-substituted phenyl; and substituted naphthyl, including naphthyl substituted at the 5, 6 and/or 7 positions.
  • Suitable aralkyl groups of compounds of the invention include single and multiple ring compounds, including multiple ring compounds that contain separate and/or fused aryl groups.
  • Typical aralkyl groups contain Tto 3 separate or fused rings and from 6 to about 1 8 carbon ring atoms.
  • Preferred aralkyl groups include benzyl and methylenenaphthyl (-CH 2 -naphthyl), and other carbocyclic aralkyl groups, as discussed above.
  • R, W and Y groups are optionally substituted.
  • a "substituted" R, W and Y group or other substituent may be substituted by other than hydrogen at one or more available positions, typically 1 to 3 or 4 positions, by one or more suitable groups such as those disclosed herein.
  • suitable groups that may be present on a "substituted" R, W and Y group or other substituent include e.g. halogen such as fluoro, chloro, bromo and iodo; cyano; hydroxyl; nitro; azido; alkanoyl such as a C ⁇ .
  • alkanoyl group such as acyl and the like; carboxamido; alkyl groups including those groups having 1 to about 12 carbon atoms, or 1 , 2, 3, 4, 5, or 6 carbon atoms; alkenyl and alkynyl groups including groups having one or more unsaturated linkages and from 2 to about 12 carbon, or 2, 3, 4, 5 or 6 carbon atoms; alkoxy groups having those having one or more oxygen linkages and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5 or 6 carbon atoms; aryloxy such as phenoxy; alkylthio groups including those moieties having one or more thioether linkages and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5 or 6 carbon atoms; alkylsulfmyl groups including those moieties having one or more sulfmyl linkages and from 1 to about 12 carbon atoms, or 1 , 2, 3, 4, 5, or 6 carbon atoms; alkylsulfonyl groups including those moieties
  • R group being a substituted or unsubstituted biphenyl moiety
  • aralkyl having 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms, with benzyl being a preferred group
  • aralkoxy having 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms, with O-benzyl being a preferred group
  • a heteroaromatic or heteroalicyclic group having 1 to 3 separate or fused rings with 3 to about 8 members per ring and one or more N, O or S atoms, e.g.
  • a treatment compound can be administered to a subject by one or a combination of ways.
  • a carbocyclic aryl alkenoic acid derivative can be administered as a prophylactic to prevent the onset of or reduce the severity of a targeted condition.
  • the compound can be administered during the course of or following recovery from a targeted condition.
  • a treatment compound can be administered to a subject, either alone or in combination with one or more therapeutic, inert or partially active agents, as a pharmaceutical composition in mixture with conventional excipient, i.e. pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral or intranasal application which do not deleteriously react with the active compounds and are not deleterious to the recipient thereof.
  • conventional excipient i.e. pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral or intranasal application which do not deleteriously react with the active compounds and are not deleterious to the recipient thereof.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl- cellulose, polyvinylpyrrolidone, etc.
  • the pharmaceutical preparations can be sterilized and if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • compositions may be prepared for use in parenteral administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; intranasally, particularly in the form of powders, nasal drops, or aerosols; vaginally; topically e.g. in the form of a cream; rectally e.g. as a suppository; etc.
  • the pharmaceutical agents may be conveniently administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical arts, e.g., as described in Remington 's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA, 1980).
  • Formulations for parenteral administration may contain as common excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like.
  • biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of certain compounds or formulations thereof.
  • parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
  • Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or citric acid for vaginal administration.
  • Other delivery systems will administer the therapeutic agent(s) directly at a targeted site, e.g., by stent, needle or related implementation.
  • a compound according to this invention can be employed in the present treatment methods as the sole active pharmaceutical agent or can be used in combination with other active ingredients, e.g., 4-PBA, hydroxyurea or other suitable compounds including one or more other carbocyclic aryl compounds. See Proc. Natl. Acad. Sci. (2000) 97: 1796-1780 for additional information relating to 4-PBA and particularly to its use a therapuetic agent.
  • concentration of one or more treatment compounds in a therapeutic composition will vary depending upon a number of factors, including the dosage of the compound to be administered, the chemical characteristics (e.g., hydrophobicity) of the composition employed, and the intended mode and route of administration.
  • one or more than one of the compounds may be provided in an aqueous physiological buffer solution containing about 0.1 to 10%) w/v of a compound for parenteral administration.
  • Suitable dose ranges may include from about l ⁇ g/kg to about lOOmg/kg of body weight per day.
  • Therapeutic compounds of the invention are suitably administered in a protonated and water-soluble form, e.g., as a pharmaceutically acceptable salt, typically an acid addition salt such as an inorganic acid addition salt, e.g., a hydrochloride, sulfate, or phosphate salt, or as an organic acid addition salt such as an acetate, maleate, fumarate, tartrate, or citrate salt.
  • a pharmaceutically acceptable salt typically an acid addition salt such as an inorganic acid addition salt, e.g., a hydrochloride, sulfate, or phosphate salt, or as an organic acid addition salt such as an acetate, maleate, fumarate, tartrate, or citrate salt.
  • Pharmaceutically acceptable salts of therapeutic compounds of the invention also can include metal salts, particularly alkali metal salts such as a sodium salt or potassium salt; alkaline earth metal salts such as a magnesium or calcium salt; ammonium salts such an ammonium or tetramethyl ammonium salt; or an amino acid addition salts such as a lysine, glycine, or phenylalanine salt.
  • metal salts particularly alkali metal salts such as a sodium salt or potassium salt; alkaline earth metal salts such as a magnesium or calcium salt; ammonium salts such an ammonium or tetramethyl ammonium salt; or an amino acid addition salts such as a lysine, glycine, or phenylalanine salt.
  • Preferred compounds of this invention feature significant activity in a standard in vitro hsc70 assay. Typically, the derivatives will inhibit or decrease, preferably decrease hsc70 levels in the assay by at least about 10%, preferably at least about 25% when
  • a compound to be tested is used.
  • carbocyclic aryl alkenoic compounds will often be preferred.
  • Exemplary assays include monitoring at least one of hsc70 transcription, translation, or protein trafficking including measurment of complexes that include hsc70 and particularly those hsc70 complexes involved in the degradation of proteins such as the CFTR.
  • a preferred assay monitors hsc70 transcription by the following nuclease protection assay: a) culturing cells capable of expressing at least one heat shock protein, preferably hsc70 in suitable medium (e.g., DMEM, LHC-8) and adding a compound to be tested, e.g., an carbocyclic aryl alkenoic acid derivative, to the medium for between from about 6 hours to about 72 hours, b) preparing a lysate from the cells and isolating RNA from same preferably under conditions that reduce or eliminate RNA degradation, c) hybridizing the RNA in the lysate to a probe capable of specifically binding a nucleic acid sequence encoding the heat shock protein, e.g.,hsc70 protein, the hybridization being capable of forming a specific binding pair, usually under high stringency conditions, after about 6 hours to about 24 hours, d) contacting the specific binding pair with a nuclease such as RNAse, the nucleas
  • hsc70 probe and RNAse protection experiments are provided in the Examples following.
  • the amount of hsc70 specific RNA or mRNA detected in the presence of the compound is compared to a suitable control, which control is treated under the same conditions as the assay culture but does not include die compound that is tested.
  • a suitable control which control is treated under the same conditions as the assay culture but does not include die compound that is tested.
  • a standard hsc70 nuclease protection assay or related phrase refers to the steps a) through e) above in which nucleic acid encoding the hsc70 protein is tested.
  • Such an assay can be readily modified, if desired, to include monitoring of other RNAs including mRNA encoding proteins such as CFTR and/or control proteins such as those specifically mentioned below.
  • Preferred assays will include a conventional qualitative technique such as densitometry in embodiments in which good measurement and data analysis is needed.
  • Further preferred compounds will exhibit an ID 50 of about 500 ⁇ M or less, still more preferably an IC50 of about 1 to about 10 ⁇ M or less in the standard in vitro hsp 70 nuclease protection assay.
  • Compounds such as the carbocyclic aryl alkenoic acid derivatives disclosed herein exhibiting such preferred activity are considered to be good trafficking inhibitor compounds.
  • nuclease protection assay can be readily adapted to monitor other nucleic acids such as those encoding other proteins besides hsc70.
  • Such an assay may be particularly useful in embodiments in which a compound of this invention is tested for capacity to increase transcription of the nucleic acid.
  • the hsp70 probe described above can be substituted with another probe that specifically binds the subject nucleic acid preferably under high stringency conditions.
  • useful probes include those useful for measuring hemoglobin, and surfactant protein expression. See e.g., Peterec, S.M et al. (1994) Physiol. Lung Cell. Mol. Physiol. 267: 12784- 12788; and Collins et al. (1995) Blood 85: 43-49.
  • Other suitable probes for use in accord with this invention can be found at the National Center for Biotechnology Information (NCBI)- Genetic Sequence Data Bank (Genbank).
  • NCBI National Center for Biotechnology Information
  • Genbank Genetic Sequence Data Bank
  • Genbank is also available on the internet at http://www.ncbi.nlm.nih.gov. See generally Benson, D.A. et al. (1997) Nucl. Acids. Res. 25: 1 for a description of Genbank.
  • Suitable probe lengths in the assays will generally vary depending on intended use but will generally be from about 50 nucleotides to about 5000 nucleotides in length, preferably about 100 to about 500 nucleotides.
  • a particular assay for monitoring hsc70 protein /// vitro is by conducting the following immunoprecipitation assay:
  • a compound to be tested e.g., a carbocyclic aryl alkenoic acid derivative
  • Preferred detection formats include Western immunoblots in which second antibodies are used to bind the immune complex.
  • Such second antibodies can be detectably labeled themselves or detectably labeled third antibodies can be used that bind, preferably specifically, the second antibody bound to the immune complex.
  • General guidance relating to performing this assay can be found in Harlow and Lane (eds) in: Antibodies: A Laboratory Manual 1988, Cold Spring Harbor Laboratory, New York. Also see Harlow, et al. for examples of strategies for detectably labeling the antibodies.
  • Suitable antibodies can be polyclonal or monoclonal as needed.
  • hsc70 immunoprecipitation assay is provided in the discussion and Examples following.
  • the amount of hsc70 protein detected in the presence of the compound is compared to a suitable control, which control is treated under the same conditions as the assay culture but does not include the compound that is tested.
  • a standard hsc70 protein assay or related phrase refers to the steps a) through e) above.
  • Such an assay can be readily modified, if desired, to include monitoring of otiier proteins such as other heat shock proteins, CFTR and/or control proteins such as those mentioned below.
  • the assay will include a conventional quantative technique such as densitometry.
  • the assay further detects CFTR. especially human CFTR.
  • the assay is suitably adapted so that CFTR is detected in the lysates.
  • step e) is modified so that presence of the CFTR in the immune complexes is detected and preferably quantified.
  • the lysate is contacted with a first antibody capable of binding CFTR. Binding of the first antibody can be readily detected by binding of a second detectably labled antibody according to conventional immunological methods.
  • This embodiment of the hsc 70 assay will be referred to herein as a "standard in vitro CFTR assay" or related term to denote supplemental or exclusive detection of the CFTR.
  • a decrease in the amount of CFTR detected in the standard in vitro CFTR assay is taken to be indicative of an increase in the level of functional or potentially functional CFTR. That is, less CFTR in the immune complex and especially those complexes that include or consist of hsc70 is taken to be indicative of less transmembrane protein available for degradation. Accordingly, CFTR expression levels increase in the assay in line with a decrease in the amount of degradative complexes. See also Rubenstein, R.C, (2000), supra and the Examples below.
  • carbocyclic aryl alkenoic acid derivatives are capable of inhibiting specific enzymes such as histone deacetylase. Additionally preferred derivatives serve as good amino acid acylating agents.
  • Methods for detecting inhibition of histone deacytlase are known in the field. See e.g., Candido, E.P et al. (1978) Cell 14: 105-1 13; and Lea, MA and N. Tulsyan (1995) Anticancer Res. 15: 879-883. Methods for detecting amino acid acylation are also known and include use of conventional chromatographic approaches, e.g., HPLC.
  • the present invention includes methods of detecting and analyzing compounds such as carbocyclic aryl alkenoic acid derivatives with therapeutic capacity to treat or prevent any of the above-mentioned diseases or disorders.
  • a disease or condition is impacted or modulated by protein expression if afflicted cells, tissue and/or organs exhibit an increase or decrease in subject protein (or proteins) of at least about 2 fold, preferably about 2 to 1000-fold, more preferably about 2- to 100-fold, and more typically about 2- to 10-fold relative to a suitable control. That control is typically the same cells, tissues and/or organs taken from a normal or unafflicted subject.
  • the change in protein expression can be measured by methods referenced herein including those in vitro and in vivo assays in which subject proteins are measured.
  • preferred carbocyclic aryl alkenoic acid derivatives modulate protein ⁇ expression particularly by increasing transcription and/or reducing unwanted protein degradation. Accordingly, the compositions and methods of this invention are particularly useful in the treatment of conditions or disorders modulated by such protein expression.
  • Preferred cells for use in the methods of this invention include those expressing heat shock proteins and particularly hsc70.
  • suitable cells can be an immortalized cell line or primary culture of cells (e.g., obtained form a tissue or organ such as the nose). More preferred cells manifest a change in protein expression following contact with a suitable molecule such as 4- PBA, i.e., at least about a 10% increase or decrease relative to a suitable control.
  • More suitable cells include those amenable to standard recombinant DNA techniques such as transformation (e.g., mediated by calcium, biolistic transfer, electroporation and the like) by a vector that encodes the subject protein.
  • transformation e.g., mediated by calcium, biolistic transfer, electroporation and the like
  • CFTR and especially human CFTR.
  • the human CFTR sequence has been disclosed. For example, see the Genbank website referenced above.
  • one or a combination of strategies can identify such cells. For example, in one approach, about 1 x 10 5 cells are seeded in petri dishes in suitable growth medium. For primary cultures of cells, a desired tissue or organ is obtained from an animal and dispersed according to standard methods (e.g., by sonication, mechanical agitation, and or exposure to dispersing agents known in the field, e.g., detergents and proteases). After one or a few days, the growth medium is removed from the petri dish and the cells washed with phosphate-buffered saline. The cells are then contacted with about 0.0 ImM to about 5mM 4-PBA or 4-phenyl- ⁇ 3-butenoic acid in the culture.
  • suitable growth medium e.g., a desired tissue or organ is obtained from an animal and dispersed according to standard methods (e.g., by sonication, mechanical agitation, and or exposure to dispersing agents known in the field, e.g., detergents and proteases
  • the medium is removed and the cells lysed in an appropriate lysis buffer such as those described herein.
  • the cells are then assayed according to any of the methods described herein for response to the added 4-PBA or 4-phenyl- ⁇ 3- butenoic acid.
  • Examples of such cells include immortalized cystic fibrosis bronchiolar epthelial cells such as those referenced below, e.g., IB3-1 cells.
  • Other examples of such primary cells include nasal epithelia.
  • a control experiment is generally tailored for use in a particular assay. For example, most control experiments involve subjecting a test sample (e.g., a population of suitable cells or lysate thereof) to medium, saline, buffer or water instead of a compound to be tested in parallel to the cells receiving an amount of test compound. A desired assay is then conducted in accordance with the present metiiods. Specific examples of suitable control experiments are described below.
  • a test sample e.g., a population of suitable cells or lysate thereof
  • medium, saline, buffer or water instead of a compound to be tested in parallel to the cells receiving an amount of test compound.
  • a desired assay is then conducted in accordance with the present metiiods. Specific examples of suitable control experiments are described below.
  • Specific techniques for use with the methods described herein may involve one or more standard laboratory manipulations such as chemilummescence tests, thin layer chromatography (TLC) separations, nucleic acid isolation and purification, SDS-PAGE gel electrophoresis, autoradiography, scintillation counting, densitometery, Northern and Western Blot hybridization, and immunoassays (e.g., RIA and EL1SA tests).
  • TLC thin layer chromatography
  • SDS-PAGE gel electrophoresis SDS-PAGE gel electrophoresis
  • autoradiography scintillation counting
  • densitometery densitometery
  • Northern and Western Blot hybridization e.g., Western Blot hybridization
  • immunoassays e.g., RIA and EL1SA tests.
  • HPLC high performance liquid chromatography
  • preferred compounds of this invention are derivatives of carbocyclic aryl alkenoic acid.
  • exemplary of such derivatives are phenylcarbocyclic aryl alkenoic acid derivatives including 4-phenyl- ⁇ 3 -transbutenoic acid. That acid can. under certain conditions, be ⁇ oxidized to phenylacetic acid which serves as an amino acid acylating agent.
  • One aspect of this invention is the discovery that phenylcarbocyclic aryl alkenoic acid, and particularly 4-Phenyl- ⁇ 3-transbutenoic acid compound can be used to modulate protein expression.
  • the particular compound has been shown to restore normal biosynthetic trafficking to the CFTR mutation ⁇ F508.
  • the mechanism centers on butyrate-mediated down-regulation of the chaperone protein Hsc70.
  • 4-Phenyl- ⁇ 3-transbutenoic acid and 4-PBA both have a second potent ability to regulate gene and/or protein expression in a number of physiologic processes.
  • 4-PBA administration increases fetal hemoglobin levels, perhaps by transcriptional up-regulation. See Dover et al. supra and Stamatoyannopoulos et al., Blood, 84:3198-3204 (1994).
  • Fetal hemoglobin levels and percent F cells increase, and it is thought that transcriptional upregulation of ⁇ globin may be explained by the observation that butyrate promotes regulation of gene expression via inhibition of histone deacetylase. Inhibition of histone deacetylation by the butyrates is reported to be associated with tumor cell differentiation and is the rationale for the use of phenylbutyrate as an adjunct chemotherapeutic agent.
  • butyrate class of chemical agents including many of the compounds of this invention may be thought of as a new kind of "gene drug” that acts by transcriptional regulation.
  • Transcriptional regulators can be harnesses to up or down-regulate redundant gene pathways that normally are relatively quiescent.
  • a new class of chemical compound is disclosed herein which in particular embodiments can treat the ⁇ F508 trafficking defect through a pharmacologic strategy.
  • One goal is the restoration of normal chloride conductance using a transcriptional regulator to correct the biosynthetic trafficking defect associated with ⁇ F508 expression and enhance the mutant protein chloride transport.
  • a preferred compound of this invention 4-Phenyl- ⁇ 3-transbutenoic acid (or trans styrylacetic acid), may be employed as a sole or adjunctive therapeutic agent for the treatment or prevention of the inherited urea cycle disorders as well as other maladies, e.g., inherited hemoglobinopathies, thalassemias and cancer. Buphenyl has been approved for use in some of these conditions. See Dover et al, Blood, 84:339- 343 (1994); Dover et al., N. Engl. J. Med., 327:569-570 (1992); Collins et al., Blood, 85:43-49 (1995); K. Smigel, J. Natl.
  • the 70 kD heat shock protein family consists of Hsp 70 (sometimes called Hsp72) which is inducible by heat shock and/or the presence of denatured intracellular proteins and Hsc70 (sometimes called Hsp73), the 70 kD heat shock cognate protein which is constitutively expressed and is involved in the uncoating of clathrin-coated endosomes.
  • Hsc70 also has a role in the lysosomal degradation of intracellular proteins, and was recently shown to be required for the ubiquitin- dependent degradation of a number of cellular proteins.
  • Examples 1-3 refer to use of 4-PBA. See also Rubenstein, R.C. and P.L. Zeitlan, (2000), supra: the disclosure of which is inco ⁇ orated herein by reference.
  • Each of the methods described in Examples 1 -3 can be modified to accomadate one or more carbocyclic aryl alkenoic acid derivatives including stereoisomers of phenylcarbocyclic aryl alkenoic acid derivatives and specifically cis and trans isomers of 4-phenyl-3 ⁇ -butenoic acid.
  • Rabbit anti-CFTR antiserum 181 (directed against CFTR amino acids 415-427 prior to the first nucleotide binding fold) was described previously (25).
  • a rabbit polyclonal antiserum specific for Hsc70 (5) was a generous gift of Drs. C. R. Brown and W. J. Welch (University of California at San Francisco).
  • a rat monoclonal antibody specific for Hsc70, clone 1B5 was a generous gift of Dr. A. Laszlo (Washington University, St. Louis, MO). This antibodyis also commercially available (Stressgen Biotechnologies, Victoria, BC, Canada).
  • a mouse monoclonal antibody directed against Hsp90 (clone AC88) and rabbit polyclonal antisera specific for Hsp40 and Hsp70 were purchased from Stressgen Biotechnologies.
  • a mouse monoclonal antibody directed against calnexin (clone AF8) (18) was a generous gift of Dr. Michael Bremier (Harvard University).
  • a mouse monoclonal antibody to Hdj2 (clone KA2A5.6) was from NeoMarkers (Union City, CA).
  • Donkey anti-rabbit IgG- horseradish peroxidase conjugate and sheep anti-mouse IgG-horseradish peroxi-dase conjugates were purchased from Amersham (Arlington Heights, IL).
  • Goat anti-rat IgG-horseradish peroxidase conjugate was purchased from Boehringer-Mannheim (Indianapolis, IN) or Amersham.
  • Detection of immunoreactivity was performed with the enhanced chemilummescence reagent (ECL, Amer-sham) and fluorography.
  • ECL enhanced chemilummescence reagent
  • Recombinant bovine Hsc70 (.95% purity) was purchased from Stressgen Bioteclmologies for use in constructing a standard curve of Hsc70 immunoreactivity. Immunoblots containing the bovine Hsc70 were probed with the Hsc70-specific polyclonal antiserum.
  • ATCC ATCC, Manassas, VA
  • EcoR I site of pSK(2) Bluescript, Stratagene, La Jolla, CA
  • the resulting plasmid was sequenced in the Genetics Core Facility at the Johns Hopkins Hospital and found to be identical to sequences in exons 8 and 9 of the human Hsc70 sequence, with sequencing using the T3 primer and T7 primer leading to sense and antisense sequence, respectively.
  • Hybridization probes were synthesized using a Maxiscript T7 kit (Ambion, Austin, TX) and [ ⁇ 32 P]UTP (Amersham or DuPont N ⁇ N, Boston, MA) according to the Maxiscript protocol.
  • Probes for internal control hybridizations were purchased from Ambion, and probes were similarly synthesized using the Maxiscript T7 kit. Probes were isolated by acid phenol-chloroform (Ambion) extraction, separated from uninco ⁇ orated nucleotide by gel filtration (Sephadex G25 RNAspin column, Boel ringer-Mannheim), and ethanol-acetate precipitated before resuspension in hybridization buffer. The concentration of radioactivity in the synthesized probes was determined by liquid scintillation.
  • Immunodetection of immunoprecipitated Hsc70 or CFTR was performed as described above.
  • Densitometric analysis of these images was performed using Alphalmager image analysis software (version 4.0, Alphalnnotech) with two-dimensional integration of the selected band. Density of the lane surrounding the band was similarly determined by two-dimensional integration and used as a baseline density for background subtraction. For comparisons within an experiment, the density of the control lane, the 100-ng lane for bovine Hsc70 standard curve experiments and the 10- ⁇ g lane for CFTR standard curve experiments, was arbitrarily set to 1.0. A one-way ANOVA was used to determine statistical significance of changes in density of fluorographic bands (SPSS software, version 7.0).
  • Example 1- 4-PBA treatment of IB3-1 cells decreases expression of Hsc70
  • IB3-1 (38). IB3-1 has the CFTR genotype ⁇ F508/W1282X and is a model system for study of the intracellular trafficking of ⁇ F508-CFTR because the W1282X allele gives rise to an unstable and therefore untranslated mRNA. This results in IB3-1 cells containing only ⁇ F508-CFTR (17). It has been shown that treatment of IB3-1 cells with 4-PBA results in restoration of appropriate intracellular trafficking of ⁇ F508- CFTR (30).
  • IB3-1 cells were treated with increasing concentrations of 4-PBA in culture for 2 days.
  • total Hsc70 immunoreactivity in whole cell lysates declined in a dose-dependent fashion with increasing concentrations of 4-PBA as detected by a Hsc70-specific rabbit polyclonal antiserum (Fig. /A, representative immunoblot; Fig. 7B, compiled densitometric analysis of Fig. 1A and 7 other immunoblots).
  • Fig. /A representative immunoblot
  • Fig. 7B compiled densitometric analysis of Fig. 1A and 7 other immunoblots.
  • Similar data were obtained when a rat monoclonal antibody to Hsc70 was used to probe the immunoblots.
  • Hsp70 (Hsp72) expression is induced by heat shock and the presence of denatured proteins within the eukaryotic cell (16).
  • the Hsp70 homologue DnaK and the Hsp40 homologue DnaJ act to promote protein folding (16).
  • Hdj2 is the member of the Hsp40 family that specifically interacts with and regulates the ATPase activity of Hsc70. Hdj2 also interacts with CFTR during CFTR translation (26).
  • FIG. 1 A-C are more specifically explained as follows.
  • Fig. 1. Dose-dependent reduction in Hsc70 expression mediated bysodium 4-phenylbutyrate (4- PBA).
  • Primary antiserum was rabbit polyclonal antiserum specific for Hsc70.
  • Density of 0 4-PBA(control) lane was set to 1, and density (means 6 SE) of other lanes is expressed relative to control. Statistical significance (P values indicated below error bars) was determined by a 1 -way ANOVA in comparison with control.
  • C standard curve construction. IB3-1 lysate protein (5 ⁇ g) or indicated amount of purified recombinant bovine Hsc70 was resolved on 8% SDS- polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose, and immunodetection of Hsc70 was performed as described above under General Comments. Primary antiserum was rabbit polyclonal antiserum specific for Hsc70. D: densitometry was performed as described under General Comments on 3 identical experiments.
  • Density of 100-ng lane was set to 1, and densities of other lanes are expressed relative to 100-ng lane. Mean relative density is shown by filled circles. Error bars (SE) are contained within symbols. Relative density of 5 ⁇ g of IB3-1 lysate is depicted by open circle and corresponds to about 35 ng of bovine Hsc70 immunoreactivity.
  • FIG. 2A-2E are more specifically explained as follows. Calnexin, Hsp90, Hsp70, Hsp40, and Hdj2 expression is unchanged by 4-PBA treatment. IB3-1 cells were treated as described for Fig. 1. Immunoblotting with 5 ⁇ g of IB3-1 SDS lysate protein was performed as described in the foregoing General Comments.
  • Example 2- 4-PBA treatment results in decreased Hsc70 mRNA expression.
  • Hsc70 mRNA was measured in lysates of IB3-1 cells by RNase protection. It was found that, in comparison to levels of 18S rRNA as an internal standard for total RNA assayed and recovered, a concentration- dependent decrease in steady-state Hsc70 mRNA levels after 4-PBA treatment (Fig. 3) that correlated with the decrease in Hsc70 immunoreactivity observed in Fig. 1. There was a maximum decrease of, 50% of control expression with continuous exposure to 5 mM 4-PBA.
  • FIGS. 3A-B are explained in more detail as follows. Dose-dependent downregulation of Hsc70 mRNA expression by 4-PBA.
  • A IB3-1 cells were treated as for Figs. 1 and 2. Hsc70 mRNA and 18S rRNA (as an internal reference) were measured by Direct Protect RNase protection as described under General Comments.
  • B densito-metric analysis on 3 independent experiments was perfo ⁇ ned as described by first normalizing density of Hsc70 hybridization by internal reference RNAhybridization (either 18S rRNAor cyclophilin A mRNAhybridization; see General Comments) to control for total RNAin each hybridization and RNA recovery during assay. This ratio for each condition was subsequently made relative to Hsc70- to-reference RNA ratio for control lane. Means 6 SE for 3 independent experiments are shown. P values (shown above respective error bars) were determined by 1 -way ANOVA in comparison with control.
  • Example 3 ⁇ F508-CFTR forms a complex with Hsc70 that is decreased by 4-PBA, low temperature, butyrate, and glycerol.
  • Immunoprecipitation with an antise m that recognizes both Hsp70 and Hsc70 results in the coimmunoprecipitation of ⁇ F508-CFTR (37).
  • a direct interaction between CFTR and Hsc70 was confirmed by testing whether specific immunoprecipitation of Hsc70 would result in coim-munoprecipitation of CFTR immunoreactivity (Fig. 4 ).
  • the relative mobility of CFTR in this experiment was — 170 kDa, which is consistent with the ER glycosylated "band B" fo ⁇ ri.
  • Recovery of Hsc70 by immunoprecipitation was ⁇ 10% of input, which is typical in this kind of experiment when recovery has been measured (26). It is assumed that this is representative of the total cellular pool of Hsc70.
  • IB3-1 cells treated with increasing concentrations of 4-PBA had decreased amounts of immunoprecipitable Hsc70 (Fig. 4 A). Widi increasing concentrations of 4-PBA, less immunoreactive CFTR was recovered in complex with Hsc70. At >1 mM 4-PBA, CFTR was undetectable in the immunoprecipitates. Similarly, CFTR was not associated with Hsc70 when IB3-1 cells were incubated at 25°C. We previously showed that treatment with >0.1 mM 4-PBA or incubation at 25°C leads to increased overall expression and the appearance of mature CFTR in IB3-1 cells (30).
  • CFTR immunoreacitivity as detected by densitometry decreases linearly as a function of decreasing protein but that the decrease in densitometric signal exceeds the decrease in CFTR protein, i.e., a decrease in IB3-1 protein from 5 to 2.5 ⁇ g leads to an approximately two-thirds decrease in densitometric signal.
  • CFTR immunoreactivity was not consistently detected in samples containing 2 ⁇ g of IB3-1 protein and was not detected in samples containing 1 ⁇ g of IB3-1 protein.
  • the slightly greater change in CFTR vs. Hsc70 densitometric signal in Fig. 4B actually reflects a similar decrease in immunoreactive protein of the two species.
  • FIGS 4A-C are more specifically explained as follows. 4-PBA-treatment of IB3-1 cells decreases amount of cystic fibrosis transmembrane conductance regulator (CFTR) immunoreac-tivity coprecipitated with Hsc70. IB3-1 cells were grown in indicated concentration of 4-PBA for 2 days at 37°C. Cells were solubilized with RIPA, and 250 ⁇ g of total protein were incubated with Hsc70-specific rabbit polyclonal antiserum as described under General Comments. Immune complexes were recovered by centrifugation after incubation with protein A-Sepharose.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • composition of precipitated immune complexes was analyzed by SDS-PAGE and protein immunoblot using anti-CFTR antiserum 181 and rabbit polyclonal anti-Hsc70 antibody as de-scribed under General Comments.
  • Total protein (10 ⁇ g) was resolved in IB3-1 SDS lysate lane, and immunoprecipitate from equivalent of 80 ⁇ g of cellular protein was analyzed in immunoprecipitation lanes.
  • Relative mobility of CFTR associated with Hsc70 was ,170 kDa.
  • B densitometric analysis of these and similar immunoblots (4 independent experiments) was performed as described for Fig. 1.
  • Mean density (6SE) relative to control for 4 independent experiments is shown.
  • P values vs. control were determined by 1-way ANOVA.
  • CFTR densitometry standard curve construction Immunodetection of CFTR in indicated amount of IB3-1 lysate protein was perfo ⁇ ned as described under General Comments. Densitometry was performed as described as described above on 4 independent concentration curves. For each independent experiment, density of CFTR immunoreactivity in 10- ⁇ g sample was set to 1 , and densities of CFTR immunoreactivity in other samples are expressed relative to 10- ⁇ g lane. Mean relative density is shown by closed symbols. Error bars (SE) are contained within symbols where not visible.
  • SE Error bars
  • Hsc70 expression If association with Hsc70 is necessary for CFTR ubiquitination, as it is for a number of other cellular proteins (3), then escape from this association may decrease the proportion of ⁇ F508-CFTR that is prematurely degraded.
  • G418 promotes read through and stabilization of the otherwise unstable mRNA derived from the W1282X missense allele present in IB3-1 cells and results in the appearance of CFTR channel activity at the IB3-1 plasma membrane (2).
  • Representative data for these immunoblot experiments are shown in Fig. 5. The results again demonstrate a reduction in Hsc70 immunoreactivity with 1 mM butyrate and 1 M glycerol but no change in Hsp90. Hsp70. or Hsp40 immunoreactivity.
  • Hsc70 expression in IB3-1 cells is decreased by butyrate (BA;1 mM) and glycerol (Glyc, 1 M) but not phenylacetate (PAA; 1 mM) and G418 (0.2 mg/ml); Hsp90, Hsp70, and Hsp40 expression are not affected by these agents.
  • BA butyrate
  • PAA phenylacetate
  • G418 0.2 mg/ml
  • Hsp90, Hsp70, and Hsp40 expression are not affected by these agents.
  • IB3-1 cells were incubated at 37°C for 2 days under indicated conditions.
  • Phenylacetate at 1 mM had little effect on Hsc70 or Hsp90-expression.
  • 4- PBA is rapidly and completely converted by ⁇ -oxidation to phenylacetate and then conjugated with glutamine to form phenacetylglutamine, which is excreted in the urine (6).
  • Phenylacetate has a different potency profile from butyrate or 4-PBA with respect to specific gene induction (8).
  • Figure 7 demonstrates that treatment of IB3-1 cells with glycerol or butyrate decreases the amount of immunoreactive CFTR copre-cipitated with Hsc70. Again, the densitometric analysis of this and similar experiments (Fig. 7B) suggests that the decrease in CFTR associated with Hsc70 resulted from decreased expression of Hsc70.
  • FIG. 6 is explained in more detail as follows. Butyrate and glycerol also decrease Hsc70 mRNA expression.
  • composition of precipitated immune complexes was analyzed by SDS-PAGE and protein immunoblot using anti-CFTR antiserum 181 and rabbit polyclonal anti-Hsc70 antibody as described above.
  • Total protein (10 ⁇ g) was resolved in EB3-1 SDS lysate lane, and immunoprecipitate from equivalent of 80 ⁇ g of cellular protein was analyzed in immunoprecipitation lanes.
  • Relative mobility of CFTR coimmunoprecipitated with Hsc70 was again ,170 kDa.
  • B densitometric analysis of these immunoblots (4 independent experiments) was performed as described for Fig. 1. Shown is mean density (6SE) relative to control for 4 independent experiments. P values vs. control were dete ⁇ nined by 1-way ANOVA.
  • ⁇ F508-CFTR The usual intracellular fate of ⁇ F508-CFTR is degradation, at least in part by the ubiquitin-proteasome system (20, 36). Therefore, 4-PBA may promote ⁇ F508- CFTR trafficking by inhibiting its recognition by the intracellular degradation pathway.
  • Hsc70 protein expression induced by 4-PBA, butyrate, and glycerol is, 40-60%.
  • Butyrate is typically thought to act as a transcriptional activator, which contrast with these examples.
  • decreased expression of surfactant proteins A and B mRNA in fetal rat lung has been reported in response to butyrate treatment (27). This is consistent with the examples suggesting a decrease in Hsc70 expression at the protein and mRNAlevels after treatment with butyrate and 4-PBA.
  • ⁇ 508-CFTR is typically degraded by the ubiquitin-proteasome system (20, 36).
  • inhibition of the proteolytic component of this system with lactacystin or N-acetyl-L-leucinyl-L-leucinyl-L-leucinal does not promote ⁇ F508-CFTR trafficking to the cell surface (20, 36).
  • Hsc70 associates with CFTR during CFTR translation, and the association with ⁇ F508-CFTR is greater and longer lived than with wild-type CFTR (26). CFTR also undergoes cotranslational ubiquitination (32), and the possibility of enhanced ubiquitin-dependent degradation of the ⁇ F508 peptide in the presence of Hsc70 is absent in the in vitro folding system (34).
  • ⁇ 1% of ⁇ F508-CFTR is trafficked via the normal pathway; >99% is targeted for and subsequently degraded (35).
  • -75% of wild-type CFTR is targeted for and subsequently degraded, whereas 25%) of wild-type CFTR is trafficked to the cell surface (35).
  • the "trafficking" pathway for ⁇ F508-CFTR is disfavored by at least 2-3 kcal/mol compared with that of wild-type CFTR (7). This results from either an intrinsic instability of the ⁇ F508- CFTR protein, as is suggested by the higher proportion of ⁇ F508-CFTR that would enter the degradative pathway compared with wild-type CFTR.
  • cystic fibrosis transmembrnae conductance regulator is recognized by hsp70 and degraded in a pre-Golgi nonlysosomal compartment. Proc. Natl. Acad. Sci. USA 90: 9480-9484, 1993.
  • 4-Phenyl- ⁇ 3-transbutenoic acid restores the normal biosynthetic trafficking of the ⁇ F508 protein in CF, and therefore, may be useful as a treatment for disorders or conditions impacted by undesired protein expression such as CF.
  • 4-Phenyl- ⁇ 3-transbutenoic acid works by down regulating Hsc70 which is involved in the degradation of the ⁇ F508 protein.
  • the ⁇ F508 protein acts by inhibiting histone descetylation leading to increased expression of other chloride channels.
  • 4-PBA has been approved by the FDA for the treatment of certain urea cycle disorders.
  • the data provided by Examples 4-12 show, among other things, that 4-Phenyl- ⁇ 3-transbutenoic acid is much more effective at lower concentrations.
  • IB3-1 ( ⁇ F508/W1282X) cells were exposed to increasing concentrations of the test compounds from l ⁇ M to 100 ⁇ M for 48 hr.
  • CFTR was immunoprecipitated and phosphorylated as described in the Methods.
  • Lane 1 shows control, untreated cells at standard cell culture conditions. Band B CFTR is barely visible consistent with low level ⁇ F508 expression.
  • the positive control condition, untreated cells grown at 28°C, is in lane 7. Band C has been induced and Band B is relatively depleted consistent with an effect of low temperature on protein trafficking.
  • Lanes 2-6 show a dose-dependent induction of Band C as well as an induction of Band B and total CFTR. This is consistent with an effect on protein production and on protein trafficking.
  • Example 6- 4-phenyl- ⁇ 3 -transbutenoic acid mediated up-regulation of band C CFTR in primary cystic fibrosis bronchial epithelial cells.
  • FIGS 10A and 10B Shown in Figures 10A and 10B are results of CF bronchial epithelial cells were harvested from the discarded lungs of a CF patient who underwent therapeutic lung transplant.
  • the genotype is homozygous ⁇ F508.
  • the methods are as described above.
  • Control cells grown in standard conditions for 0 or 48 hrs are in lanes 6 and 7. Bands B and C are barely detectable.
  • the positive control condition, 28°C is in lane 1 and shows up-regulations of both bands B and C.
  • the testxompound in doses from l ⁇ M to 1 mM for 48 hr is shown in decreasing order in lanes 2-5.
  • the primary patient-derived cells are much more sensitive to the effects and show dramatic induction of bands B and C by 1 ⁇ M with maximum induction by 1 mM.
  • Shown in Figure 1 1 is an experiment in which cells were exposed to control or test conditions or study drug for 48 hrs and then analyzed by immunoblotting for Hsc70, a 70 kD heat shock chaperone protein. This semi-quantitative blot demonstrates at least a 50% reduction in Hsc70 at 100 ⁇ M and 1 mM concentrations (lanes 4 and 5). Hsc70 is not as dramatically down regulated by growth at 27°C.
  • Example 9- 4-phenyl- ⁇ 3-transbutenoic acid mediates downregulation of Hsc70 in the primary CF cells studied in Figures 9 and 10A.
  • Shown in Figure 12 is an experiment performed along lines as shown in Figure 10A in the same primary cell line. Hsc70 under control conditions is strongly visible in lane 6. Study drug at l ⁇ M or higher, and low temperature, are associated with more than 50% reduction in Hsc70.
  • IB3-1 cells were exposed to the test conditions and 1 mM study drug for the indicated periods of time. CFTR was detected by immunoprecipitation and phosphorylation. Control cells at 0 and 32 hrs have barely visible CFTR bands B and C. Exposure to low temperature induces band C. 1 mM 4- phenyl- ⁇ 3-transbutenoic acid begins to induce band C and 4 hr and appears maximal by 16 hrs.
  • Example 11- Induction of CFTR band C and of Hsp70 chaperone protein by 4-phenylbutyrate in IB3-1 cells were exposed to increasing concentrations of 4-phenylbutyrate and then immunoblotted for CFTR with anti-CFTR antibody 181 (upper panel) or for Hsp70 (lower panel). Control cells at 37°C show band B CFTR, but virtually undetectable band C CFTR. These cells as expected show very little Hsp70, the heat shock inducible chaperone. At 27°C, a lower temperature, there is very little Hsp70 as expected, but as expected, there has been induction of band C CFTR. 4-Phenylbutyrate induces Hsp70 in a dose-dependent manner and also promotes appearance of band C CFTR.
  • Example 12- Overexpression of Hsp70 by transient transfection with Hsp70 cDNA induces CFTR in IB3-1 cells.
  • IB3-1 cells were transfected with Hsp70 or a control plasmid using lipofectamine by standard methodologies. After 48 hrs, the cells were collected and immunoblotted with anti-CFTR antibody 181. The arrows indicate the CFTR, however the mobility is somewhere from 160-180 kD. The induction of Hsp70 is associated with a dramatic ove ⁇ roduction of CFTR as compared to control plasmid transfection at 37°C or growth at 27°C.
  • Example 13 Assay for Determining Nasal Potential Differences in No ⁇ nal and CF Patients using 4-PBA
  • Figure 16A shows a nasal potential difference pattern from an individual with cystic fibrosis.
  • the methodology employed involves superfusion of the inferior turbinate with Ringer's solution and measurement of the electrical potential with reference to a subcutaneous electrode.
  • This CF pattern shows a typical hype ⁇ olarization, large amiloride inhibition, and minimal repolarization to low chloride or isoproterenol maneuvers.
  • Figure 16B shows a nasal potential difference pattern in a non-CF individual.
  • the baseline potential is less polarized than in CF.
  • the amiloride inliibition is much lower.
  • the low chloride and isoproterenol exposure induce a sizeable repolarization.
  • Figure 16C shows a nasal potential difference pattern of a patient with CF (deltaF508 homozygous) who has taken 20 gm 4-phenylbutyrate daily for 7 days.
  • the low chloride/isoproterenol exposures induce chloride transport. This is consistent with the presence of a functional CFTR in the nasal epithelial cell surface.
  • Figure 16D the same individual as in Figure 16D after 18 days on 4- phenylbutyrate.
  • the low chloride/isoproterenol exposure continues to sustain chloride transport.

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Abstract

L'invention concerne, d'une manière générale, des maladies modulées par une expression de protéines indésirable. Dans un aspect, les méthodes selon l'invention permettent d'administrer à un mammifère, et notamment à un humain, une quantité thérapeutiquement efficace d'un composé aryl acénoïque capable de moduler cette expression de protéines indésirable. L'invention concerne également des dosages permettant de détecter des composés présentant une propriété thérapeutique voulue.
PCT/US2000/006377 1999-03-12 2000-03-11 Modulation de l'expression de proteines au moyen de derives d'acide carbocyclique aryl alcenoique Ceased WO2000053174A1 (fr)

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EP2103942A1 (fr) 2008-03-20 2009-09-23 Universitätsklinikum Freiburg Procédé de détermination d'un type d'infection rhumatismale dans des synovies
US12042476B2 (en) 2022-02-04 2024-07-23 Mcmaster University Methods for the treatment of lysosomal storage diseases

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6048896A (en) * 1995-11-03 2000-04-11 Pharmacia & Upjohn S.P.A. 4-phenyl-4-oxo-2-butenoic acid derivatives with kynurenine-3-hydroxylase inhibiting activity

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6048896A (en) * 1995-11-03 2000-04-11 Pharmacia & Upjohn S.P.A. 4-phenyl-4-oxo-2-butenoic acid derivatives with kynurenine-3-hydroxylase inhibiting activity

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2103942A1 (fr) 2008-03-20 2009-09-23 Universitätsklinikum Freiburg Procédé de détermination d'un type d'infection rhumatismale dans des synovies
WO2009115190A1 (fr) * 2008-03-20 2009-09-24 Universitätsklinikum Freiburg Procédé de détermination du type d'une maladie rhumatismale inflammatoire dans le liquide synovial
US12042476B2 (en) 2022-02-04 2024-07-23 Mcmaster University Methods for the treatment of lysosomal storage diseases

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