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Title:  Methods for treating cell death diseases and inflammation

United States Patent:  6,552,071

Issued:  April 22, 2003

Inventors:  Yuan; Junying (Newton, MA); Kobori; Masuko (Ibaraki, JP); Yang; Zhen (Brookline, MA)

Assignee:  President and Fellows of Harvard College (Cambridge, MA)

Appl. No.:  735848

Filed:  December 13, 2000


The present invention features methods and compounds for treating or preventing a cell death disease or inflammation, and methods for synthesizing wedelolactone.


The Role of Caspase-11 in Modulating Cell Death and Inflammation

Previous studies have indicated that caspase-11 plays a role in mediating both cell death and inflammatory responses (Wang et al., J. Biol. Chem. 271:20580-20587, 1996; Wang et al., Cell 92:501-509, 1998). For example, overexpression of caspase-11 in Rat-1 and HeLa cells induces apoptosis. In contrast, caspase-11 knockout mice are resistant to ischemic brain injury-induced apoptosis. In addition, caspase-11 mutant embryonic fibroblast cells are resistant to apoptosis induced by overexpression of the cell death promoting gene ICE. These results demonstrate that caspase-11 is involved in mediating cell death.

These same studies revealed that caspase-11 is involved in mediating inflammatory responses. For example, caspase-11 promotes pro-interleukin 1 beta processing by ICE, and its expression is induced upon stimulation with LPS. In addition, caspase-11 knockout mice are resistant to the LPS model of septic shock, indicating that this gene is involved in mediating inflammatory responses.

Induction of Caspase-11 is Mediated Through the NF-.kappa.B Pathway

NF-.kappa.B is a dimeric transcription factor consisting of two subunits, Rel A (p65) and NF-.kappa.B 1 p50. NF-.kappa.B exists in the cytoplasm in an inactive form by virtue of its association with an inhibitor I.kappa.B.alpha.. Upon stimulation by appropriate signals, I.kappa.B.alpha. is selectively phosphorylated by the I.kappa.B kinase complex and is subjected to degradation through a ubiquitin/proteasome pathway. Upon degradation of I.kappa.B.alpha., NF-.kappa.B undergoes rapid nuclear translocation and induces gene expression. A myriad of pathological stimuli have been shown to induce NF-.kappa.B activity. This list includes bacterial endotoxins, proinflammatory cytokines, viral infections, parasites, UV irradiation, chemotherapeutic agents, oxidative stress and DNA damage. Abberant regulation of NF-.kappa.B has been associated with pathogenesis of several diseases, including cancer. The pharmaceutical industry has made considerable efforts to identify novel inhibitors of NF-.kappa.B activation.

Expression of caspase-11 is also induced by TNF-.alpha. and IL-1.beta. (Y. Jung and J. Yuan, unpublished data). TNF-.alpha. and IL-1.beta. are known to induce multiple biological responses through NF-.kappa.B. Recently, it has been shown that NF-.kappa.B is activated and plays a key role in mediating cell death in mouse focal cerebral ischemia (Schneider et al., Nat. Med. 5:554-559, 1999). These observations have led to the examination of the possible involvement of NF-.kappa.B in caspase-11 induction. NF-.kappa.B (+/+) wild type, p65 (-/-) mutant (Beg et al., Nature 376:167-170, 1995), and p65 (-/-)/p50 (-/-) double mutant 3T3 cells (Sha et al., Cell 80:321-330, 1995) were examined for the regulation of caspase-11 induction. Treatment of wild type 3T3 cells with TNF-.alpha., IL-1.beta., or LPS induced caspase-11 expression. A mutation in NF-.kappa.B p65 blocked the ability of TNF-.alpha. but not that of IL-1.beta. and LPS, to induce caspase-11 expression; whereas inactivation of both NF-.kappa.B p65 and p50 subunits are needed to inhibit the caspase-11 expression induced by IL-1.beta. and LPS. These results suggest that NF-.kappa.B is essential for the induction of caspase-11 by cytokines; furthermore, the effect of TNF-.alpha. may be mediated through p65 only, whereas signals of LPS and IL-1.beta. may be mediated through either p65 or p50.

Identification of Modulators of Caspase-11 Expression, Cell Death, and Inflammatory Responses

Given the role that caspase-11 plays in modulating cell death and inflammatory responses, it is believed that compounds that modulate the expression or activity of caspase-11 would also be useful in treating or preventing a cell death disease or inflammation.

To identify modulators of caspase-11 expression or activity, a tissue culture model of caspase-11 induction was generated. In this model, BalbC/3T3 cells normally express undetectable levels of caspase-11 biological activity, as measured by Western blot analysis. However, upon contacting the cells with LPS for 6 hours, caspase-11 biological activity was clearly induced. A caspase-11 modulatory compound may be administered to the cells prior to administration of LPS, and then caspase-11 biological activity may be measured at a specific time after addition of LPS to the culture media of the cells. A caspase-11 modulatory compound that suppresses the LPS-induced biological activity of caspase-11 is considered to be a compound that modulates not only caspase-11 expression, but also cell death or inflammation.

Alternatively, the biological activity of caspase-11 may be measured using other standard techniques. For example, caspase-11 levels may be measured by immunoprecipitation techniques. In addition, the measurement of biological activity may include the measurement of caspase-11 nucleic acid levels, the effect of caspase-11 on a target molecule, or the effect of caspase-11 on cell proliferation or cell death. For example, standard Northern blot analysis (Ausubel et al., supra) using a caspase-11 cDNA (or cDNA fragment) as a hybridization probe can be used to measure levels of caspase-11 expression. The level of caspase-11 RNA expression in the presence of the candidate molecule is compared to the level measured for the same cells in the same culture medium but in the absence of the candidate molecule.

Caspase-11 modulatory compounds may be purified (or substantially purified) molecules or may be one component of a mixture of compounds (e.g., an extract or supernatant obtained from cells; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1994). In a mixed compound assay, caspase-11 expression is tested against progressively smaller subsets of the candidate compound pool (e.g., produced by standard purification techniques, such as HPLC or FPLC) until a single compound or minimal compound mixture is demonstrated to modulate caspase-11 expression.

Alternatively, or in addition, candidate caspase-11 modulatory compounds may be screened for those that modulate caspase-11 -mediated inhibition of cell proliferation. In this approach, the degree of cell proliferation in the presence of a candidate compound is compared to the degree of cell proliferation in its absence, under equivalent conditions. Again, such a screen may begin with a pool of candidate compounds, from which one or more useful modulatory compounds are isolated in a step-wise fashion. Cell proliferation activity may be measured by any standard assay, such as the mixed tumor transplantation (MTT) assay.

The identification of modulators of caspase-11, cell death, or inflammation can also be achieved by measuring the level of cell death that occurs in a population of cells contacted with a candidate compound. Cell death can be measured by determining cellular ATP levels, wherein a cell that is undergoing cell death has a decreased level of cellular ATP compared to a control cell that is not exposed to the test compound. Cell death may also be measured by staining with a vital dye, for example, trypan blue, wherein a dead cell will be stained with the vital dye, and a living cell will not be stained with the dye. Cell death may also be measured using any technique known to those skilled in the fields of molecular and cell biology.

Since caspase-11 induction is mediated through the activation of the NF-.kappa.B pathway, as discussed above, an alternative for the identification of modulators of caspase-11, cell death, or inflammation approach is to directly screen for inhibitors of the NF-.kappa.B pathway. This can be done, for example according to the following method, based on the T-Rex tetracycline inducible system (Invitrogen). To generate an NF-.kappa.B responsive system, the NF-.kappa.B response element is introduced into pcDNA6/TR vector upstream from the tetracycline repressor gene (TetR) (Invitrogen) replacing the constitutively active CMV promoter. In this new pNFkB-TR vector, expression of TetR protein is controlled by NF-.kappa.B induction. Luciferase activity is selected as an optimal output for the screen due to its high sensitivity and low background. Therefore, the Luc gene from the pGL3 vector (Promega) is introduced into the tetracycline operator-containing pTO vector (Invitrogen). Overall, in this system induction of NF-.kappa.B activity in response to LPS results in a reduction in luciferase expression. Activation of NF-.kappa.B results in an induction of TetR expression, which binds to the tetracycline operator in pTO-Luc vector and inhibits luciferase expression. Attenuation of NF-.kappa.B induction by a small molecule inhibitor results in an increase in luciferase expression providing a positive readout, rather than a negative readout. This allows for the distinction between selective NF-.kappa.B repressors and generally toxic compounds that occur in a system with a negative readout.

A compound that is identified as being a modulator of caspase-11, cell death, or inflammation can be tested in animal models. For example, a candidate compound can be injected intraperitonally into animals (e.g., mice) with a lethal dose of LPS (for example, 40 mg/kg). The animals are then monitored for symptoms of septic shock, including shivering, fever, lethargy, watery eyes and ultimately death. A candidate compound that results in mice exhibiting less severe septic shock symptoms, compared to control mice (receiving LPS, but no candidate compound) is a compound that can be used to treat cell death diseases and inflammation.

Structural Derivatives of Compounds That Modulate Caspase-11 Expression or Activity, Cell Death, or Inflammation

The synthesis of wedelolactone has been the subject of many investigations (Pandey et al., Tetrahedron 45:6867-6874, 1989). Our approach to the synthesis of wedelolactone employs a similar conventional route disclosed by Pandey et al. (supra) that features tyrosinase oxidation of catechol to synthesize the o-quinone from catechol. The o-quinone can react with different substituted 4-hydroxyl-coumarins to give the wedelolactone related molecules. These methods are described in detail in Example 5. Alternatively, wedelolactone may be synthesized according to the methods described in detail in Example 6.

In addition, the use of both solution-phase synthesis and combinatorial synthesis of structural derivatives of wedelolactone may be used and integrated in the search for ideal mimics of wedelolactone that are more efficacious. The probability of finding interesting mimics of wedelolactone with improved pharmacological profiles from a large number of structural derivatives is quite high and the testing for efficacy is readily performed, as described herein.


A compound identified as able to modulate caspase-11 biological activity or to decrease cell death, wherein the cell death is not caused by hepatotoxicity, or to decrease inflammation using any of the methods described herein may be administered to patients or animals with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. The chemical compounds for use in such therapies may be produced and isolated by any standard technique known to those in the field of medicinal chemistry. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the identified compound to patients suffering from a cell death disease or inflammation. Administration may begin before the patient is symptomatic.

Any appropriate route of administration may be employed. For example, the therapy may be administered either directly to the site of a predicted cell death or inflammation event (for example, by injection) or systemically (for example, by any conventional administration technique). Administration of the compound may also be parenteral, intravenous, intraarterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmalic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, by suppositories, or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols. The dosage of the therapeutic compounds in a pharmaceutically acceptable formulation depends on a number of factors, including the size and health of the individual patient. The dosage to be delivered may be determined by one skilled in the art.

Methods well known in the art for making formulations are found, for example, in "Remington: The Science and Practice of Pharmacy" ((19th ed.) ed. A. R. Gennaro AR., 1995, Mack Publishing Company, Easton, Pa.). Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for compounds that decreases necrosis include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

If desired, treatment with a compound identified according to the methods described above, may be combined with more traditional therapies for a disease characterized by cell death, for example, tacrine hydrochloride for the treatment of Alzheimer's disease, or interferon .beta.-1a for the treatment of multiple sclerosis. Similarly, treatment with a compound identified according to the methods described above, may be combined with more traditional therapies for inflammation, for example, nonsteroidal anti-inflammatory drugs or indomethicin.

Preventative Anti-cell Death Therapy

In a patient diagnosed with a cell death disease (e.g., a neurodegenerative disease, such as Alzheimer's disease, stroke, or Huntington's disease), any of the above therapies may be administered before the occurrence of the disease phenotype. In particular, compounds shown to decrease cell death may be administered by any standard dosage and route of administration (as described above).

The methods of the instant invention may be used to prevent or treat a cell death disease or inflammation, as described herein, in any mammal, for example, humans, domestic pets, or livestock.

Claim 1 of 16 Claims

What is claimed is:

1. A method for treating or preventing a cell death disease in a subject, said method comprising administering wedelolactone, or a derivative or salt thereof, to said subject, wherein said cell death disease is not caused by hepatotoxicity.

If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.



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