WO1996040982A1

WO1996040982A1 - Therapeutic inhibition of phospholipase a2 in neurodegenerative disease

Info

Publication number: WO1996040982A1
Application number: PCT/US1996/009772
Authority: WO
Inventors: Russel E. Rydel; Michael S. Dappen; Varghese John; Lawrence Yung-Sun Fang; Eugene D. Thorsett
Original assignee: Athena Neurosciences, Inc.
Priority date: 1995-06-07
Filing date: 1996-06-07
Publication date: 1996-12-19
Also published as: AU6381496A

Abstract

The invention provides methods and compositions for treating neurodegeneration in mammalian cells by administering a phospholipase A2 inhibitor.

Description

THERAPEUTIC INHIBITION OF PHOSPHOLIPASE A2 IN NEURODEGENERATIVE DISEASE

BACKGROUND

Cell death is an important aspect during the embryonic or post-natal development of major organ systems.

Apoptosis, or programmed cell death, also plays a critical role in maintaining homeostasis in many adult tissues.

Apoptosis is a term used to refer to the process(es) of programmed cell death and has been described in several cell types (Waring et al. (1991) Med. Res. Rev. 11: 219; Williams

GT (1991) Cell 65: 1097; Williams GT (1992) Trends Cell Biol. 2 : 263; Yonisch-Rouach et al. (1991) Nature 352: 345) .

Apoptosis is likely involved in controlling the amount and distribution of certain differentiated cell types, such as lymphocytes and other cells of the hematopoietic lineage as well as other somatic and germ cells. The mechanism(s) by which apoptosis is produced in cells is incompletely understood, as are the regulatory pathways by which the induction of apoptosis occurs.

Apoptosis Mechanism(s Apoptosis was .first described as a morphologic pattern of cell death characterized by cell shrinkage, membrane blebbing and chromatin condensation culminating in cell fragmentation (Kerr et al. , 1972) . One hallmark pattern early in the process of cell death is internucleosomal DNA cleavage (Wyllie, 1980) . The death-sparing effects of interrupting RNA and protein synthesis and the stereotyped patterns of cell death during development were consistent v;ith a cell autonomous genetic program for cell death (Wyllie et al. (1980) Int. Rev. Cvtol. 68: 251; Sulston, J. and Horvitz, H. (1977) Develop. Biol. 56: 110; Abrams et al. (1993)

Development 117 : 29) . The isolation of mutants defective for developmental cell death in the nematcde Caenorhabditis elegans supported this view (Ellis, K. and Horvitz, H. (1986) Cell 44: 817; Hengartner et al. (1992) Nature 356: 494). Control of apoptosis may be a regulatory feature of a variety of diseases, such as aging, AIDS, and autoimmune diseases, among others. Despite the identification of genes necessary for cell death and the ability to regulate apoptosis by known genes, the essential biochemical events in apoptotic death remain largely unknown.

Cell Proliferation Control and Neoplasia Many pathological conditions result, at least in part, from aberrant control of cell proliferation, differentiation, and/or apoptosis. For example, neoplasia is characterized by a clonally derived cell population which has a diminished capacity for responding to normal cell proliferation control signals. Oncogenic transformation of cells leads to a number of changes in cellular metabolism, physiology, and morphology. One characteristic alteration of oncogenically transformed cells is a loss of responsiveness to constraints on cell proliferation and differentiation normally imposed by the appropriate expression of cell growth regulatory genes. Despite progress in developing a more defined model of the molecular mechanisms underlying the transformed phenotype and neoplasia, few significant therapeutic methods applicable to treating cancer beyond conventional chemotherapy have resulted.

Thus, it is desirable to identify agents which can modify apoptosis activity so as to modulate cell proliferation, differentiation, and/or apoptosis for therapeutic or prophylactic benefit. Further, such agents can serve as commercial research reagents for control of cell proliferation, differentiation, and/or apoptosis in experimental applications, and/or for controlled proliferation and differentiation of predetermined stem cell populations in vitro, in ex vivo therapy, or jLn vivo. A variety of neurodegenerative diseases are characterized by cell death of neurons by a mechanism that is not presently distinguishable from many known models of apoptosis. Some of these neurodegenerative diseases appear to be related to excess accumulation of certain proteins. Examples of such amyloidosis-related neurodegenerative diseases include thiεe caused by the prion protein (PrP) which is associated with transmissible spongiform encephalopathy (Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, scrapie, and kuru) , and those caused by excess cystatin C accumulation (hereditary cystatin C angiopathy) , among others. Although much current effort is being expended on certain neuronal diseases associated with senile dementia, there is less effort being devoted to neurodegenerative diseases which are related to excess accumulation of certain proteins other than the A/3 peptide.

It would be desirable to have compounds and methods for treating non-Alzheimer's type neurodegenerative diseases wherein neuron loss is produced by programmed cell death which can be circumvented by suitable therapeutic intervention with pharmaceuticals.

Alzheimer's Disease

Alzheimer's disease (AD) is a progressive disease known generally as senile dementia. Broadly speaking the disease falls into two categories, namely late onset and early onset. Late onset, which occurs in old age (65 + years) , may be caused by the natural atrophy of the brain occurring at a faster rate and to a more severe degree than normal. Early onset AD is much more infrequent but shows a pathologically identical dementia with brain atrophy which develops well before the senile period, i.e. , between the ages of 35 and 60. Alzheimer's disease is characterized by the presence of numerous amyloid plaques and neurofibrillary tangles (highly insoluble protein aggregates) present in the brains of AD patients, particularly in those regions involved with memory and cognition. While in the past there was significant scientific debate over whether the plaques and tangles are a cause or are merely the result of AD, recent discoveries indicate that amyloid plaque is a causative precursor or factor. In particular, it has been discovered that the overproduction of /3-amyloid peptide ("A/3"), a major constituent of the amyloid plaque, can result from mutations in the gene encoding amyloid precursor protein, a protein which when normally processed will not produce the Aj8 peptide. One hypothesis regarding the pathogenesis of the disease is that deposition of A/3 peptide, which is the major macromolecular component of amyloid plaques, is the causative agent of the characteristic AD pathological changes leading to formation of neurofibrillary tangles, neuronal cell loss, vascular damage, and, ultimately, dementia (Hardy and Higgins (1992) Science 256: 184) . Amyloid precursor protein (APP) is encoded by a single gene in humans. RNA transcripts of the APP gene are alternatively spliced to encode several APP protein isoforms; the predominant APP isoform in brain lacks a serine protease inhibitor domain that is present in other tissues. A/3 is a proteolytic cleavage product arising from the carboxy region of various APP isoforms, including the predominant APP isoform in the brain (U.S. Patent No. 4,666,829; Glenner and Wong (1984) Biochem. Biophys. Res. Commun. 120: 1131; Kitaguchi et al. (1988) Nature 331: 530; Ponte et al., ibid. , p.525; R.E. Tanzi, ibid. , p.528; Kang and Muller-Hill (1990) Biochem. Biophys. Res. Commun. 166: 1192; Yoshioka et al. (1991) Biochem. Biophys. Res. Commun. 178: 1141; Johnson et al. (1990) Science 248: 854; Neve et al.

(1990) Neuron 5_: 329) . The accumulation of extracellular A/3 results in insoluble amyloid deposits and may be neurotoxic, leading to neuronal death and neurofibrillary tangle formation. Moreover, A/3 peptide appears to be toxic to brain neurons, and neuronal cell death is associated with the disease (Schubert et al. (1995) Proc. Natl. Acad. Sci. (USA) 92: 1989; Lorenzo and Yankner (1994) Proc. Natl. Acad. Sci. (USA) £1: 12243; Yankner et al. (1990) Science 250: 279; Kowall et al. (1991) Proc. Natl. Acad. Sci. (USA) 88: 7247; and Pike et al. (1993) J. Neurosci. 13: 1676) . Mattson et al. (1992) J. Neurosci. 12: 376 and Mattson et al. (1993) Trends in Neuroscience 16: 409, report that A/3 and fragments thereof can destabilize calcium (Ca⁺²) homeostasis in cultured human cortical neurons, and can render the neurons more susceptible to calcium ionophore-induced neurotoxicity. Meda et al. (1995) Nature 374: 647 report that A/3 and IFN-γ activates cultured microglial cells, leading to neuronal cell injury in co-cultured neurons. Both Meda et al. (1995) op.cit and Schubert et al. (1995) op.cit report the likely involvement of reactive free radical species, such as reactive nitrogen intermediates and reactive oxygen species.

Reports show that soluble A/3 peptide is produced by healthy neuronal cells in culture media (Haass et al. (1992) Nature 359: 322) and is present in human and animal cerebrospinal fluid (Seubert et al. (1992) Nature 359: 325). Thus, the mere presence of soluble A/3 peptide may not be sufficient for explaining the onset and progression of AD. However, aggregation and formation of insoluble complexes of A/3 have been implicated as having enhanced neurotoxicity to cultured neuronal cells. To date, the exact molecular mechanisms which result in the characteristic pathology and neuronal deficits of Alzheimer's disease have not been described in the art. The development of experimental models of Alzheimer's disease that can be used to define further the underlying biochemical events involved in AD pathogenesis would be highly desirable. Such models could presumably be employed, in one application, to screen for agents that alter the degenerative course of Alzheimer's disease. For example, a model system of the biochemical events which contribute to the pathology of Alzheimer's disease could be used to screen for drugs or therapeutic regimens that reverse, arrest, or slow the pathogenesis and progression of AD. Presumably, such models could be employed to develop pharmaceuticals that are effective in preventing, arresting, or reversing AD. Currently, there are no human pharmaceuticals which are known to be effective in inhibiting the development or progression of the degenerative CNS neuropathology of Alzheimer's Disease. U.S. Patent 5,192,753 report that certain non-steroidal anti-inflammatory drugs useful in treating rheumatoid arthritis (e.g., indomethacin) are allegedly useful in reducing symptomatic progression in a selected group of five AD patients, but no effects on neuropathological progression were noted and the sample size and experimental methodology employed were insufficient to conclusively demonstrate efficacy. U.S. Patent 5,137,873 disclose the use of tachykinin agonists to treat AD, although this approach has not proven successful in producing substantial amelioration of the progression of AD, and significantly more effective therapeutic agents are desired in the art.

There is a need in the art for pharmaceuticals which have therapeutic use to treat or prevent Alzheimer's Disease and A/3-related neurodegenerative diseases which have similar pathogenic mechanisms. A more thorough understanding of the molecular events underlying the development and progression of such A/3 related neurodegenerative diseases would facilitate development of such pharmaceuticals. Identification of critical biochemical events involved in these A/3-related neurodegenerative diseases can provide a basis for development of methods and model systems for screening compound banks to identify such pharmaceuticals, as well as providing a basis for the design of therapeutic methods and treatment modalities for Alzheimer's disease.

It would be desirable to have methods and model systems for screening test compounds for the ability to inhibit or prevent or inhibit neuronal toxicity produced by neurotoxic forms of a pathogenic A/3 peptide. In particular, it would be desirable to base such methods and systems on metabolic pathways and/or signal transduction pathways which have been found to be involved in such pathogenesis, where the test compound would be able to interrupt or interfere with the metabolic pathway or signal transduction pathway which leads to damage of neuronal and/or glial cells in the presence of pathogenic forms of A/3. Such methods and systems should provide rapid, economical, and suitable means for screening large numbers of test compounds.

Based on the foregoing, it is clear that a need exists for identification of metabolic pathways and/or signal transduction pathways which have been found to be involved in the pathogenesis of A/3-mediated and non-A/3-mediated neurodegenerative diseases, and the development of methods of treatment and pharmaceutical screening assays based on the identification of these pathways. The present invention fulfills these and other needs in the art.

The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

SUMMARY OF THE INVENTION

A basis of the present invention is the unexpected finding that neuronal cell degeneration is mediated by a biochemical cascade which requires activity of phospholipase A₂, ("PLA₂") , an enzyme which catalyzes the hydrolysis of the fatty acid ester bond at the sn-2 position of membrane phospholipids to produce arachidonic acid and its metabolites, and in the case of one cytosolic form of PLA₂, cPLA₂, also produces lysophospholipids. Agents which selectively block PLA₂ activity in neurons and/or glial cells and/or astrocytes or monocytes can be used to inhibit A/3-mediated neuronal degeneration and other forms of apoptotic cell death and neurodegeneration. Such active agents can reduce neuronal cell death such as that which results from exposure of neuronal cells to pathogenic forms and amounts of A/3 as occurs in Alzheimer's Disease and from exposure of neuronal cells to other types of apoptotic stimuli, such as exposure to amyloidogenic peptides which produce neurodegenerative conditions. These selective PLA₂-blocking agents can be used to inhibit neuronal degeneration. In one aspect, the invention provides a method for identifying active agents which significantly inhibit neuronal degeneration induced by amyloidogenic polypeptides, aberrant cytokine expression, and/or pathogenic A/3 peptides or their analogs, either directly or via their effects on secondary cell types such as glial cells, astrocytes, macrophages, or other non-neuronal cells which interact with central or peripheral neurons and which can manifest toxicity in response to exposure to Aβ or a non-A/3 amyloidogenic peptide. The method comprises administering an agent to a cell population comprising neurons, wherein said cell population is exposed to an amount of pathogenic A/3 or non-A/3 amyloidogenic polypeptide capable of inducing neuronal degeneration in the cell population, and determining whether the presence of said agent produces inhibition of PLA₂ activity and, typically, also produces a detectable reduction in the amount and/or rate of neuronal degeneration in the cell population; if said agent produces PLA₂ inhibition in neurons and/or inhibits neuronal degeneration, the agent is thereby identified as an active agent. Preferably, the method is used to demonstrate that the active agent inhibits PLA₂ activity and also inhibits neuronal degeneration in neuronal cells exposed to an apoptotic stimulus, pathogenic A/3 or non-A/3 amylopidogenic protein.

In a variation of the method, the agent is initially selected from a bank (or library) of compounds on the basis of the agent's capacity or selectivity for inhibiting PLA₂ in vitro, such as by its ability to inhibit PLA₂ enzymatic activity in an iri vitro assay employing a predetermined amount of a standardized preparation of PLA₂; an agent which is thus initially selected is administered to a cell population comprising neurons, wherein said cell population is exposed to an amount of pathogenic Aβ or amyloidogenic protein capable of inducing neuronal degeneration in the cell population, and the capacity of said agent to produce a detectable reduction in the amount and/or rate of neuronal degeneration in the cell population is determined, with agents capable of reducing neuronal degeneration being thereby identified as active agents. In this variation, the capacity of the agent to selectively or specifically inhibit PLA₂ in a cultured cell population comprising neurons can optionally be determined. In an aspect, the invention also provides a method for identifying an active agent which significantly inhibits neuronal degeneration in a transgenic animal model of Alzheimer's Disease or other neurodegenerative disease; such active agents can be sold commercially as reagents to control the disease phenotype of such transgenic animals for any purpose desired by an end-user of such animals, and can serve as candidate pharmaceuticals for therapy of neurodegenerative disease, among other uses. The method comprises initially selecting an PLA₂-inhibiting agent from a bank (or library) of compounds on the basis of: (1) the agent's capacity, selectivity, or specificity for inhibiting PLA₂ in vitro, such as by its ability to inhibit PLA₂ enzymatic activity in an in vitro assay employing a predetermined amount of a standardized preparation of PLA₂, and/or (2) the capacity of the agent to selectively inhibit PLA₂ in a cultured cell population comprising neurons; and administering the selected agent to a transgenic animal capable of developing detectable pathology characteristic of the neurodegenerative disease, and determining whether administration of the selected agent inhibits or retards development of said detectable pathology as compared to a substantially identical identifying control transgenic animal which lacks the agent; an agent which retards or inhibits development of pathology is thereby identified as an active agent.

In an aspect, the invention provides a method for reducing or retarding neurodegeneration in a cell population comprising neurons or neuronal cell lines and exposed to an apoptotoic stimulus, such as exposure to an amount of pathogenic A/3 or non-A/3 amyloidogenic polypeptide sufficient to produce neurodegeneration; said method comprising administering an efficacious dose of a PLA₂ inhibitor predetermined to retard or inhibit neuronal degeneration. In one embodiment, the cell population may reside in the central nervous system of a mammal and the PLA₂ inhibitor is administered in vivo. The invention also provides the use of a PLA₂ inhibitor to treat neurodegenerative disease pathology in a mammal. In an aspect, the invention provides a method for retarding or inhibiting neurodegeneration a cell population comprising neurons and exposed to an amount of pathogenic Aβ or amyloidogenic protein sufficient to produce neurodegeneration; said method comprising administering to the cell population an efficacious dose of an antisense polynucleotide capable of inhibiting expression of PLA₂, typically by reducing transcription and/or translation of the PLA₂ gene sequences. In one embodiment, the cell population may reside in the central nervous system of a mammal and the PLA₂ inhibitor is administered in vivo. The invention also provides the use of a PLA₂ antisense polynucleotide to treat neurodegenerative disease pathology in a mammal. In an embodiment, the antisense polynucleotide is produced by transcription of a transgene or gene therapy vector incorporated into a cell or animal; alternatively, antisense oligonucleotides can be administered in soluble form, formulated in liposomes, or by other suitable delivery format.

In an aspect, the invention provides a transgenic animal, such as a transgenic mouse, which harbors a transgene encoding a functional PLA₂ enzyme and capable of transcription and translation in neuronal and/or astrocytes and/or glial cells ln vivo. Typically, the transgene comprises a gene encoding a human PLA₂ enzyme operably linked to a transcriptional regulatory sequence which is transcriptionally active in neural cell types, and is preferably inducible. In one variation, the 5' flanking portion of the murine or human PLA₂ or APP gene, including the promoter (and frequently including an upstream portion and/or intronic portion(s) often having enhancer activity) and sufficient to drive transcription of linked sequences in the brain of a transgenic animal, serves as the transcriptional regulatory sequence of the PLA₂-encoding transgene. Such transgenic animals can overexpress PLA₂, either constitutively or inducibly, and can serve as models of accelerated A/3-mediated neurodegenerative disease; such animals can be sold for toxicological and pharmaceutical applications for evaluation of compounds or agents (physical or chemical) which modulate PLA₂-mediated neurodegeneration.

The invention also provides, in an aspect, a knockout animal comprising a genome having a homozygous pair of functionally disrupted endogenous PLA₂ alleles, such that substantially no endogenous PLA₂ is expressed. In a variation, the knockout animal genome also comprises a transgene encoding a heterologous PLA₂ enzyme (e.g., a PLA₂ knockout mouse having a transgene encoding human PLA₂) , which is expressed under the control of an operably linked transcriptional regulatory sequence, such as the naturally occurring mouse PLA₂ promoter and 5' flanking sequence.

In a variation, the invention provides a knockout mouse having a genome comprising a homologous pair of functionally inactivated mouse PLA₂ alleles and a transgene encoding and expressing a pathogenic human APP gene product, such as a human Swedish mutation APP transgene, human APP717 mutant APP transgene, or the like. Optionally, the mouse genome may further comprise a transgene encoding a mammalian PLA₂ which is transcribed under the control of a transcriptional regulatory sequence which is inducible or repressible in neuronal cells. Optionally, a naturally- occurring mouse model of neurological disease (e.g., Shaker and the like) can be employed.

In an aspect of the invention, an agent is selected from a compound library on the basis of its detectable inhibition of PLA₂ activity in an jLn vitro PLA₂ enzyme assay and/or in a cell culture PLA₂ assay system; the agent is administered to a transgenic animal of the invention which is expressing PLA₂ in neuronal tissue to thereby generate a treated transgenic animal refractory to neurodegenerative pathology and/or evaluate the suitability of the selected agent for iri vivo administration. Also provided by the invention is a method for inhibition of neuronal cell death in a cell population comprising mammalian glial cells and neuronal cells. The method comprises delivering an effective dosage of an PLA₂ inhibitor to a cell population comprising cells stimulated to exhibit neurotoxicity and neuronal cell death. Typically, the cell population is a co-cultured cell population of human cortical or hippocampal neurons and human microglia and/or human astrocytes and/or monocytes. In some variations, transgenic animals may serve as the source of the glial and/or neuronal cells. The cell population also may reside in a mammalian central nervous system in vivo.

The invention provides pharmaceutical compositions comprising an effective dose of an active agent, which is a PLA₂ inhibitor capable of reducing neurodegeneration, in a pharmaceutically acceptable form suitable for administration to a human or non-human animal. Often, such active agents are provided in a form suitable for delivery to CNS tissues to produce efficacious concentrations in the CSF or parenchyma of the brain of an intact mammal.

In an aspect of the invention, pharmaceutical compositions are provided which have potent antineurodegenerative properties and which comprise a PLA₂ inhibitor as an active agent. The pharmaceutical compositions of the invention comprise an efficacious dosage of at least one species of such an active agent. In one embodiment, the pharmaceutical composition comprises an active agent of a type known to inhibit PLA₂, which include arachidonic acid derivatives and analogs (e.g., arachidonyl trifluoromethyl ketone) , benzenesulfonamides, aminosteroids, bromoenol lactone, manoalide, p-bromophenacyl bromide, minocycline, doxycycline, 7,7,-dimethyl-5,8-eicosadienoic acid, quinacrine, and the like, among others known in the art. These pharmaceutical compositions possess the activity of inhibitng PLA₂ activity and, advantageously, are found to inhibit neurodegeneration. The pharmaceutical compositions are effective at reducing pathological damage related to neuonal injury and degenerative processes.

The invention also provides methods for treating a neurodegenerative disease comprising administering to a mammal (e.g., a human or veterinary patient) an efficacious dose of an active agent capable of inhibiting neurodegeneration. Several active agents and their structural formulae are disclosed herein for use in the method. In an aspect, the method comprises administering a pharmaceutical composition comprising an active agent of a type known to inhibit PLA₂, which include arachidonic acid derivatives and analogs (e.g., arachidonyl trifluoromethyl ketone) , benzenesulfonamides, aminosteroids, bromoenol lactone, manoalide, p-bromophenacyl bromide, minocycline, doxycycline, 7,7,-dimethyl-5,8- eicosadienoic acid, quinacrine, and the like, among others known in the art and those specifically disclosed herein and in the appended figures.

The invention also comprises a kit comprising a composition of a PLA₂ inhibitor which is an active agent and instructions for administering an efficacious dosage to a patient having a neurodegenerative disease. In an aspect, the neurodegenerative disease is Alzheimer's disease. In an aspect, the neurodegenerative disease is a non-Alzheimer's disease neurodegenerative disease. In one embodiment, the instructions recite treatment of Alzheimer's disease or another neurodegenerative disease as the specific indication for use of the pharmaceutical composition of the PLA₂ inhibitory active agent.

Other aspects of the invention will be evident by reference to the specification and examples provided herein. All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 (panels A and B) shows the expression of cytokine induced by Aβ peptide in human microglial cells. Panel A shows IL-1/3 and IL-6 cytokine release. Panel B shows TNFα release.

Figure 2 (panels A-D) shows ELISA results for TNFα or IL-1/3 released from A/3-stimulated microglial cells treated with AN 20606 or AN 20628.

Figure 3 shows survival curves for neuronal cultures exposed to A/3 peptide and varying concentrations of AN 20628 for three days.

Figure 4 shows survival curves for neuronal cultures exposed to Aβ peptide and varying concentrations of AN 20606 for three days. Figure 5 shows survival curves for neuronal cultures exposed to A/3 peptide and varying concentrations of AN 20628, AN20602, or AN 17935 for three days.

Figure 6 shows survival of cultured human cortical neurons pretreated with AN 20579, AN 20606, or AN 20628 for two hours prior to exposing the neuronal cells to a pathogenic concentration of A/3 peptide for three days.

Figure 7 shows the structural formulae of compounds listed in Table 1.

Figure 8 shows the structural formulae of compounds listed in Table 2.

Figure 9 shows the effect of the PLA₂ inhibitor AN20579 on cortical or hippocampal neurons contacted with mellitin.

Figure 10 shows the effect of the PLA₂ inhibitor AN20606 on PC12 neuronal cells induced to undergo apoptosis by serum withdrawal.

Figure 11 shows a generic synthetic route to obtain a benzenesulfonamide PLA₂ inhibitors of the invention. Figure 12 shows a synthetic route to obtain a preferred benzenesulfonamide PLA₂ inhibitor of the invention. Figure 13 shows the biological and biochemical activities of selected benzenesulfonamides. Figure 14 shows effects of AN20606, AN22669, and AN22831 o survival of rat sympathetic neurons treated with anti-NGF to induce cell death.

Figure 15 shows a synthetic pathway for synthesis of compounds of the invention.

Figure 16 shows the structures of various compounds of the invention identified by AN number.

Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

The term "A/3 peptide-mediated neurodegeneration" as used herein refers to degeneration of neuronal cells (e.g., cortical or hippocampal neurons, primary neuron cultures, neuronal cell lines) which is causally linked to accumulation of neurotoxic Aβ peptide; such toxicity may be manifested in the neuronal cells by direct interaction with toxic A/3 peptide or via indirect effects resulting from interaction of A/3 peptide with neuronal-associated cells (e.g., astrocytes, astrocytoma cells, microglial cells, monocytes, etc.) . Such indirect effects may involve nitric oxide formation, excitatory amino acid mimetics, and/or cytokine production by the non-neuronal calls, whereby such compounds produce neuronal cell damage. A/3 peptide-mediated neurodegenerative diseases are exemplified, but not limited to, Alzheimer's Disease. Some neuropathologies may be causally associated with aberrant forms or amounts of other fragments or isoforms of the APP gene besides A/3; these neuropathologies are also defined herein as Aβ peptide-mediated neurodegenerative diseases for purposes of this specification. The term "apoptotic neurodegeneration" as used herein refers to degeneration of neuronal cells (e.g., CNS or PNS neurons, primary neuron cultures, neuronal cell lines) which is causally linked to an apoptotic stimulus such as accumulation of a neurotoxic substance (e.g., an amyloidogenic polypeptide other than Aβ) or a reduction of a necessary growth factor (e.g., NGF, BDNF, CTNF, etc.) which effects neuronal apoptosis. Such apoptosis may be manifested in the neuronal cells by direct interaction with an amyloidogenic polypeptide other than Aβ peptide or via indirect effects resulting from interaction of an amyloidogenic polypeptide othe than Aβ on neuronal-associated cells (e.g., astrocytes, astrocytoma cells, microglial cells, monocytes, etc.) , or loss of trophic factors. Such indirect effects may involve nitric oxide formation, excitatory amino acid mimetics, and/or cytokine production by the non-neuronal calls, whereby such compounds produce neuronal cell damage. Some neuropathologies may be causally associated with aberrant forms or amounts of extracellular proteins other than A/3; these neuropathologies are also defined herein as amyloidogenic polypeptide-mediated neurodegenerative diseases for purposes of this specification; for illustration, an example of such a type of neuropathology is Creutzfeldt-Jakob disease.

The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring. As used herein, laboratory strains of rodents which may have been selectively bred according to classical genetics are considered naturally-occurring animals.

The term "active agent" is used herein to refer to an agent which is identified by one or more screening method(s) of the invention as an agent which inhibits PLA₂ activity and retards or reduces neurodegeneration. Active agents can be sold as commercial reagents for standardizing toxicological or pharmaceutical evaluations which employ neuron cultures or transgenic animals which exhibit neurodegenerative pathology. Some active agents will have therapeutic potential as drugs for human use, such as being administered to AD patients or individuals ascertained to be predisposed to developing AD or AD-type pathology (e.g., Down's Syndrome patients or familial AD) . Some active agents will have therapeutic potential as drugs for human use, such as being administered to patients or individuals ascertained to be predisposed to developing degenerative neuropathology

(e.g., Creutzfeldt-Jakob disease, Huntington's disease, stroke patients). Active agents are often small (<3,000 Daltons) organic molecules, but may be macromolecules (e.g., polypeptides, polynucleotides, etc.), inorganic compounds, including metal salts. A selective inhibitor of PLA₂ produces a preferential inhibition of PLA₂ as compared to inhibition of other mammalian phospholipases; such that the concentration required to produce inhibition of 50% of PLA₂ catalytic activity is at least one order of magnitude lower than the concentration required to produce inhibition of 50% of the catalytic activity of phospholipases other than PLA₂. A selective inhibitor of cPLA₂ produces a preferential inhibition of cPLA₂ as compared to inhibition of other mammalian PLA₂ enzymes. The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents are evaluated for potential activity as active agents by inclusion in screening assays described hereinbelow. Agents may be selected from a combinatorial compound library for the capacity to interact with and/or inhibit PLA₂. The agent library may be naive or may be composed of structural analogs of known PLA₂ inhibitors, or a combination of both. Example agents of a type known to inhibit PLA₂ would include arachidonic acid derivatives and analogs (e.g., arachidonyl trifluoromethyl ketone) , benzenesulfonamides, aminosteroids, bromoenol lactone, manoalide, p-bromophenacyl bromide, minocycline, doxycycline, 7,7,-dimethyl-5,8-eicosadienoic acid, quinacrine, and the like, among others known in the art and those disclosed specifically herein and in the appended drawings. Examples are: N-cycloheptyl-4-[N-methyl-N-[ (E)-3-(4- ethylsulfonylpheny1) -2-propenoy1]amino]benzenesulfonamide (AN20606) and N-cyclohepyl-4-[N-methyl-N[ (E) -3-(4- cyanopheny10-2-propenoy1]amino] benzenesulfonamide (AN 20628) and their derivatives.

The term "PLA₂" as used herein refers to a naturally-occurring mammalian PLA₂ polypeptide having enzymatic activity. A paradigmatic PLA₂ can be considered to be human cPLA₂ substantially equivalent to that such as that described in U.S. Patent 5,354,677 and 5,328,842; Clark et al. (1991) Cell 65: 1043, and Sharp et al. (1991) J. Biol. Chem. 266: 14850, or the cognate cPLA₂ enzyme in a non-human mammalian species. PLA₂ activity is present in a variety of cytosolic and extracellular PLA₂ polypeptide species. A preferred PLA₂ polypeptide of the invention is a cytosolic PLA₂, such as cPLA₂, and typically a calcium-activable cPLA₂ which is activated (exhibits enhanced catalytic activity) by the presence of calcium ions (Ca⁺²)

The term "pathogenic Aβ peptide" refers to polypeptides comprising a peptide sequence encoded by the APP gene which have the property of producing neurotoxicity on neuronal cell cultures and/or primary neurons, typically in the presence of microglial cells and/or astrocytic cells and/or monocytes, or directly; generally a pathogenic Aβ peptide comprises at least residues 25-35 of the amyloid β protein, and often consists of residues 1-40 or 1-42 of the amyloid β peptide. Generally, polypeptide sequences encoded by the APP gene which are flanking the 1-42 Aβ sequence -DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGWIA- are absent. Preferred Aβ peptides are: A/31-40, amino acid sequence = DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGW A/31-42, amino acid sequence = DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGWIA

Neurotoxicity of any A/3 peptide can be determined by assay for neuronal cell viability according to the methods of the invention and according to methods known in the art.

Typically, neurotoxicity of a pathogenic A/3 peptide will be dose-dependent. Furthermore, aggregation state of the A/3 peptide is believed to affect toxicity.

The term "amyloidogenic polypeptide" refers to polypeptides which form extracellular deposits and/or intracellular inclusions, and/or which have the property of producing neurotoxicity on neuronal cell cultures and/or primary neurons, typically in the presence of microglial cells and/or astrocytic cells and/or monocytes, or directly; as used in this disclosure, amyloidogenic polypeptides are not products of the APP gene or fragments thereof (e.g., Aβ peptide) . Sipe, JD (1992) Ann. Rev. Biochem. 61: 947 provides a review of several known amyloidogenic polypeptides. For illustration, mellitin is an amyloidogenic polypeptide for purposes of the invention as its contact with neuronal cell cultures induces apoptotic neurodegeneration of the neuronal cells, even if detectable amyloid deposits are not formed.

Neurotoxicity of any amyloidogenic polypeptide can be determined by assay for neuronal cell viability according to the methods of the invention and according to methods known in the art. Typically, neurotoxicity of an amyloidogenic polypeptide will be dose-dependent.

The term "cognate" as used herein refers to a gene sequence that is evolutionarily and functionally related between species. For example but not limitation, in the human genome, the human CD4 gene is the cognate gene to the mouse CD4 gene, since the sequences and structures of these two genes indicate that they are the most highly homologous match between the two species and both genes encode a protein which functions similarly (e.g., in signaling T cell activation through MHC class II-restricted antigen recognition) . As used herein, the term "xenogenic" is defined in relation to a recipient mammalian host cell or nonhuman animal and means that an amino acid sequence or polynucleotide sequence is not encoded by or present in, respectively, the naturally-occuring genome of the recipient mammalian host cell or nonhuman animal. Xenogenic DNA sequences are foreign DNA sequences; for example, a human cPLA₂ gene is xenogenic with respect to murine ES cells; also, for illustration, a human cystic fibrosis-associated CFTR allele is xenogenic with respect to a human cell line that is homozygous for wild-type (normal) CFTR alleles. Thus, a cloned murine nucleic acid sequence that has been mutated (e.g., by site directed mutagenesis) is xenogenic with respect to the murine genome from which the sequence was originally derived, if the mutated sequence does not naturally occur in the murine genome. As used herein, a "heterologous gene" or "heterologous polynucleotide sequence" is defined in relation to the transgenic nonhuman organism producing such a gene product. A heterologous polypeptide, also referred to as a xenogeneic polypeptide, is defined as a polypeptide having an amino acid sequence or an encoding DNA sequence corresponding to that of a cognate gene found in an organism not consisting of the transgenic nonhuman animal. Thus, a transgenic mouse harboring a human cPLA₂ gene can be described as harboring a heterologous cPLA₂ gene. A transgene containing various gene segments encoding a heterologous protein sequence may be readily identified, e.g. by hybridization or DNA sequencing, as being from a species of organism other than the transgenic animal. For example, expression of human cPLA₂ amino acid sequences may be detected in the transgenic nonhuman animals of the invention with antibodies specific for human cPLA₂ epitopes encoded by human cPLA₂ gene segments. A cognate heterologous gene refers to a corresponding gene from another species; thus, if murine cPLA₂ is the reference, human cPLA₂ is a cognate heterologous gene (as is porcine, ovine, or rat cPLA₂, along with cPLA₂ genes from other species) . A mutated endogenous gene sequence can be referred to as a heterologous gene; for example, a transgene encoding a murine cPLA₂ comprising a mutation (which is not known in naturally- occurring murine genomes) is a heterologous transgene with respect to murine and non-murine species. The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GTATA" .

The following terms are used to describe the sequence relationships between two or more polynucleotides: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, such as a polynucleotide sequence of Fig. 1, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window", as used herein, refers to a conceptual segment of at 22 least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2,: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr. , Madison, WI) , or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected. The term

"sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. The reference sequence may be a subset of a larger sequence.

As used herein, the term "transcriptional unit" or "transcriptional complex" refers to a polynucleotide sequence that comprises a structural gene (exons) , a cis-acting linked promoter and other cis-acting sequences necessary for efficient transcription of the structural sequences, distal regulatory elements necessary for appropriate tissue-specific and developmental transcription of the structural sequences, and additional cis sequences important for efficient transcription and translation (e.g., polyadenylation site, mRNA stability controlling sequences) .

As used herein, "linked" means in polynucleotide linkage (i.e., phosphodiester linkage). "Unlinked" means not linked to another polynucleotide sequence; hence, two sequences are unlinked if each sequence has a free 5' terminus and a free 3' terminus.

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.

As used herein, the term "targeting construct" refers to a polynucleotide which comprises: (1) at least one homology region having a sequence that is substantially identical to or substantially complementary to a sequence present in a host cell endogenous gene locus, and (2) a targeting region which becomes integrated into an host cell endogenous gene locus by homologous recombination between a targeting construct homology region and said endogenous gene locus sequence. If the targeting construct is a "hit-and-run" or "in-and-out" type construct (Valancius and Smithies (1991) Mol. Cell. Biol. 11: 1402; Donehower et al. (1992) Nature 356: 215; (1991) J. NIH Res. 2: 59; Hasty et al. (1991) Nature 350; 243, which are incorporated herein by reference), the targeting region is only transiently incorporated into the endogenous gene locus and is eliminated from the host genome by selection. A targeting region may comprise a sequence that is substantially homologous to an endogenous gene sequence and/or may comprise a nonhomologous sequence, such as a selectable marker (e.g., neo, tk, gpt) . The term "targeting construct" does not necessarily indicate that the polynucleotide comprises a gene which becomes integrated into the host genome, nor does it necessarily indicate that the polynucleotide comprises a complete structural gene sequence. As used in the art, the term "targeting construct" is synonymous with the term "targeting transgene" as used herein. The terms "homology region" and "homology clamp" as used herein refer to a segment (i.e., a portion) of a targeting construct having a sequence that substantially corresponds to, or is substantially complementary to, a predetermined endogenous gene sequence, which can include sequences flanking said gene. A homology region is generally at least about 100 nucleotides long, preferably at least about 250 to 500 nucleotides long, typically at least about 1000 nucleotides long or longer. Although there is no demonstrated theoretical minimum length for a homology clamp to mediate homologous recombination, it is believed that homologous recombination efficiency generally increases with the length of the homology clamp. Similarly, the recombination efficiency increases with the degree of sequence homology between a targeting construct homology region and the endogenous target sequence, with optimal recombination efficiency occurring when a homology clamp is isogenic with the endogenous target sequence.

The terms "functional disruption" or "functionally disrupted" as used herein means that a gene locus comprises at least one mutation or structural alteration such that the functionally disrupted gene is incapable of directing the efficient expression of functional gene product. The invention encompasses knockout animals, such as mice, which are homozygous for a functionally disrupted PLA₂ gene, typically a cPLA₂ gene. For example but not limitation, an endogenous cPLA₂ gene that has a neo gene cassette integrated into an exon (e.g., the second exon) of a cPLA₂ gene, is not capable of encoding a functional protein (isoform) that comprises the inactivated exon, and is therefore a functionally disrupted cPLA₂ gene locus. Also for example, a targeted mutation in the exons of an endogenous cPLA₂ gene may result in a mutated endogenous gene that can express a truncated PLA₂ protein. Functional disruption can include the complete substitution of a heterologous cPLA₂ gene locus in place of an endogenous cPLA locus, so that, for example, a targeting transgene that replaces the entire mouse cPLA₂ locus with a human cPLA₂ allele, which may be functional in the mouse, is said to have functionally disrupted the endogenous murine cPLA₂ locus by displacing it. Preferably, at least one exon which is incorporated into the mRNAs encoding most or all of the cPLA₂ isoforms are functionally disrupted. Deletion or interruption of essential transcriptional regulatory elements, polyadenylation signal (s), splicing site sequences will also yield a functionally disrupted gene. Functional disruption of an endogenous cPLA₂ gene, may also be produced by other methods (e.g., antisense polynucleotide gene suppression). The term "structurally disrupted" refers to a targeted gene wherein at least one structural (i.e., exon) sequence has been altered by homologous gene targeting (e.g., by insertion, deletion, point mutation(s), and/or rearrangement).

Typically, cPLA₂ alleles that are structurally disrupted are consequently functionally disrupted, however cPLA₂ alleles may also be functionally disrupted without concomitantly being structurally disrupted, i.e., by targeted alteration of a non- exon sequence such as ablation of a promoter. An allele comprising a targeted alteration that interferes with the efficient expression of a functional gene product from the allele is referred to in the art as a "null allele" or "knockout allele".

The term "alkyl" refers to a cyclic, branched, or straight chain alkyl group containing only carbon and hydrogen, and unless otherwise mentioned, contain one to twelve carbon atoms. This term is further exemplified by groups such as methyl, ethyl, n-propyl, isobutyl, t-butyl, pentyl, pivalyl, heptyl, adamantyl, and cyclopentyl. Alkyl groups can either be unsubstituted or substituted with one or more substituents, e.g., halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality. The term "lower alkyl" refers to a cyclic, branched or straight chain monovalent alkyl radical of one to six carbon atoms. This term is further exemplified by such radicals as methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl (or 2-methylpropyl) , cyclopropylmethyl, i-amyl, n-amyl, and hexyl.

The term "aryl" or "Ar" refers to a monovalent unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl) , which can optionally be unsubstituted or substituted with, e.g., halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality. The term "substituted alkoxy" refers to a group having the structure -O-R, where R is alkyl which is substituted with a non-interfering substituent. The term "arylalkoxy" refers to a group having the structure -O-R-Ar, where R is alkyl and Ar is an aromatic substituent. Arylalkoxys are a subset of substituted alkoxys. Examples of preferred substituted alkoxy groups are: benzyloxy, napthyloxy, and chlorobenzyloxy.

The term "aryloxy" refers to a group having the structure -O-Ar, where Ar is an aromatic group. A preferred aryloxy group is phenoxy.

The term "heterocycle" refers to a monovalent saturated, unsaturated, or aromatic carbocyclic group having a single ring (e.g., morpholino, pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzo[b]thienyl) and having at least one heteroatom, defined as N, O, P, or S, within the ring, which can optionally be unsubstituted or substituted with, e.g., halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality. The term "heteroaryl" or "HetAr" refers to an aromatic heterocycle.

"Arylalkyl" refers to the groups -R-Ar and -R-HetAr, where Ar is an aryl group, HetAr is a heteroaryl group, and R is straight-chain or branched-chain aliphatic group. Examples of arylalkyl groups include benzyl and furfuryl. Arylalkyl groups can optionally be unsubstituted or substituted with, e.g., halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.

As used herein, the term "halo" or "halide" refers to fluoro, bromo, chloro and iodo substituents.

As used in the structures that follow, the term "OBn" means benzyloxy. As used herein, the term "amino" refers to a chemical functionality -NR'R", where R' and R" are independently hydrogen, alkyl, or aryl. The term "quaternary amine" refers to the positively charged group -N⁺R'R"R"', where R' , R", and R'" are independently selected and are alkyl or aryl. A preferred amino group is -NH₂.

The term "pharmaceutical agent or drug" as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw- Hill Dictionary of Chemical Terms (ed. Parker, S. , 1985) ,

McGraw-Hill, San Francisco, incorporated herein by reference).

DETAILED DESCRIPTION

Overview A basis of the present invention is the unexpected finding that neuronal cell degeneration is mediated by a biochemical cascade which requires PLA₂ activity, an enzyme which catalyzes the hydrolysis of the fatty acid ester bond at the sn-2 position of membrane phospholipids to produce arachidonic acid and its metabolites, and in the case of the cytosolic form, cPLA₂, also produces lysophospholipids. Agents which selectively block PLA₂ activity in neurons (such as neuronal cell lines and cultured neurons) and/or glial cells (including glial cell lines) can be used to inhibit neuronal degeneration, such as that which results from exposure of such cells to pathogenic forms of A/3 or amyloidogenic protein as occurs in Alzheimer's Disease and other neurodegenerative conditions. These selective PLA₂- blocking agents can be used to inhibit and/or retard neuronal degeneration.

Cellular models of Alzheimer's Disease (AD) neuropathology are based on the ability of the Alzheimer's Disease-associated Aβ peptide to induce biological changes (e.g., microglia and astrocyte activation, monocyte activity, neuronal degeneration) in cultured human and rodent cell populations (neurons, neuronal cell lines, microglia, microglial cell lines, astrocytes, astrocytoma cells and cell lines, monocytes and monocytic cell lines) and neuronal and glial cell lines, wherein the biological changes mimic the neuropathological changes associated with Alzheimer's Disease. A basis of the invention is the unexpected observation that several structurally dissimilar inhibitors of PLA₂ were capable of inhibiting A/3-mediated, NGF withdrawal- mediated, or amyloidogenic protein-mediated microglial activation and A/3-mediated neuronal degeneration in such cellular models of AD. Based on this observation and others made by Applicants, it is believed that PLA₂ activity is an essential component of A/3-mediated and amyloidogenic-mediated neuropathological changes, such as those which occur in AD, Down's Syndrome, and other neurodegenerative diseases. Active agents which inhibit PLA₂ activity are expected to inhibit neuropathological changes.

Phospholipase A₂ Phospholipases A₂ (PLA₂s; EC 3.1.1.4) are enzymes that hydrolyze the 2-acyl ester bond of phosphoglycerides generating free fatty acids and lysophospholipids (for review, see, Kramer, RM (1993) Advances in Second Messenger and

Phosphoprotein Research 28: 81; Glaser et al. (1993) TiPS 14:

92; Dennis EA (1994) J. Biol. Chem. 269: 13057) . PLA₂s are a diverse class of enzymes with regard to function, localization, regulation, mechanism, sequence, structure, and role of divalent metal ions.

In general, PLA₂ enzymes catalyze the hydrolysis of the fatty acid ester bond at the sn-2 position of membrane phospholipids to produce arachidonic acid and its metabolites. A variety of polypeptide species can exhibit PLA₂ activity; for purposes of this specification, these polypeptides are considered PLA₂ isozymes.

Group I, II, and III PLA₂s are extracellular enzymes of approximately 14-18 kD in humans, and are designated sPLA₂s, in recognition of their secretion. sPLA₂s are found in many extracellular fluids and have a broad substrate specificity for many types of phospholipids. Group IV PLA₂ is a cytosolic enzyme of approximately 85 kD (based on deduced cDNA coding sequence) to 110 kD (based on SDS-PAGE of purified protein) , and is designated cPLA₂ to indicate its cytosolic location. Unlike sPLA₂s, the cPLA₂ enzyme exhibits preferential catalysis of phospholipids which contain arachidonic acid, and is most likely the enzyme responsible for arachidonic acid release which is the rate- limiting step for subsequent eicosanoid biosynthesis of pro- inflammatory lipid mediators (prostaglandins, leukotrienes, lipoxins, and platelet-activating factor: "PAF") .

Other P A₂ activities, both cytosolic and extracellular, are less well-characterized with regard to macromolecular identification and polypeptide sequences. cPLA₂ is present in the cytosol of a variety of species and cell types, including human U937 cells

(monocytes) , platelets, kidney, and macrophages, among others, and is implicated in controlling arachidonic acid metabolism and eicosanoid production.

Human cPLA₂ has been cloned as a cDNA isolated from mRNA of a human monocytic cell line (U.S. Patent 5,354,677 and 5,328,842; Sharp et al. (1991) op.cit; Clark et al. (1991) op.cit) and the mRNA encodes a protein of 749 amino acids which has little detectable homology with the secreted sPLA₂s or any other protein in known sequence databases. The cPLA₂ cDNA identifies a single copy gene in the human genome, with no detectable closely related genes based on Southern blotting experiments. cPLA₂ contains an amino-terminal domain which binds calcium and similar divalent cations, and cPLA₂ binds to membrane vesicles at submicromolar concentrations of Ca⁺² in a calcium-dependent fashion. cPLA₂ can translocate to membranes when activated in the presence of calcium. Presumably, cPLA₂ associates with membrane components in vivo under suitable calcium concentrations. Agents that stimulate the release of arachidonic acid (ATP, thrombin, phorbol ester, calcium ionophore) can cause increased serine phosphorylation of cPLA₂ which increases the enzymatic activity of cPLA₂ (Lin et al. (1993) Cell 72: 269) . Phosphorylation is believed to contribute to the control of cPLA₂ activity in vivo (Lin et al. (1992) Proc. Natl. Acad. Sci. (USA) ji9: 6147; Lin et al. (1993) Cell 12_.'. 269; Qiu et al. (1993) J. Biol. Chem. 268: 24506; Kramer et al. (1993) J. Biol. Chem. 268: 26796).

Antibodies have been raised against human cPLA₂ and crossreact with cPLA₂ from a variety of animals, indicating conservation of structure between species. Anti-cPLA₂ antibodies identify the presence of cPLA₂ in lung, brain, testis, kidney, spleen, liver, and heart, although the precise role of cPLA₂ in the metabolism of each of these tissues is not known.

The art generally recognizes the physiologic role of cPLA₂ to be in the mediation of inflammation via its role in arachidonic acid metabolism and lipid/lipoprotein metabolism, such as cell membrane homeostasis. Roshak et al. (1994) J. Biol. Chem. 269: 25999 used antisense oligonucleotides complementary to the cPLA₂ mRNA to inhibit prostaglandin production in LPS-induced monocytes, indicating a potential role for cPLA₂ in generating inflammatory regulators in monocytes. Verity MA (1993) Ann. N.Y. Acad. Sci. 679: 110 speculates that "abusive activation" of PLA₂ via uncontrolled Ca⁺² influx might produce irreversible cell injury of neurons via extensive localized lipid peroxidation and subsequent membrane disintegration. U.S. Patent 5,354,677 and 5,328,842 indicates that cPLA₂ inhibitors are expected to be used to treat inflammatory conditions, such as psoriasis, asthma, and arthritis (see, col. 15), and prophesizes that such anti- inflammatory compounds can be identified as cPLA₂ inhibitors. A number of inhibitors of PLA₂ activity have been reported. Trifluoromethyl ketones (e.g., palmitoyl trifluoromethyl ketone, arachidonyl trifluoromethyl ketone) have been reported to be capable of inhibiting a Ca⁺²- independent PLA₂ activity (Ackermann et al. (1995) J. Biol. Chem. 270: 445) as well as cPLA₂ (Street et al. (1993) Biochemistry 32: 5935) . Several benzenesulfonamide derivatives have also been reported to be capable of inhibiting PLA₂ activity (European Patent Application 468 054; Oinuma et al. (1991) J. Med. Chem. 34: 2260) .

Reynolds et al. (1994) Anal. Biochem. 217: 25 describe a convenient microtiter plate assay for cPLA₂. Currie et al. (1994) Biochem. J. 304: 923, describe a cPLA₂ assay for assaying cPLA₂ activity from activated whole cells. This assay can be adapted for assay of related PLA activity, whether from cPLA₂ or other PLA₂ enzymes having similar catalytic activities. A suitable source of cPLA₂ can be obtained, if desired, by expression of a recombinant expression vector in a suitable host cell, as described in U.S. Patent 5,354,677, or by conventional biochemical purification from mammalian cells, as is known in the art.

Methods for Identifying Neurodegeneration Inhibitors

One method to identify active agents which inhibit the development of neuropathology is simply brute force screening of all possible chemical structures in a suitable cellular and/or animal model of apoptotic neurotoxicity. Unfortunately, the complexity and structural potential of chemistry makes a thorough search of all of the chemical structural space impossible, even if facile synthetic methods were available for all potential compounds. Because an exhaustive search of chemical space is not possible, it is exceedingly important to identify properties of likely inhibitors of neurodegenerative processes involved in A/3 peptide neurotoxicity or amyloidogenic polypeptide neurotoxicity and related diseases. In order to expedite the screening of compound libraries and to increase the probability of obtaining active agents which inhibit neurodegeneration, it is desirable to preselect compounds which are known or suspected inhibitors of PLA₂ (based on structural homology to substrates or inhibitors) , and preferably are selective inhibitors of cytosolic PLA₂. The PLA₂ inhibitors are typically identified by initially employing a PLA₂ assay, which may comprise an in vitro PLA₂ enzyme assay using a standardized amount of a purified or recombinantly produced mammalian PLA₂, such as human cPLA₂, and/or may comprise a whole cell assay, or a combination thereof. For example and not limitation, a primary PLA₂ assay can be performed essentially according to Reynolds et al. (1994) Anal. Biochem. 217: 25, with an agent added to test assay reactions and compared to a control reaction lacking an added agent. Agents which are found to inhibit PLA₂ activity in the assay are then selected for subsequent testing in a secondary assay. An alternative primary assay can optionally comprise a whole cell PLA₂ assay, such as that disclosed in Currie et al. (1994) Biochem. J. 304: 923. Other suitable assays for measuring the capacity of an agent to inhibit PLA₂ will be apparent to those in the art in view of Applicants' specification. The primary PLA₂ assays can also be multiplexed, so that agents which are positively identified in one primary assay are verified as bona fide PLA₂ inhibitors in another type of primary assay. Preferably, the PLA₂ activity is a cytosolic PLA₂ enzyme, most typically a calcium-dependent cPLA₂.

Agents selected in the primary assay(s) as PLA₂ inhibitors are evaluated for their capacity to inhibit neuronal degeneration and/or microglial and/or astrocyte (astrocytoma cells) and/or monocyte activation in mammalian cortical or hippocampal cell cultures or neuronal cell line cultures, mixed neuronal/glial cultures, or the like, treated with an amyloidogenic polypeptide, A/3 peptide or variant thereof, or growth factor withdrawal as described in the Examples, or by other suitable neurotoxicity assays for measuring apoptotic neurodegeneration. These secondary assays measure the ability of a selected agent to inhibit neurodegeneration in neurodegenerative disease models. Typically, a secondary assay is performed using a primary rat or human cortical or hippocampal neuron culture and/or a rat or human cortical or hippocampal astrocyte/microglia culture, as described herein; alternatively, a neuronal cell line can be employed, typically with (1) primary glial cells and/or a glial cell line, and/or (2) primary astrocytes and/or an astrocytic cell line (astrocytoma cells) . However, other suitable neurodegeneration models can be employed, such as transgenic mice expressing an amyloidogenic polypeptide or Aβ and exhibiting neuropathology (e.g., a PrP transgenic mouse, APP717 transgenic mouse, APP Swedish mutation transgenic mouse) . A plurality of secondary assays may also be multiplexed, so that for example agents which score positive as in a neuronal cell culture neurodegeneration inhibition assay can be tested in a mammalian model of neurodegenerative disease (e.g., a transgenic mouse PrP model or Alzheimer's model) , and vice versa.

Thus, a primary screening assay to identify PLA₂ inhibitors is performed prior to a secondary screening assay to identify neurodegeneration inhibitors. An advantage of this approach is that is substantially reduces the chemical structure space which needs to be searched to identify neurodegeneration inhibitors. Furthermore, several structural families of PLA₂ inhibitors are known, whereas there is no known inhibitor of neurodegeneration which is substantially effective as a human therapeutic; thus the agent search can be focused to compounds likely to be PLA₂ inhibitors based on their structural homology to identified PLA₂ inhibitors or to PLA₂ inhibitors prophesized by rational design based on the known PLA₂ protein structures, such as human cPLA₂.

Agents A bank or library of agents is selected at the discretion of the practitioner. Typical agents will be structural congeners of known PLA₂ inhibitors, or compounds rationally predicted to have PLA₂ inhibition activity. In some, embodiments random or pseudorandom agent libraries can be employed, as can combinatorial chemistry libraries, peptide/peptoid libraries, and the like. In general, agents such as halogenated methylketones of arachidonic acid or palmitic acid, or the like, can be suitable PLA₂ inhibitors. Aminosteroids (e.g., 21- aminosteroids; lazaroids) and bromoenol lactone are suitable for use as agents, especially to obtain irreversible PLA inhibitors which may have an advantageous duration of action. Benzenesulfonamides and various arylsulfonamides are also suitable agents to include in a compound library of the invention. Typically, such compounds are selected from the group of known chemical compounds known in the chemical and pharmaceutical literature; from described compound libraries; from natural compounds which may comprise undetermined structures; and from other suitable sources of chemical diversity.

Essentially any type of agent desired by the practitioner may be evaluated using the method, although agents believed likely to have PLA₂ inhibition activity are typically preferred.

Examples of the types of compounds believed to be preferable for inclusion in agent libraries include: BIRM 270 (Farina et al. (1994) J. Pharmacol Exp. Therap. 271: 1418; Ro23-9358 (LeMahieu et al. (1993) J. Med. Chem. 36: 3029; U73122 (Chen et al. (1995) Life Sciences 56: 103); BMS-181162 (Tramposch et al. (1994) J. Pharmacol. Exp. Therap. 271: 852; Burke et al. (1995) J. Biol. Chem. 270: 274) ; and "Compound 1" (Abdullah et al. (1995) Bioorganic and Medicinal Chem. Let. 5 : 519; Hazen et al. (1991) J. Biol. Chem. 266: 7227), among others.

Agents which are identified as active agents for PLA₂ inhibition and inhibition of neurodegeneration are administered to cell populations comprising neuronal cells to reduce or arrest neuronal cell death via PLA₂-dependent pathways.

The agents can be any molecule, compound, or other substance which can be added to the cell culture or administered to a test animal without substantially interfering with cell or animal viability. Suitable test agents may be small molecules, biological polymers, such as polypeptides, polysaccharides, polynucleotides, and the like. The test compounds will typically be administered to transgenic animals at a dosage of from 1 ng/kg to 10 mg/kg, usually from 1 μg/kg to 1 mg/kg.

Preferably, active agents are able to cross the blood-brain barrier of a human to produce a therapeutically efficacious concentration in cerebrospinal fluid and CNS tissues (e.g., cortical or hippocampal neurons) . Other approaches to enhancing delivery of drugs, particularly across the blood-brain barrier, utilize pharmacologic-based procedures involving drug latentiation or the conversion of hydrophilic drugs into lipid-soluble drugs. The majority of the latentiation approaches involve blocking the hydroxyl, carboxyl and primary amine groups on the drug to make it more lipid-soluble and therefore more easily transported across the blood-brain barrier. Pardridge and Schimmel, U.S. Patent 4,902,505, disclose chimeric peptides for enhancing transport by receptor-mediated transcytosis.

Disease Model Systems Generally, the nomenclature used hereafter and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, cell culture, and transgene incorporation (e.g., electroporation, microinjection, lipofection) . Generally enzymatic reactions, oligonucleotide synthesis, and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references which are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference. Chimeric targeted mice are derived according to

Hogan, et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. , IRL Press, Washington, D.C, (1987) which are incorporated herein by reference.

Embryonic stem cells are manipulated according to published procedures (Teratocarcinomas and Embryonic Stem

Cells: A Practical Approach. E.J. Robertson, ed. , IRL Press, Washington, D.C. (1987); Zjilstra et al., Nature 342:435-438 (1989); and Schwartzberg et al., Science 246:799-803 (1989), each of which is incorporated herein by reference) . Oligonucleotides can be synthesized on an Applied

Bio Systems oligonucleotide synthesizer according to specifications provided by the manufacturer.

In one aspect of the invention are provided nonhuman animals harboring at least one copy of a transgene comprising a polynucleotide sequence which encodes a heterologous PLA₂ polypeptide operably linked to a transcription regulatory sequence capable of producing expression of the heterologous PLA₂ polypeptide in the transgenic nonhuman animal. Said heterologous PLA₂ polypeptide is expressed in cells which normally express the naturally-occurring endogenous PLA₂ gene (if present) . Typically, the nonhuman animal is a mouse and the heterologous PLA₂ gene is a human PLA₂ gene, such as the human cPLA₂ gene. Such transgenes typically comprise a PLA₂ expression cassette, wherein a linked promoter and, preferably, an enhancer drive expression of structural sequences encoding a heterologous PLA₂ polypeptide in neuronal cell types. Often, the mouse cPLA₂ gene is the inactivated target gene and optionally includes a transgene encoding a human cPLA₂ polypeptide having PLA₂ activity. The invention also provides transgenes comprising a gene encoding a human PLA₂, said gene operably linked to a transcription regulatory sequence functional in the host transgenic animal (e.g., a neural-specific promoter). Such transgenes are typically integrated into a host chromosomal location by nonhomologous integration. The transgenes may further comprise a selectable marker, such as a neo or gpt gene operably linked to a constitutive promoter, such as a phosphoglycerate kinase (pgk) promoter or HSV tk gene promoter linked to an enhancer (e.g., SV40 enhancer).

The invention further provides nonhuman transgenic animals, typically nonhuman mammals such as mice, which harbor at least one copy of a transgene or targeting construct of the invention, either homologously or nonhomologously integrated into an endogenous chromosomal location so as to encode a human PLA₂ polypeptide. Such transgenic animals are usually produced by introducing the transgene or targeting construct into a fertilized egg or embryonic stem (ES) cell, typically by microinjection, electroporation, lipofection, or biolistics. The transgenic animals express the human PLA₂ gene of the transgene (or homologously recombined targeting construct) , typically in brain tissue. Such animals are suitable for use in a variety of disease model and drug screening uses, for sales to commercial laboratories conducting toxicological evaluation of compounds believed likely of producing chronic neuronal toxicity, as well as other applications. The invention also provides nonhuman animals and cells which harbor at least one integrated targeting construct that functionally disrupts an endogenous PLA₂ gene locus, typically by deleting or mutating a genetic element (e.g., exon sequence, splicing signal, promoter, enhancer) that is required for efficient functional expression of a complete gene product.

The invention also provides transgenic nonhuman animals, such as a non-primate mammal, that have at least one inactivated endogenous PLA₂ allele, and preferably are homozygous for inactivated P A₂ alleles, and which are substantially incapable of directing the efficient expression of endogenous (i.e., wildtype) PLA₂. For example, in a preferred embodiment, a transgenic mouse is homozygous for inactivated endogenous PLA₂ alleles and is substantially incapable of producing murine PLA₂ encoded by a endogenous (i.e., naturally-occurring) PLA₂ gene. Such a transgenic mouse, having inactivated endogenous PLA₂ genes, is a preferred host recipient for a transgene encoding a heterologous PLA₂ polypeptide, preferably a human PLA₂ polypeptide. For example, human PLA₂ may be encoded and expressed from a heterologous transgene(s) in such transgenic mice. Such heterologous transgenes may be integrated in a nonhomologous location in a chromosome of the nonhuman animal, or may be integrated by homologous recombination or gene conversion into a nonhuman PLA₂ gene locus, thereby effecting simultaneous knockout of the endogenous PLA₂ gene (or segment thereof) and replacement with the human PLA₂ gene (or segment thereof) . A preferred PLA₂ gene is the cPLA₂ gene.

Such animals are suitable for use in a variety of disease model and drug screening uses, for sales to commercial laboratories conducting toxicological evaluation of compounds believed likely of producing chronic neuronal toxicity, as well as other applications.

Particular techniques for producing transgenic mice which express the Swedish form of /3APP, APP codon 717 variants, and other AD-associated transgenic disease models are described elsewhere in the art. It will be appreciated that the preparation of other transgenic animals expressing the Swedish human jSAPP and/or APP codon 717 mutants may easily be accomplished, including rats, hamsters, guinea pigs, rabbits, and the like. The effect of test compounds on PLA₂ activity in /3APP-transgenic test animals may be measured in various specimens from the test animals.

Particular techniques for producing transgenic mice which express an amyloidogenic polypeptide, and other neurodegenerative disease animal models are described elsewhere in the art. It will be appreciated that the preparation of other transgenic animals expressing an amyloidogenic polypeptide may be accomplished, including rats, hamsters, guinea pigs, rabbits, and the like. The effect of test compounds on PLA₂ activity in test animals may be measured in various specimens from the test animals.

An animal model of Parkinson's disease involving iatrogenic hydroxyl radical generation by MPTP (Chiueh et al. (1992) Synapse 11: 346, incorporated herein by reference) was used to evaluate the protective effect of C7 on free radical- induced damage. The neurotoxin, MPTP, has been shown to lead to the degeneration of dopaminergic neurons in the brain, thus providing a model of experimentally induced Parkinson's disease (e.g., iatrogenic toxicity) . This model is now widely accepted in the art and is used for evaluating potential therapeutic agents for this disease.

Antisense Polynucleotides Additional embodiments directed to modulation of

PLA₂ activity include methods that employ specific antisense polynucleotides complementary to all or part of the human or mouse PLA₂ sequences, such as antisense polynucleotides to the human cPLA gene or mRNA. Such complementary antisense polynucleotides may include nucleotide substitutions, additions, deletions, or transpositions, so long as specific hybridization to the relevant target sequence corresponding to human or mouse PLA₂ cDNA is retained as a functional property of the polynucleotide. Complementary antisense polynucleotides include soluble antisense RNA or DNA oligonucleotides which can hybridize specifically to PLA₂ mRNA species and prevent transcription of the mRNA species and/or translation of the encoded polypeptide (Ching et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 10006; Broder et al. (1990) Ann. Int. Med. 113: 604; Loreau et al. (1990) FEBS Letters 274: 53; Holcenberg et al., W091/11535; U.S.S.N. 07/530,165; WO91/09865; WO91/04753; WO90/13641; and EP 386563, each of which is incorporated herein by reference) . The antisense polynucleotides therefore inhibit production of PLA₂ polypeptides. Since PLA₂ protein expression is associated with activation and enzymatic activity, antisense polynucleotides that prevent transcription and/or translation of mRNA corresponding to PLA₂ polypeptides may inhibit PLA₂ activity and/or reverse the degeneration of neuronal and/or microglial cells and/or astrocytic cells and/or monocytic cells. Compositions containing a therapeutically effective dosage of PLA₂ antisense polynucleotides may be administered for treatment of neurodegenerative diseases, if desired. Antisense polynucleotides of various lengths may be produced, although such antisense polynucleotides typically comprise a sequence of about at least 25 consecutive nucleotides which are substantially identical to a naturally-occurring PLA₂ polynucleotide sequence, and typically which are identical to a human PLA₂ sequence, such as human cPLA₂.

Antisense polynucleotides may be produced from a heterologous expression cassette in a transfectant cell or transgenic cell. Alternatively, the antisense polynucleotides may comprise soluble oligonucleotides that are administered to the external milieu, either in the culture medium in vitro or in the cerebrospinal fluid or direct brain application in vivo. Soluble antisense polynucleotides present in the external milieu have been shown to gain access to the cytoplasm and inhibit translation of specific mRNA species and/or transcription of specific genes. In some embodiments the antisense polynucleotides comprise methylphosphonate moieties or are polyamide nucleic acids (PNAs) . For general methods relating to antisense polynucleotides, see Antisense RNA and DNA. (1988), D.A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) .

Apoptotic Neurodegenerative Disease Apoptotic neurodegenerative diseases are believed to comprise a broad variety of neurodegenerative diseases. For example and not to limit the invention, apoptotic neurodegenerative diseases are exemplified, but not limited to: Lewy Body disease, degeneration resulatant from cerebral ischemia, ALS, prion-related disease (Creutzfedlt-Jakob, kuru, etc.), Parkinson's disease, multiple sclerosis, hereditary ataxia, Shy Drager Syndrome, Progressive Supranuclear Palsy, Huntington's disease, spinal muscular atrophy (Types I, II, and III), Reye's Syndrome, status epilepticus, progressive multifocal leukoencephalopathy, viral encephalitis, normal pressure hydrocephalus, subacute sclerosing panencephalitis, head and spinal cord trauma post-injury degeneration, frontal lobe dementia, poliomyelitis and postpolio neuropathy, glaucoma, and various neuropathies (autonomic, Guillan-Barre, diabetic, porphyria, autoimmune, vasculitis, among others.

Apoptotic neurodegenerative diseases associated with amyloidogenic polypeptides include but are not limited to:

Prion-related diseases (e.g., Creutzfeldt-Jakob disease, scrapie, Kuru) ;

Transthyretin (TTR) -induced polyneuropathies (including, but not limited to: Portuguese, Japanese, Swedish, Illinois-German, Swiss-Indiana, Maryland-German, Appalachian-Israel) ;

ApoAl-induced polyneuropathy (e.g., Iowa variant); Gelsolin-induced polyneuropathy (e.g., Finnish variant); Icelandic Hemorrhage angiopathy due to cystatin C disease;

Serum AA amyloid polyneuropathy as seen in familial Mediterranean fever, and other conditions that case AA amyloidosis, such as leprosy, tuberculosis, rheumatoid arthritis; and Immunoglobulin/light chain amyloid polyneuropathy as seen in multiple myeloma and primary amyloidosis; among others. It is beleived that these diseases and other neurodegenerative diseases involvoing neuronal apoptotis can be treated by administration of a therapeutically efficacious dose of a suitalble PLA₂ inhibitor. Such administration will often require chronic dosing. Other neurodegenerative diseases are described in: 19th Edition: Cecil Textbook of Medicine. Wyngaarden, Smith and Bennett, eds. pp. 1141-1145, 1992, W.B. Saunders, Philadelphia, PA.

Compositions of Neurodegeneration Inhibitors Active agents which are PLA₂ inhibitors and also inhibit neuronal degeneration in disease models can be used to retard or reduced neuropathology in vivo. Thus, the present invention further comprises pharmaceutical compositions incorporating a compound selected by the above-described method and including in a pharmaceutically acceptable carrier. Such pharmaceutical compositions should contain a therapeutic or prophylactic amount of at least one compound identified by the method of the present invention. The pharmaceutically acceptable carrier can be any compatible, non-toxic substance suitable to deliver the compounds to an intended host. Sterile water, alcohol, fats, waxes, and inert solids may be used as the carrier. Pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like may also be incorporated into the pharmaceutical compositions. Preparation of pharmaceutical conditions incorporating active agents is well described in the medical and scientific literature. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 16th Ed., 1982, the disclosure of which is incorporated herein by reference.

The pharmaceutical compositions just described are suitable for systemic administration to the host, including both parenteral, topical, and oral administration, including intracranial administration. Thus, the present invention provides compositions for administration to a host, where the compositions comprise a pharmaceutically acceptable solution of the identified PLA₂-inhibitory compound in an acceptable carrier, as described above. Such formulations can be used therapeutically on mammals having AD-type neuropathology or disease progression of a related neurodegenerative disease. Compositions containing the present PLA₂ inhibitors can be administered for prophylactic and/or therapeutic treatments of neurodegenerative disease. In therapeutic application, compositions are administered to a patient already affected by the particular neurodegenerative disease, in an amount sufficient to cure or at least partially arrest the condition and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose" or "efficacious dose." Amounts effective for this use will depend upon the severity of the condition, the general state of the patient, and the route of administration, but generally range from about 1 mg to about lOg of PLA₂ inhibitor per dose, with dosages of from 10 mg to 2000 mg per patient being more commonly used. Suitable concentrations (i.e., efficacious dose) can be determined by various methods, including generating an empirical dose-response curve, predicting potency and efficacy of a congener by using QSAR methods or molecular modeling, and other methods used in the pharmaceutical sciences. The compositions for parenteral administration will commonly comprise a solution of an active agent or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier or organic solvent (e.g., DMSO, solvated PEG, etc.). Since many of the active agents of the invention can be lipophilic or latentiated, it is preferable to include in the carrier a hydrophobic base (e.g., polyethylene glycol, Tween 20) . A variety of aqueous carriers can be used, e.g. , water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the active agent in these formulations can vary widely, i.e.. from less than about 1 nM, usually at least about O.lmM to as much as 100 mM and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Most usually, the active agent is present at a concentration of 0.1 mM to 5 M. For example, a typical formulation for intravenous or intracranial injection comprises a sterile solution of an active agent at a concentration of 1-500 mM in Ringer's solution. The generally hydrophobic nature of some of the active agents indicates that a hydrophobic vehicle may be used, or that an aqueous vehicle comprising a detergent or other lipophilic agent (e.g., Tween, NP-40, PEG) ; alternatively, the active agents may be administered as a suspension in an aqueous carrier, or as an emulsion.

Thus, a typical pharmaceutical composition for intramuscular injection could be made up to contain 10 ml sterile buffered water, and about 1-1000 mg of active agent. A typical composition for intravenous infusion can be made up to contain 250 ml of sterile Ringer's solution, and about 100- 5000 mg of active agent. Lipophilic agents may be included in formulations. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science. 15th Ed. , Mack Publishing Company, Easton, Pennsylvania (1980) , which is incorporated herein by reference.

The invention also provides the use of a PLA₂ inhibitor to slow, arrest, or reverse the development of a neurodegenerative disease such as Alzheimer's disease or

Down's Syndrome in a human patient; an efficacious amount of the PLA₂ inhibitor is administered to the patient to inhibit progression of the disease.

The invention also provides the use of a PLA₂ inhibitor to slow, arrest, or reverse the development of a neurodegenerative disease in a human patient; an efficacious amount of the PLA₂ inhibitor is administered to the patient to inhibit progression of the disease.

The following examples are provided for illustration and are not intended to limit the invention to the specific example provided.

EXPERIMENTAL EXAMPLES EXAMPLE 1: Aθ-MEDIATED NEURODEGENERATION General Methods Pathogenic Aβ peptide: The following A/3 peptides were synthesized and used, typically after being dissolved in water. The Aβ peptides typically aggregate and/or change the folding state of the peptide over time into conformations having varying pathogenicity/neurotoxicity. Each batch of Aβ peptide stock solution is checked for toxicity on neuronal cell cultures or mixed neuronal/glial cell cultures, according to methods described herein and methods known to those skilled in the art.

A/31-40, amino acid sequence =

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGW A/31-42, amino acid sequence = DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGWIA

Primary Rat Cortical or Hippocampal Neurons:

Cultures of rat cortical neurons were established from 18 day rat fetuses. Cortical tissue was dissociated by incubation in a trypsin/EDTA solution (0.05% trypsin + 0.53 mM EDTA in HBSS; Gibco) for 20 minutes at 37°C. The trypsin was then inactivated by resuspending the cells in serum-containing medium (DMEM/FBS) : Dulbecco's Modified Eagle's Medium (DMEM) containing 4.5 g/L glucose, 1 mM sodium pyruvate, 1 mM glutamine, 100 Units/ml penicillin, 100 μg/ml streptomycin, and supplemented with 10% heat-inactivated fetal bovine serum (Gibco) . Cells were then pelleted by centrifugation and resuspended in a chemically-defined medium (DMEM/B27) : DMEM containing B27 supplement (Gibco) in place of FBS. Polyethyleneimine (PEI) -coated 6.4-mm (96-well) dishes were rinsed once with DMEM/FBS, and then seeded at 0.75 - 1.25 X 10⁵ cells per well in 0.1 ml DMEM/B27. Cultures were maintained in a H₂0)-saturated incubator with an atmosphere of 90% air/10% C0₂ at 37°C. Cell viability was visually assessed by phase contrast microscopy and quantified by measuring the reduction of alamarBlue™ (Alamar Biosciences, Inc.) as described below. Serum replacement with B27 supplement yields nearly pure neuronal cultures as judged by immunocytochemistry for glial fibrillary acidic protein and neuron-specific enolase (Brewer et al. (1993), J. Neurosci. Res. 35(5) :567- 576.

Primary Human Cortical or Hippocampal Neurons: Cultures of human cortical neurons were prepared using a modification of the procedure described in P. Seubert et al. (1992), Nature 359:325-327. Cortical tissue was dissociated by incubation in a trypsin/EDTA solution (0.05% trypsin + 0.53 mM EDTA in HBSS; Gibco) for 20 minutes at 37°C. The trypsin was then inactivated by resuspending the cells in serum- containing medium (MEM/FBS) : Modified Eagle's Medium (MEM) containing 1% glucose, 1 mM sodium pyruvate, 1 mM glutamine, and supplemented with 10% fetal bovine serum (Gibco) . Cells were then pelleted by centrifugation and resuspended in a chemically-defined medium (MEM/B27) : MEM containing B27 supplement (Gibco) in place of FBS. Polyethyleneimine (PEI)- coated 6.4-mm (96-well) dishes were rinsed once with MEM/FBS, and then seeded at 0.75 - 1.25 X 10⁵ cells per well in 0.1 ml MEM/B27. Cultures were maintained in a H₂0-saturated incubator with an atmosphere of 95% air/5% C0₂ at 37°C. The culture medium was exchanged twice weekly.

Primary Human Cortical or Hippocampal Astrocytes and Microglia: Cultures of human cortical astrocytes and microglia were prepared using a modification of the procedure described for cortical neurons. Cortical tissue from fetuses of 16 to 20 weeks of gestation was washed 3 times in Ca²⁺/Mg²⁺ free Hanks balanced salt solution (CMF HBSS) and then dissociated by repeated pipetting. The solution was brought to a final volume of 80 ml CMF HBSS for approximately 10 ml of tissue. DNase (Sigma) was added to a final concentration of 0.05 mg/ml. 20 ml of the solution was passed through one 100 μm nylon cell strained (Falcon) . The cells were then centrifuged for 5 minutes at 200 X G in an IEC Clinical Centrifuge and resuspended in a trypsin/EDTA solution (0.05% trypsin + 0.53 mM EDTA in HBSS; Gibco) and incubated for 20 minutes at 37°C (10 ml of trypsin was added per 2-3 ml of tissue) . The trypsin was then inactivated by adding (MEM/FBS): Modified Eagle's Medium (MEM) containing 1% glucose, 1 mM sodium pyruvate, 1 mM glutamine, and supplemented with 10% fetal bovine serum (JRH) . After adding a final concentration of 0.05 mg/ml DNase the cells were resuspended and then pelleted by centrifugation and resuspended in MEM/FBS. 1.6 X 10⁸ cells were seeded in a T- 150 tissue culture flask coated with polyethyleneimine (PEI) . (10% PEI (Sigma) was diluted 1:10 in H₂0, filtered through a 45 mm unit and then diluted into 150 mM sodium borate pH 8.5 at 1:100. The flasks were coated overnight at room temperature, washed two times in PBS and coated with 20 ml/flask of MEM/FBS at 37°C for at least one hour prior to plating cells.) Cultures were maintained in a H₂0-saturated incubator with an atmosphere of 95% air/5% C0₂ at 37°C. The culture medium was changed one and four days after plating and the cultures were then left undisturbed for at least one week. After approximately two weeks in vitro, the flasks were gently shaken and floating microglia were collected and centrifuged for 5 minutes at 200 X G in an IEC Clinical Centrifuge. The microglia were reseeded in 96 well tissue culture plates at a density of 5,000-40,000 cells/well in 125 μl in MEM/FBS. Astrocyte cultures were prepared by multiple passaging of the established mixed brain cell cultures. Each T-150 was incubated for 3-4 minutes at 37°C with a trypsin/EDTA solution (see above) . The trypsin was then inactivated by adding MEM/FBS. The cells were triturated and then pelleted by centrifugation and resuspended in MEM/FBS. The cells from one T-150 were seeded at a 1:30 to 1:5 dilution in T-150's not coated. Just prior to confluency the cells were repassaged by trypsinization as described above. This process was repeated until the cultures were >98% pure astrocytes.

Experimental treatments and analysis of neuronal survival: Amyloid-/3 (A/3) stock solutions were prepared as 1 mM stocks in sterile ddH₂0 immediately prior to addition to cultures. Rat cortical neurons were exposed to Aβ by removing the culture medium and replacing it with DMEM/N2 or DMEM/B27 containing A/31-40. Human cortical neurons were exposed to Aβ by removing the culture medium and replacing it with MEM, MEM/N2, or MEM/B27 containing A/31-40. Cultures were maintained for 2-4 days before neuronal survival was quantified using alamarBlue™.

Neurotoxicity Assay using alamarBlue™: The alamarBlue™ assay incorporated a proprietary fluorometric/colorimetric metabolic indicator (Alamar

Biosciences, Inc.). Viable cells convert alamarBlue™ from an oxidized (non-fluorescent, blue) form to a reduced (fluorescent, red) form. Assays were performed by replacing the culture media with a 10% alamarBlue™ solution in DMEM (rat cortical cultures) or MEM (human cortical cultures) . Reduction of alamarBlue™ was determined spectrofluorometrically using a Millipore Cytofluor 2350 Scanner (excitation, 560 mM; emission, 590 nm) and CytoCalc™ software (Millipore Corporation) . Neuronal viability as assessed by alamarBlue™ was comparable to that obtained by measuring the fluorogenic probe calcein AM, the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) , or the reduction of the tetrazolium salt, 2,3-bis (2-methoxy-4-nitro- 5-sulfophenyl) -2H-tetrazolium-5-carboxanilide (XTT) . Assays of phospholipase A₂ activity

Arachidonic acid release assay: Cortical neurons or microglia are labelled overnight with ³H-arachidonic acid. Cultures wells are rinsed several times with medium containing fatty acid free serum albumin and then treated with an activator of phospholipase A₂. Released ³H-arachidonic acid is measured after various time-points. The amount of released ³H-arachidonic acid is an indirect measurement of the activity of phospholipase A₂ to cleave arachidonic acid from the sn-2 position of membrane phospholipids. Fatty acid free serum albumin serves as a trap for released ³H-arachidonic acid.

Cytosolic phospholipase A₂ activity can be determined indirectly by measuring phospholipase A₂-mediated release of eicosanoids (prostaglandins, thromboxanes, oxygenated metabolites of arachidonic acid, and leukotrienes) [e.g., Currie et al., Biochem. Journal (1994) 304: 923], platelet activating factor, or lysophosphatidic acid. Cytosolic phospholipase A₂ activity can also be measured indirectly by measuring the extent of cPLA₂ phosphorylation [Lin et al. (1993), Cell 72:269-78.

A3 Induces Cytokine Release in Microglia

Cultured microglial cells were treated with 50 μM of A/31-40 or vehicle only (Control). The levels of IL-1/3, IL-6, and TNF-α released into the culture medium were determined by ELISA assay (R&D Systems) according to manufacturer's instructions. Figure 1 shows the results, indicating that A/31-40 stimulates release of IL-1/3, IL-6, and TNF-α.

Benzenesulfonamide Effect on Microglia

The effect of two benzenesulfonamide inhibitors of PLA₂ (European Patent Application 468 054) were examined on the activation of microglial cells by A/31-40 as in Example 1. N-cycloheptyl-4-[N-methyl-N-[ (E) -3-(4-methylsulfonylphenyl)-2- propenoyl]amino]benzenesulfonamide (AN20606) and N-cyclohepyl- 4-[N-methyl-N[ (E)-3-(4-cyanophenyl0-2-propenoyl]amino] benzenesulfonamide (AN 20628) at levels of 0 μM, 5 μM, 10 μM, and 20 μM were added to microglial cultures that had been stimulated with 50μM AjSl-40 for one day; control cultures were not stimulated with A/31-40. The amount of TNF-α and IL-1/3 released into the culture medium was measured by ELISA. Figure 2 shows the results graphically. Both cPLA₂-inhibitory compounds resulted in a marked and dose-dependent decrease in the amount of IL-1/3 and TNF-α released into the medium as a consequence of A/31-40 treatment, indicating that these PLA₂- inhibitors also inhibit the induction of cytokine secretion by microglial cells exposed to A/3.

Selectivity of Aβ Toxicity

The effects of the benzenesulfonamide PLA₂ inhibitor AN20606 and the selective cytosolic PLA₂ inhibitor arachidonyl trifluoromethyl ketone (AN20579) were examined in cultured human cortical microglia for their selectivity for inhibiting microglia activation mediated by amyloid-/3 peptide and lipopolysaccharides (LPS) . LPS are a major constituent of the cell wall of gama-negative bacteria and are extensively used for generating inflammatory responses in cultured cells and in vivo. As show in Table 3, AN20606 and AN20576 selectively inhibited A/31-40-mediated IL-1/3 and TNFα release. LPS- mediated cytokine release was actually enhanced in the presence of AN20606 and AN20579. The results are shown in Table l.

This data indicates that PLA₂ inhibitors are not general anti-inflammatory agents in human microglia, but are selective inhibitors of A/3-mediated inflammation.

Table 1

Treatment IL-1 Released (pg/ml) TNF Released (pg/ml)

Control ND ND

50 μM Aβl-40 273 ± 65.5 105 ± 41

50 μM AB1-40 + 20 μM AN20606 16.5 ± 6 (6%) 3.7 ± 1.4 (4%)

50 μM AB1-40 + 20 μM AACOCF3 36 ± 7.2 (13%) 54.8 ± 9.4 (52%)

Control MD ND

10 ng/ml LPS 22.7 ± 5.2 488 ± 4

10 ng/ml LPS + 20 μM AN20B06 37 ± 0.3 (163%) 632 ± 211 (130%)

10 ng ml LPS + 20 μM AACOCF3 51 ± 6.4 (225%) 785 ± 50 (161%) Dose-Dependence of Benzenesulfonamide Activity

The effect of the two benzenesulfonamide inhibitors of PLA₂ in Example 2 were examined to determine the dose- dependence of their effect on neuronal survival in human cortical neuron cultures exposed to 0 μM, 25μM, or 50 μM of AjSl-40 and varying doses of the benzenesulfonamide.

Figure 3 shows that N-cyclohepyl-4-[N-methyl-N[ (E)- 3-(4-cyanophenyl0-2-propenoyl]amino] benzenesulfonamide (AN 20628) produces a dose-dependent increase in neuronal survival in the presence of pathogenic A/3 peptide. Figure 4 shows that N-cycloheptyl-4-[N-methyl-N-[ (E) -3-(4-methylsulfonylphenyl)-2- propenoyl]amino]benzenesulfonamide (AN20606) also produces a dose-dependent increase in neuronal survival in the presence of pathogenic A/3 peptide. This demonstrates that two PLA₂ inhibitors reduce neuronal toxicity associated with Aβ in a dose-dependent relationship.

Specificity of Action for PLA₂ Inhibition

To determine the specificity of action of the benzenesulfonamide PLA₂ inhibitor as resulting from selective inhibition of PLA₂, we compared the effect of two other phospholipase inhibitors: l-[6-[ [ (17/3) -3-methoxyestra- 1,3,5(10)-trien-17-yl]amino]hexyl]-lH-pyrrole-2,5-dione (Chen et al. (1995) Life Sciences 56; 103), which inhibits both PLA₂ and PC-PLC (phosphatidyl choline-phospholipase C) and potassium 8(9)-tricyclo[5.2.1.0²'⁶]decyl xanthate (AN20602) , which is a specific PC-PLC inhibitor. Figure 5 graphically shows the dose-dependent effect of each of these phospholipase inhibitors on survival of neurons in human cortical neuron cultures exposed to A31-40. As is shown in Figure 5, the effect on enhancing neuronal survival is consistent with specific inhibition of PLA₂ activity, and inhibition of PLC is relatively ineffective in enhancing neuronal survival after exposure to Aβ . Pretreatment with PLA₂ Inhibitors The effect of pretreating human cortical neuron cultures with the benzenesulfonamide cPLA₂ inhibitors and arachidonyl trifluoromethyl ketone prior to exposure to a neurotoxic concentration of A/31-40 was determined. Figure 6 shows that pretreatment with any of the three PLA₂ inhibitors produced substantially decreased neuronal death resulting from A/31-40 exposure.

Effect of Various PLA2 or PLC Inhibitors on Aβ Neurotoxicity in Human Cortical Neurons

Human cortical neurons were treated with 50 μM A/3l- 40 and various inhibitors of PLA₂ or PLC. Compounds were added at the time of A/31-40 treatment at concentrations 390 nM to 100 μM. Compounds were also added to neuronal cultures in the absence of A/31-40. Neuronal survival was determined after 3 days of treatment by alamarBlue®. IC50 is the dose of compound that inhibited A/3-mediated neurotoxicity by 50%. TD10 and TD50 are the concentrations of compound that resulted in 10% and 50% loss of neuronal survival, respectively, when compound was added to neuronal cultures for 3 days in the absence of A/31-40. Data represents the mean ± SD (n = 3) . ND, not detected at the concentration range used. Results are shown in Table 2. Compound structures are shown in Fig. 7.

Table 2

Pretreatment with Inhibitor Human cortical neurons were treated with 50 μM A/3l- 40 and various inhibitors of PLA₂ or PLC. Compounds were added 2 hours prior to A/31-40 treatment at concentrations of 390 nM to 100 μM, and were then re-added at the time of A/31-40 treatment. Compounds were also added in an identical manner to neuronal cultures in the absence of A/31-40. Neuronal survival was determined after 3 days of treatment by alamarBlue®. IC50 is the dose of compound that inhibited A/3- mediated neurotoxicity by 50%. TD10 and TD50 are the concentrations of compound that resulted in 10% and 50% loss of neuronal survival, respectively, when compound was added to neuronal cultures for 3 days in the absence of A/31-40. Data represents the mean ± SD (n = 3) . ND, not detected at the concentration range used. The results are shown in Table 3. Compound structures are shown in Fig. 8.

Table 3

EXAMPLE 2: INHIBITION OF NON-A3 APOPTOTIC STIMULATED NEURODEGENERATION

Tissue Culture Methods PC12 cell cultures Stock cell cultures were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum. Experiments in serum-free medium were performed as previously described (Rukenstein et al. (1991) , J. Neurosci. 11:2552-2563) except the cells were plated at a density of 25-100 X 10³ cells per well in 96-well plastic culture dishes (surface area ^" 0.4 cm²). The bottom surfaces of the wells were precoated with rat-tail collagen as described elsewhere (Green and Tischler (1982) , Adv. Cell Neurobiol. 3:373-414) , and the volume of the medium per well was 100 μl.

Rat sympathetic neurons

Postnatal day 1-3 rat superior cervical ganglion (SGC) cell were dissociated and plated as previously described (Lee et al. (1980), Neuroscience 5:2239-2245) at a density of 0.5 ganglion per well in 96-well plastic culture plates (surface area 0.4 cm²). Neurons were maintained in 200 μl of culture medium (Eagles' MEM with Earle's salts, 10% fetal bovine serum, 2 mM L-glutamine, 100 μg/ml streptomycin, and 100 U/ml penicillin, and 100 ng/ml nerve growth factor (NGF) . The growth of nonneuronal cells (i.e., fibroblasts and Schwann cells) was inhibited by the addition of 20 μM uridine and 20 μM fluorodeoxyuridine) . Neurons were deprived of NGF by replacing the NGF-containing medium with the same medium, except that a polyclonal goat anti-NGF antiserum was substituted for NGF. Neuronal survival was determined 24-30 hours after NGF deprivation by visual inspection using phase- contrast light microscopy and the metabolic indicator AlamarBlue™. Rat Cortical Neurons

Primary rat cortical cultures were established from 18 day rat fetuses. Cortical tissue was dissociated by incubation in a trypsin/EDTA solution (0.05% trypsin + 0.53 mM EDTA in HBSS; Gibco) for 20 minutes at 37°C. The trypsin was then inactivated by resuspending the cells in serum-containing medium (DMEM/FBS): Dulbecco's Modified Eagles's Medium (DMEM) containing 4.5 g/L glucose, 1 mM sodium pyruvate, 1 mM glutamine, 100 Units/ml penicillin, 100 μg/ml streptomycin, and supplemented with 10% heat-inactivated fetal bovine serum (Gibco) . Cells were then pelleted by centrifugation and resuspended in a chemically-defined medium (DMEM/B27) : DMEM containing B27 supplement in place of FBS(Gibco). Polyethyleneimine (PEI) -coated 6.4 mm (96-well) dishes were rinsed with PBS, coated with DMEM/FBS, and then seeded at 0.75 - 1.25 X 10⁵ cells per well in 0.1 ml DMEM/B27. Cultures were maintained in a H₂0-saturated incubator with an atmosphere of 90% air/10% C0₂ at 37°C. Serum replacement with B27 supplement yields nearly pure neuronal cultures as judged by immunocytochemistry for glial fibrillary acidic protein and neuron-specific enolase (Brewer et al., J. Neurosci. Res. 35(5) :567-576, 1993).

Human Cortical Neurons Primary human cortical neuron cultures were established using dissociated human cerebral cortical tissue at 16-20 weeks gestation. The protocol for obtaining postmortem fetal neural tissue complied with all federal guidelines for fetal research and with the Uniformed Anatomical Gift Act. Cortical tissue was dissociated by incubation in a trypsin/EDTA solution (0.05% trypsin + 0.53 mM EDTA in HBSS; Gibco) for 20 minutes at 37°C. The trypsin was then inactivated by resuspending the cells in serum-containing medium (MEM/FBS) : Modified Eagles's Medium (MEM) containing 1% glucose, 1 mM sodium pyruvate, 1 mM glutamine, and supplemented with 10% fetal bovine serum (Gibco) . Cells were then pelleted by centrifugation and resuspended in a chemically-defined medium (MEM/B27) : MEM containing B27 supplement in place of FBS (Gibco). Polyethyleneimine (PEI)-coated 6.4-mm (96-well) dishes were rinsed once with MEM/FBS, and then seeded at 0.75- 1.25 X 10⁵ cells per well in 0.1 ml MEM/B27. Cultures were maintained in a H₂0-saturated incubator with an atmosphere of 95% air/5% C0₂ at 37°C. The culture medium was exchanged twice weekly and 10 μM ara-C (an antimitotic) was added with every other feeding. The use of B27 supplement and treatment with ara-C allowed the establishment of highly enriched human neuronal cultures.

Cell Survival Assays AlamarBlue™ assay

The AlamarBlue™ assay incorporates a proprietary fluorometric/colorimetric metabolic indicator (Alamar

Biosciences, Inc.) . Viable cells convert alamarBlue™ from an oxidized (non-fluorescent, blue) form to a reduced (fluorescent, red) form. Assays were performed by replacing the culture media with a 10% alamarBlue™ solution in RPMI (PC12 cells) , MEM (rat sympathetic neurons) , or DMEM (rat cortical neurons) . Reduction of alamarBlue™ was determined spectrofluorometrically using a Millipore Cytofluro 2350 Scanner (excitation, 560 nm, emission, 590 nm) and CytoCalc™ software (Millipore Corporation) . Neuronal viability as assessed by alamarBlue™ was comparable to that obtained by measuring the fluorogenic probe Calcein AM, the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) , or the reduction of the tetrazolium salt, 2,3-bis(2-methoxy-4-nitro- 5-sulfophenyl)-2H-tetrazolium-5-carboxanilde (XTT) (Rydel et al., unpublished data).

Lactate dehydrogenase (LDH) kinetic assay

The LDH kinetic assay utilized LD-L 10 reagent (Sigma #228-10) . 20 μl of culture supernatant was assayed using 200 μl of reconstituted LD-L 10 reagent. Samples were read every 30 seconds over a 5 minute time period using a kinetic microplate reader (UV_max, Molecular Devices) and SOFTmax® version 2.32 FPU software. Absorbance at 340 nm minus 650 nm was used to determine the rate of formation of reduced nicotinamide adenine dinucleotide (NADH) . The reaction rate was linear over this time period. The rate of reduced NADH formation is directly proportional to LDH activity in the sample. Fluorescent values were converted to U/ml by the inclusion of a LDH standard curve on each assay plate using LDH controls (Sigma # S2005 and S1005) .

Mellitin-Induced Neurodegeneration

Melittin is a 26 amino acid peptide derived from bee venom and is an activator of PLA₂ (Shier, .T. (1979) Proc. Natl. Acad. Sci. USA 76:195-199). We sought to determine if PLA₂ activation was toxic to rat cortical neurons and if this toxicity was inhibited by arachidonyl trifluoromethyl ketone, a PLA₂ inhibitor that is selective for calcium-dependent and calcium-independent cytosolic PLA₂s (Street et al. (1993) , Biochemistry 32:5936-5940; Ackermann et al. (1995), J. of Biol. Chem. 270:445-450). As shown in Fig. 9, panel (A), Melittin at a concentrations between 0.1 and 10 μM is toxic to rat cortical neurons over a 4 hour time period. As shown in Fig. 9, panel (B) , arachidonyl trifluoromethyl ketone inhibits melittin toxicity as measured by alamarBlue. As shown in Fig. 9, panel (C) , arachidonyl trifluoromethyl ketone (AN20579) inhibits melittin toxicity as measured by the release of the cytoplasmic enzyme LDH, a widely used measurement of neurodegeneration in cultured cortical neurons (Koh and Choi (1987), J. Neurosci. Methods 20:83) . Figure 9 shows the effect of the PLA₂ inhibitor AN20579 on cortical neurons contacted with mellitin.

Serum Withdrawal Apoptosis

Serum withdrawal from cultured PC12 cells is model system used to study neuronal apoptosis and the ability of neurotrophic factors and other agents to inhibit this form of neurodegeneration (Rukenstein et al. (1991), J. Neurosci. 11:2552-2563). Cultured PC12 cells were plated in the absence of serum and various concentrations of an inhibitor of PLA₂ (AN20606) (Compound 25 in Oinu a et al. (1991) J. Med. Chem. 34:2260-2267). Cell survival was determined 1 day later using the alamarBlue assay™ and was compared to sister cultures maintained in serum-containing medium. Treatment with 25 μM AN20606 resulted in 46% inhibition of cell death. Figure 10 shows the effect of the PLA₂ inhibitor AN20606 on PC12 neuronal cells induced to undergo apoptosis by serum withdrawal.

NGF Deprivation-Induced Apoptosis

Sympathetic neurons in culture die by apoptosis when deprived of NGF (Martin et al. (1988), J. Cell Biol. 106:829- 844) . Rat sympathetic neurons were deprived of NGF in the presence of 20 μM AN20579, AN20606, or AN20628. Neuronal viability was assessed 24-30 hours later by their appearance and using phase-contrast light microscopy and compared to neurons maintained in the absence or presence of 100 ng/ml NGF. Neurons maintained in the presence of NGF had a soma that was smooth and round to oval in shape, and possessed neurites with a relative uniform diameter and smooth appearance. Neurons deprived of NGF were characterized by neurite fragmentation, a shrunken and collapsed soma, and cell lysis. Neurons deprived of NGF in the presence of 20 μM AN20606 had intact neurites and a smooth soma. Neurons deprived of NGF in the presence of 20 μM AN20628 had intact neurites, a smooth to shrunken soma, but little evidence of cell lysis. Neurons deprived of NGF in the presence of 20 μM AN20579 showed signs of neurite fragmentation and cell lysis, but contained significantly more intact neurites and cell bodies than neurons deprived of NGF alone. AN20628 = Compound 23 in Oinuma et al. (1991), J. Med. Chem. 34:2260-2267: N- cyclohepyl-4-[N-methyl-N[ (E) -3-(4-cyanophenyl0-2- propenoyl]amino] benzenesulfonamide (AN 20628) . Rat sympathetic neurons

Rat sympathetic neurons were deprived of NGF in the absence or presence of 25 μM AN20606, AN22669, or AN22831. Neuronal viability was assessed 48 hours later using the alamarBlue™ assay, and compared to neurons maintained in the presence of 100 ng/ml NGF. As shown in Figure tracy , neurons deprived of NGF showed a 56 ± 2.5% loss of viability as compared to NGF-treated cultures as measured by alamarBlue™. Neurons deprived of NGF in the presence of 25 μM AN20606, AN22669, or AN22831 showed significantly less neuronal cell death (39 ± 1.4%, 26 ± 0.7%, and 11 ± 0.2% loss of viability, respectively) .

EXAMPLE 3: CHEMICAL SYNTHESES OF ACTIVE AGENTS: PLA₂ INHIBITORS

Benzenesulfonamides Benzenesulfonamides of the following structural formulae are suitable agents, typically can inhibit PLA₂ activity, and are candidate active agents for inhibiting neurodegneration and which may be suitable for therapeutic administration if in pharmaceutically acceptable form.

Structural Formula I

wherein a plurality of R¹ groups each independently stand for a hydrogen atom, a cyano, nitro, or hydroxyl group, a halogen atom, a lower alkoxy group, an acyloxy group, a group represented by the formula: -S0₂-R⁸ (wherein R⁸ stands for a lower alkyl group) , a heteroaryl or glycyloxy group or a group represented by the formula: -0-(CH₂) -COOH (wherein p is an integer of 1 to 3), and n is an integer of 1 to 4; R² stands for a hydrogen atom or pyridyl group; R³ stands for a hydrogen atom or lower alkyl, cyano, or pyridyl group;

R⁴ stands for a hydrogen atom or lower alkyl group; R⁵ and R⁶ may be the same or different from each other and stand for a hydrogen atom, lower alkyl group, a group represented by the formula: -(CH2)q-A [wherein q is 1-4 and A is a hydroxyl group, a group represented by the formula:

(wherein R⁹ and R¹⁰ may be the same or different from each other and stand for a hydrogen atom or lower alkyl group) , a group represented by the formula:

" it I

(wherein R¹¹ stands for a hydrogen atom or a lower alkyl group or a group represented by the formula:

.

(wherein s is an integer of 2-5) ] , an unsubstituted or substitutued cycloalkyl group, a bicycloalkyl, tricycloalkyl, or azabicycloalkyl group, or a group represented by the formula:

(wherein g and h are each an integer of 1 to 4, and B stands for a lower alkyl group, a substituted or unsubstituted arylalkyl group or a substitututed or unsubstitutued pyridylalkyl group) , or alternatively R⁵ and R⁶ may be combined together to form a 6- or 7- membered ring which may contain a nitrogen or an oxygen atom in addition to the nitrogen atom to which R⁵ and R⁶ are bonded, and said 6- or 7- membered ring may be substituted with a lower alkyl, arylalkyl, cycloalkylalkyl, or heteroarylalkyl group; a plurality of R⁷ groups each independently stand for a hydrogen atom, a lower alkyl group, a lower alkoxy group, or a halogen atom; and r is an integer of 1 or 2, provided that when r is 2, the two R⁷ groups may form a ring together with two adjacent carbon atoms constituting the benzeno ring; and m is an integer of 1 or 2, or a pharmacologically acceptable salt thereof.

A preferred genus of benzenesulfonamides suitable for use in the invention are given by Structural Formula II:

Structural Formula II

wherein R is at position 4 and is selected fro t he group consisting of: -CH₂S0₂, -CN, hydrogen, acetoxy, or hydroxy; and R² is cycloheptyl.

Preparation process: The benzenesulfonamides of Structural Formula I are prepared by the general procedure involving the reaction of the cinnamoyl chloride moiety (A) with the sulfonamide moiety (B) as shown schematically in Fig. 11 and shown by example for synthesis of AN20606 in Fig. 12 and for synthesis of AN36653 in Fig. 15. Synthesis of AN20606 : N-cvcloheDtyl-4- [N-methyl-N- f (E) -3- (4-methγlεulfonylphenγl ) -2-propenoγl ] amino7 benzene sulfonamide

With regard to Fig. 12, the syntheses of the intermediate and final products were performed as follows:

AN20518 p-Methylsulfonyl benzaldehyde

A mixture of 4.09 g (20 mmole) of p-methylsulfonyl benzyl chloride and 1.68 g (20 mmole) of sodium bicarbonate in 30 ml of DMSO was heated at 120°C for 16 hours (hrs) . Worked up by partition between water and dichloromethane (DCM) , the crude (3.14 g) subjected to column chromatographic purification afforded 1.84 g (50%) of AN20518 as a white solid, mp 150-3°C, TLC Rf=0.51 by 10% MeOH in DCM. ¹H NMR (CDC1₃, ppm) 10.15 (s, IH, CHO), 8.25-8.05 (m, 4H, ArH) , 3.13 (s, 3H, CH₃S0₂)

AN20519 Methyl p-methylsulfonyl cinnamate

A mixture of 1.47 g (8 mmole) of AN20518 and 2.98 g (8.8 mmole) of methyl (triphenylphosphoranylidene) acetate in 60 ml of dry toluene was heated at 90°C for 2 hrs. Stripping of solvent followed by column purification gave 1.02 g (53%) of

AN20519 as a pale white solid. mp 107-9°C, TLC Rf=0.66 by 10% MeOH in DCM. ^λE NMR (CDCI₃, ppm) 7.96-7.93 (d, 2H, ArH), 7.72-7.67 (d, IH, CH=) , 7.70-7.67 (d, 2H, ArH), 6.57-6.51 (d, IH, =CH) , 3.81 (s,

3H, COOCH3), 3.06 (s, 3H, CH3S02).

¹³C NMR (ppm) 167.15, 142.88, 142.01, 140.12, 129.26, 128.58,

122.17, 52.61, 44.99.

AN20537 p-Methylsulfonyl cinnamic acid

A solution of 0.85 g (3.54 mmole) of AN20519 in 60 ml of MeOH was added 1.07 ml of ION aq NaOH and stirred at room temperature (rt) for 16 hrs. Stripping of solvent followed by partition between EtOAc and water, then acidified the aqueous layers with concentrated HCl, filtration of the precipitates afforded 0.42 g (52%) of AN20537 as a white solid, mp 291-3°C, TLC Rf=0.09 by 10% MeOH in DCM. ^λE NMR (DMSO-d6, ppm) 7.95 (s, 4H, ArH) , 7.69-7.64 (d, IH,

CH=) , 6.74-6.69 (d, IH, =CH) , 3.24 (s, 3H, CH3S02) .

¹³C NMR (ppm) 167.54, 142.26, 141.91, 139.52, 129.28, 127,81,

123.14, 43.72.

LF865.94 p-Methylsulfonyl cinnamoyl chloride 0.39 g (1.72 mmole) of AN20537 in 30 ml of chloroform was treated 0.28 g (2.24 mmole) of oxalyl chloride and cat. amount of dry DMF, the mixture allowed to stir for 16 hrs at rt. Worked up by stripping of solvents and co-evaporation with hexane three times afforded 0.42 g (98%) of acid chloride as a yellowish solid (corrosive) .

AN20517 p-Acetaminobenzenesulfonyl chloride At -20°C, slowly added 2.98 g (20 mmole) of N-methyl acetanilide to a stirred solution of 1.65 g (100 mmole) of chlorosulfonic acid for 10 minutes, removed cold bath and gradually heat the mixture to 70°C for 2 hrs until no HCl fumes evolved. Cooled the syrupy liquid and poured into a mixture of 90 g ice and 10 ml of water with stirring. Filtered the precipitates and washed with water twice, dissolved in toluene and dried over anhydrous sodium sulfates, stripping of solvent in vacuo provided 1.8 g (35%) of AN20517 as a light tan solid. mp 124-6°C, TLC Rf=0.3 (10% MeOH in DCM).

¹H NMR (CDC1₃, ppm) 8.11-8.08 (d, 2H, ArH), 7.50-7.47 (d, 2H, ArH), 3.37 (s, 3H, NCH3), 2.07 (s, 3H, CH3CO) .

AN20607 N-Cycloheptyl-4-(methylamino)benzenesulfonamide At 0°C, 1.78 g (7.2 mmole) of AN20517 was slowly added to a mixture of 0.9 g (8 mmole) of cycloheptylamine and 1.97 g (24 mmole) of sodium acetate in 30 ml of ethanol with stirring. The reaction allowed to last 4-6 hrs, then diluted with water and extracted with DCM, washed with IN HCl, water, brine, and dried, stripping of solvent gave 2.2 g of syrupy intermediate, which redissolved in 30 ml of ethanol and added 1 ml of ION aq NaOH and refluxed for 16 hrs. Reaction worked up by neutralizing with IN HCl, extracted with DCM and washed with water and brine, stripped of solvent to give 1.27 g crude, which was subject to column purification and afforded 0.99 g (49%) of AN20607 as a white solid. mp 114-6°C, TLC Rf=0.7 (10% MeOH in DCM). H NMR (CDC1₃, ppm) 7.49-7.46 (d, 2H, ArH), 7.13-7.11 (d, IH, NH) , 6.59-6.55 (d, 2H, ArH), 6.46-6.45 (d, IH, NH) , 3.01-2.99 (M, IH, CH) , 1.59-1.19 (m, 12H, (CH₂)₆).

AN20606 N-Cvcloheptyl-4--rN-methyl-N-r fE^ -3- (4-methyl sulfonylphenyl) -2-propenovπamino)benzenesulfonamide To a stirred solution of 0.4 g (1.4 mmole) of AN20607 in 10 ml of DCM and 3 ml of pyridine was added dropwise 0.41 g (1.68 mmole) of LF865-94 in 5 ml of DCM at 0°C, the reaction allowed to stir at rt for 2 hrs. Quenched with water and extracted ith ethyl acetate, washed with IN HCl, water, bicarb, brine and dried, stripped of solvent to give 0.63 g crude, which was purified on a column to afford 0.56 g (84%) of AN20606 as a white solid. mpl32-4°C, TLC Rf=0.33 by 10% MeOH in DCM.

^XH NMR (CDCI₃, ppm) 7.97-7.94 (d, 2H, ArH), 7.85-7.82 (d, 2H, ArH), 7.74-7.68 (d, IH, CH=) , 7.49-7.46 (d, 2H, ArH), 7.37- 7.34 (d, 2H, ArH), 6.46-6.41 (d, IH, =CH) , 5.27 (m, IH, NH) , 3.43 (s, 3H, CH3S02), 3.40-3.37 (m, IH, CH) , 3.02 (s, 3H, NCH3), 1.77-1.73 (m, 2H, CH2), 1.52-1.42 (m, 8H, (CH₂)₄), 1.36- 1.31 (m, 2H, CH2) .

¹³C NMR (ppm) 165.66, 147.26, 141.54, 141.12, 141.01, 140.59, 129.01, 128.53, 128.46, 122.34, 60.98, 55.59, 44.97, 38.15, 36.39, 28.48, 24.08, 21.65, 14.76. Mass Spec (m/e) 491 (M=H)

Preparation of AN20628 : N-Cγcloheptyl-4-{N-methγl-N- f (E) - 3- (4-cyano phenyl ) -2 -propenoyl ] amino ) benzenesulfonamide To a stirred solution of 0.4 g (1.4 mmole) of AN20607 in 10 ml of DCM and 3 ml of pyridine was added dropwise 0.41 g (1.7 mmole) of 4-cyanocinnamoyl chloride in 5 ml of DCM at 0°C, the reaction allowed to stir at rt for 2 hrs. Quenched with water and extracted ith ethyl acetate, washed with IN HCl, water, bicarb, brine and dried, stripped of solvent to give 0.67 g crude, which was purified on a column to afford 0.35 g (58%) of AN20628 as a white solid. mpl57-9°C, TLC Rf=0.48 by 10% MeOH in DCM

¹H NMR (CDC1₃, ppm) 7.88.7.85 (d, 2H, ArH), 7.80-7.77 (d, 2H, ArH), 7.72-7.70 (d, IH, CH=) 7.65-7.63 (d,2H, ArH), 7.56-7.53 (d, 2H, ArH), 6.66-6.61 (d, IH, =CH) , 3.36 (s, 3H, NCH3), 3.18 (m, IH, CH) , 1.60-1.58 (m, 2H, CH2), 1.41-1.33 (m, 18H, CH₂)₄), 1.26-1.18 (m, 2H, CH2).

¹³C NMR (ppm) 164.81, 146.59, 140.68, 139.64, 133.03, 128.79, 128.03, 127.76, 123.01, 118.94, 112.04, 54.57. 37.35, 35.41, 35.34, 28.08, 23.49, 23.35R. Mass Spec (m/e) 438 (M+H)

Synthesis of AN22669 : N-Cγcloheptyl-4-{N-methγl-N- f (E)-3- phenyl ) -2 -propenoyl ] amino } benzenesulfonamide

To a stirred solution of 0.16 g (0.57 mmole) of AN20607 in 10 ml of DCM and 3 ml of pyridine was added dropwise 0.11 g (0.68 mmole) of LF865-94 in 5 ml of DCM at 0°C, the reaction allowed to stir at rt for 2 hrs. Quenched with water and extracted ith ethyl acetate, washed in IN HCl, water, bicarb brine and dried, stripped of solvent to give 0.27 g crude, which was purified on a column to afford 0.18 g (77%) of AN22669 as a white solid. mpl53-5°C, TLC Rf=0.43 by 10% MeOH in DCM.

^XH NMR (DMS0-d6, ppm) 7.89-7.86 (d, 2H ArH), 7.74-7.71 (d, IH, CH=) , 7.57 (m, IH, ArH) , 7.55-7.52 (d, 2H, ArH), 7.43-7.40 (d, 2H, ArH), 7.36-7.35 (d, 2H, Arh) , 6.44-6.41 (d, IH, =CH) , 3.36 (s, 3H, NCH3), 3.22 (m, IH, CH) , 1.62-1.59 (m, 2H, CH2), 1.48- 1.34 (m, 8H, (CH₂)₄), 1.25 (m, 2H, CH2) . Mass Spec (m/e) 413 (M+H)

Synthesis of AN22757: N-Cγcloheptyl-4 fN-methyl-N- ϊ (E) -3- (4-acetoxy phenyl ) -2 -propenoyl ] amino} benzenesulfonamide To a stirred solution of 0.11 g (0.38 mmole) of

AN20607 in 10 ml of DCM and 3 ml of pyridine was added dropwise 0.1 g (0.45 mmole) of 4-acetoxycinnamoyl chloride in 5 ml of DCM at 0°C, the reaction allowed to stir at rt for 2 hrs. Quenched with water and extracted ith ethyl acetate, washed with IN HCl, water, bicarb, brine and dried, stripped of solvent to give 0.14 g crude, which was purified on a column to afford 0.09 g (51%) of AN22757 as a white solid. mpl07-9°C, TLC Rf=0.5 by 10% MeOH in DCM. ^lηϋ NMR (CDC1₃, ppm) 7.96-7.93 (dd, 2H, ArH), 7.73-7.67 (d, IH, CH=) , 7.38-7.3 (d+d, 4H, ArH), 7.04-7.02 (d, 2H, ArH), 6.32- 6.27 (d, IH, =CH) , 4.78-4.75 (m, IH, NH) , 3.45 (s, 3H, CH3S02), 3.43 ( , IH, CH) , 2.29 (s, 3H, NCH3), 1.83-1.78 (m, 2H, CH2), 1.53-1.36 (m, 10H, (CH₂)₅).

¹³C NMR (ppm) 169.77, 166.39, 152.33, 147.44, 142.42, 140.68, 133.06, 129.60, 128.98, 128,05, 122.63, 118.67, 55.58, 38.04, 36.50, 28.49, 24.09, 21.72. Mass Spec (m/e) 471.2 (M+H)

Synthesis of AN22831: N-Cycloheptyl-4-{N-methγl-N- f (E) - 3 (4-hydroxy phenyl ) -2-propenoyl 1 amino} benzenesulfonamide

To a stirred solution of 80 mg (0.13 mmole) of AN22757 in 10 ml of MeOH was added 53 mg (0.39 mmole) of potassium carbonate in 2 ml of water, the reaction continued to stir at rt for 2 hrs. Stripping of solvent and partition between DCM and IN HCl, followed by washing with water, brine and dried to give 60 mg crude which was triturated with ether and filtered to afford 35 mg (64%) of AN22831 as a white solid. mpl75-7°C, TLC Rf=0.27 by 10% MeOH in DCM.

¹H NMR (CDCI₃, ppm) 7.88-7.85 (d, 2H, ArH), 7.70-7.68 (d, 2H, ArH), 7.58-7.56 (d, IH, CH=) , 7.44-7.42 (d, 2H, ArH), 7.33- 7.340(d, 2H, ArH), 6.26-6.23 (d, IH, =CH) , 3.20-3.17 (m IH, CH) , 3.09 (s, 3H, NCH3), 1.67-1.63 (m, 2H, CH2), 1.44-1.18 (m, 10H (CH₂)₅). Mass Spec (m/e) 429 (M+H) .

Synthesis of AN-36653: N-cγcloheptγl-4- [N-methyl-N- f (E) -3- (2-furanγl ) -2 -propenoyl ] ami nol benzene sulfonamide

With regard to synthesis of AN-36653, the synthetic scheme shown in Fig. 15 is referred to. A mixture of 460 mg (1 mmole) of the phosphonate (AN-31936) in 5 mL THF and 44 mg (1.1 mmol) sodium hydride was strirred under nitrogen till a clear yellow solution was obtained. To this solution was added 105 mg (1.1 mmol) of furan-2-carboxaldehyde and reaction stirred at room temperature for 2 hours. Worked up by partitioning between IN HCl and ethyl acetate (EtOAc) , the crude (600 mg) orange solid subjected to purification on a preparative palte (Uniplate 2000 microns) afforded 229 mg white solid (57% yield) of AN-36653. mp 152-5 °C, TLC Rf= 0. 8 by 3 % methanol (MeOH) in methylene chloride (DCM) .

^!H NMR (CDC13, ppm) 8.0 (d, 2H, ArH); 7.5 (d, IH, =CH); 7.4 (d, 2H, ArH), 7.38 (s, IH, furan), 6.59 (s, IH, furan), 6.48(s, IH, furan), 6.30 (s, IH, =CH), 4.73 (d, IH, NH), 3.51(s, 3H, CH3), 1.6 (m, 13H, cycloheptyl) ¹³C NMR (CDC13, ppm) 166.5, 1515.8, 147.8, 144.7, 140.5, 130, 128.9, 128.2, 116, 115, 112.8, 55.6, 37.9, 36.5, 28.5, 24. Mass Spec (m/e) 403 (M+H)

Synthesis of AN-36654

N-cycloheptyl-4- [N-methyl-N- [ (E) -3- (2-pyridyl ) -2-propenoyl ] ami nojbenzene sulfonamide

A mixture of 460 mg (1 mmole) of the phosphonate (AN-31936) in 5 mL THF and 52 mg (1.3 mmol) sodium hydride was strirred under nitrogen till a clear yellow solution was obtained. To this solution was added 128 mg (1.2 mmol) of pyridine-2-carboxaldehyde and reaction stirred at room temperature for 2 hours. Worked up by partitioning between IN HCl and ethyl acetate (EtOAc) , the crude (600 mg) orange solid subjected to purification on a preparative palte (Uniplate 2000 microns) afforded 111 mg white solid (26% yield) of AN-36654. mp 148-150 °C, TLC Rf= 0. 6 by 3 % MeOH in DCM.

^]H NMR (CDC13, ppm) 8.5 (m, IH, C5H5N), 8.0 (d, 2H, ArH); 7.8 (d, IH, =CH), 7.7 (m, IH, C5H5N), 7.5 (d, 2H, ArH), 7.4 (d, IH, C5H5N) , 7.2 (m, IH,

C5H5N), 7.0 (d, IH, =CH) , 4.5 (d, IH, NH), 3.51(s, 3H, CH3), 1.6 (m, 13H, cycloheptyl)

13C NMR (CDC13, ppm) 171, 152, 128.5, 127.4, 36.53, 28.50, 24.06. Mass

Spec (m/e) 414 (M+H) Synthesis of AN-36655 : N-cycloheptγl-4- [N-methyl-N- [ (E) -3- ( 5 -bromo-2 -thiophen ) -2-pro penoyl ] amino7 benzene sulfonamide

A mixture of 460 mg (1 mmole) of the phosphonate (AN-31936) in 5 mL THF and 52 mg (1.3 mmol) sodium hydride was strirred under nitrogen till a clear yellow solution was obtained. To this solution was added 229 mg (1.2 mmol) of 5-bromo-thiophene-2-carboxaldehyde and reaction stirred at room temperature for 2 hours. Worked up by partitioning between IN HCl and ethyl acetate (EtOAc) , the crude (626 mg) black oil subjected to purification on a preparative palte (Uniplate 2000 microns) afforded 285 mg yellow solid (50% yield) of AN-36655. mp 148-150 °C, TLC Rf= 0. 6 by 3 % MeOH in DCM. ^JH NMR (CDC13, ppm) 8.0 (d, 2H, ArH); 7.8 (d, IH, =CH) , 7.5 (d, 2H,

ArH),7.0 (m, 2H, thiohene) , 7.0 (d, IH, =CH), 4.7 (d, IH, NH), 3.51(s, 3H, CH3), 1.6 (m, 13H, cycloheptyl)

13C NMR (CDC13, ppm) 142, 135, 131.6, 131.5, 129, 128.1, 117.89, 55.6, 37.92, 36.53, 28.55, 24.1. Mass Spec (m/e) 499 (M+H)

Synthesis of AN-36657: N-cγcloheptγl-4- [N-methyl-N- [ (E) -3- (3- ( 1-methyl indole ) -2-prope noyl 7 amino7 benzene sulfonamide

A mixture of 460 mg (1 mmole) of the phosphonate (AN-31936) in 5 L THF and 52 mg (1.3 mmol) sodium hydride was strirred under nitrogen till a clear yellow solution was obtained. To this solution was added 207 mg (1.2 mmol) of l-methylindole-3-carboxaldehyde and reaction stirred at room temperature for 2 hours. Worked up by partitioning between IN HCl and ethyl acetate (EtOAc) , the crude (448 mg) yellow oil subjected to purification on a preparative palte (Uniplate 2000 microns) afforded 150 mg yellow solid of AN-36657. mp 196-199 °C, TLC Rf= 0. 7 by 3 % MeOH in DCM.

Η NMR (CDC13, ppm) 8.0 (m, 3H) ; 7.4 (m, 2H, ArH), 7.2 (m, 3H, indole), 7.1 (m, IH), 6.45(d, IH, =CH), 4.7 (d, IH, NH), 3.98(s, 3H, indole-N-CH3) , 3.51(s, 3H, CH3), 1.6 ( , 13H, cycloheptyl)

13C NMR (CDC13, ppm) 128.7, 128.1, 123.4, 120.4, 110.6, 55.6, 36.53, 28.52, 24.04. Mass Spec (m/e) 466 (M+H) Synthesis of AN-36690 N-cycloheptyl-4- [N-methyl-N- [ (E) -3- (2-imidazolyl ) -2-propenoγl7 aminojbenzene sulfonamide

A mixture of 460 mg (1 mmole) of the phosphonate (AN-31936) in 5 mL THF and 52 mg (1.3 mmol) sodium hydride was strirred under nitrogen till a clear yellow solution was obtained. To this solution was added 192 mg (2 mmol) of imidazole-2-carboxaldehyde and reaction stirred at room temperature for 2 hours. Worked up by partitioning between IN HCl and ethyl acetate (EtOAc) , the crude (423 mg) oil subjected to purification on a preparative palte (Uniplate 2000 microns) afforded 100 mg white solid of AN-36690. mp 114 - 6 °C, TLC Rf= 0.5 by 3 % MeOH in DCM.

^JH NMR (CDC13, ppm) 8.0 (d, 2H, ArH); 7.5 (d, IH, =CH); 7.4 (d, 2H, ArH), 7.2 (d, 2H, imidazole), 6.60 (d, IH, =CH), 4.7 (m, IH, NH), 3.50(s, 3H, CH3), 1.6 (m, 13H, cycloheptyl)

13C NMR (CDC13, ppm) 166, 129.05, 36.67, 28.59, 24.09. Mass Spec (m/e) 403 (M+H)

Synthesis of AN-36721 :

N-cvcloheptyl-4- IN-methyl-N- f (E)-3- (2-thiazolyl ) )-2-propenoyl ? amino 7 benzene sulfonamide

A mixture of 460 mg (1 mmole) of the phosphonate (AN-31936) in 5 mL THF and 52 mg (1.3 mmol) sodium hydride was strirred under nitrogen till a clear yellow solution was obtained. To this solution was added 226 mg (2 mmol) of thiazole-2-carboxaldehyde and reaction stirred at room temperature for 2 hours. Worked up by partitioning between IN HCl and ethyl acetate (EtOAc) , the crude (523 mg) brown oil subjected to purification on a preparative palte (Uniplate 2000 microns) afforded 139 mg white solid of AN-36721. mp 151-3 °C, TLC Rf= 0. 32 by 3 % MeOH in DCM.

Η NMR (CDC13, ppm) 8.0 (d, 2H, ArH), 7.9 (m, IH, thiazole) , 7.8 (d, IH, =CH); 7.4 (d, 3H) , 6.80 (d, IH, =CH), 4.6(m, IH, NH), 3.50(s, 3H, CH3) , 1.6 (m, 13H, cycloheptyl)

¹³C NMR (CDC13, ppm) 145.2, 134.35, 129.11, 128.03, 122.99, 121.76, 55.60, 38.16, 36.517, 28.52, 24.06. Mass Spec (m/e) 420 (M+H) Synthesis of AN-36722: N-cvcloheptyl-4- IN-methyl-N- f (E ) -3- ( 5- ( 3-methyl-lH-pyrazolyl ) )-2-propenoyl 7 amino I benzene sulfonamide

A mixture of 460 mg (1 mmole) of the phosphonate (AN-31936) in 5 L THF and 52 mg (1.3 mmol) sodium hydride was strirred under nitrogen till a clear yellow solution was obtained. To this solution was added 220 mg (2 mmol) of 3-methyl-lH-pyrazole-5-carboxaldehyde and reaction stirred at room temperature for 2 hours. Worked up by partitioning between IN HCl and ethyl acetate (EtOAc), the crude (402 mg) yellow solid subjected to purification on a preparative palte (Uniplate 2000 microns) afforded 331 mg yellow solid of AN-36722. • mp 211-13 °C, TLC Rf= 0. 5 by 3 % MeOH in DCM. 'H NMR (CDC13, ppm) 8.0 (d, 2H, ArH), 7.8 (d, IH, =CH) ; 7.4 (d, 2H, ArH), 6.40 (d, IH, =CH), 6.1 (s, IH, pyrazole), 4.7 (m, IH, NH) , 3.50 (s, 3H, N-CH3), 2.3 (s, 3H, CH3), 1.6 (m, 13H, cycloheptyl)

13C NMR (CDC13, ppm) 132, 131.985, 59.39, 41.89, 40.24, 32.42, 28.03. Mass Spec (m/e) 417 (M+H)

Synthesis of AN-36724: N-cycloheptyl-4- [N-methyl-N- [ (E) -3- (2- (2 ^• -bisthiophene) ) -2-pro penoγl] amino] benzene sulfonamide

A mixture of 460 mg (1 mmole) of the phosphonate (AN-31936) in 5 mL THF and 52 mg (1.3 mmol) sodium hydride was strirred under nitrogen till a clear yellow solution was obtained. To this solution was added 389 mg (2 mmol) of

2 , 2 ' -bithiophenecarboxaldehyde and reaction stirred at room temperature for 2 hours. Worked up by partitioning between IN HCl and ethyl acetate (EtOAc) , the crude (804 mg) yellow oil subjected to purification on a preparative palte (Uniplate 2000 microns) afforded 237 mg yellow solid of AN-36724. mp 165-7 °C, TLC Rf= 0. 45 by 3 % MeOH in DCM.

'H NMR (CDC13, ppm) 8.0 (d, 2H, ArH), 7.8 (d, IH, =CH) ; 7.4 (d, 2H, ArH), 7.2 (m, 4H, thiophene), 6.1 (d, IH, =CH) , 4.5 (m, IH, NH) , 3.50 (s, 3H, N-CH3), 1.6 (m, 13H, cycloheptyl) 13C NMR (CDC13, ppm) 132.63, 128.99, 128.56, 128.1, 126.1, 125.2, 124.91, 55.5, 36.52, 28.46, 24.09. Mass Spec (m/e) 571 (M+H)

Synthesis of AN-36831: N-cγcl oheptyl -4- [N-methyl -N- [ (E)-3-(2-(3 -methγlbenzothiophene ) ) -2-propenoγl ] aminol benzene sulfonamide A mixture of 460 mg (1 mmole) of the phosphonate (AN-31936) in 5 mL THF and 52 mg (1.3 mmol) sodium hydride was strirred under nitrogen till a clear yellow solution was obtained. To this solution was added 264 mg (1.5 mmol) of 3-methylbenzothiophene-2-carboxaldehyde and reaction stirred at room temperature for 2 hours. Worked up by partitioning between IN HCl and ethyl acetate (EtOAc) , the crude (366 mg) yellow solid subjected to purification on a preparative palte (Uniplate 2000 microns) afforded 275 mg offwhite powder of AN-36831. mp 120-4 °C, TLC Rf= 0. 62 by 3 % MeOH in DCM.

Η NMR (CDC13, ppm) 8.16(d, IH, benzothiophene), 8.0 (d, 2H, ArH), 7.7 (m, 3H); 7.4 (d, 2H, ArH), 7.3 (m, 2H, benzothiophene), 6.2 (d, IH, =CH) , 4.5 (m, IH, NH), 3.50 (s, 3H, N-CH3), 2.6 (s, 3H, CH3) , 1.6 (m, 13H, cycloheptyl)

13C NMR (CDC13, ppm) 129, 128.1, 126.9, 125, 123.2, 122.7, 55.6, 37.9, 36.54, 28.59, 24.13. Mass Spec (m/e) 483 (M+H)

Synthesis of AN-36832 : N-cγcloheptγl-4- [N-methyl-N- [ (E) -3- (2- ( 3-methylbenzothiophene) ) -2 - propenoyl 7 amino7 benzene sulfonamide

A mixture of 460 mg (1 mmole) of the phosphonate (AN-31936) in 5 mL THF and 52 mg (1.3 mmol) sodium hydride was strirred under nitrogen till a clear yellow solution was obtained. To this solution was added 308 mg (1.5 mmol) of N-(4-chlorophenyl)pyrrole-2-carboxaldehyde and reaction stirred at room temperature for 2 hours. Worked up by partitioning between IN HCl and ethyl acetate (EtOAc) , the crude (630 mg) yellow oil subjected to purification on a preparative palte (Uniplate 2000 microns) afforded 150 mg of light pink powder of AN-36832. mp 172-5 °C, TLC Rf= 0. 60 by 3 % MeOH in DCM.

•H NMR (CDC13, ppm) 8.0 (d, 2H, ArH), 7.49 (m, 3H), 7.3 (m, 2H, p-chlorophenyl) , 7.2 (d, 2H, ArH), 6.8 (s, IH, pyrrole), 6.5 (s, IH, pyrrole), 6.3 (s, IH, pyrrole), 6.0 (d, IH, =CH) , 4.6 (d, IH, NH) , 3.50 (s, 3H, N-CH3), 1.6 ( , 13H, cycloheptyl)

13C NMR (CDC13, ppm) 166.86, 147.99, 140.38, 131.7, 130.22, 128.83, 128.11, 128.04, 114.8, 112.7, 111.1, 55.58, 37.82, 36.53, 28.51, 24.08. Mass Spec (m/e) 513 (M+H) Preparation of 4- (N-Acety 1- -methyl amino ) benzene sulfonyl chloride (AN-20517 )

A solution of ice cold chlorosulfonic acid (90mL ) is slowly added 39.8 g (267 mmol) of N-methyl acetanilide. After addition complete the reaction is heated to 70oC. for 2 hours then cooled to room temperature. The slurry is slowly added into a solution of ice/water with rapid stirring. The white solid formed is filtered and washed with water and hexanes to give 50.1 g (80% yield) of the sulfonyl chloride. mp 122-6 °C, TLC Rf= 0. 17 by 50 % ethyl acetate in hexane.

^•H NMR (CDC13, ppm) 8.0 (d, 2H, ArH), 7.49 (m, 3H) , 7.3 (m, 2H, p-chlorophenyl) , 7.2 (d, 2H, ArH), 6.8 (s, IH, pyrrole), 6.5 (s, IH, pyrrole), 6.3 (s, IH, pyrrole), 6.0 (d, IH, =CH) , 4.6 (d, IH, NH) , 3.50 (s, 3H, N-CH3), 1.6 (m, 13H, cycloheptyl) 13C NMR (CDC13, ppm) 129.11, 38, 23.4

Synthesis of AN -20607 : N-cγcl oheptyl -4- [N-methyl -amino ] benzene sulfonamide

A mixture of 50.1 g (212 mmol) of sulfonyl chloride and 24 g (212 mmol) of cycloheptyl amine and 61 g of sodium acetate were stirred in ethanol at OoC under. an atmosphere of nitrogen. Worked up by partitioning between water and ethyl acetate, the organic layer is separated washed with brine and dried over sodium sulfate to give an colorless oil. The oil is dissolved in ethanol and add NaOH (4 g) reflux solution 16 hours. The reaction cooled to OoC and adjust pH to 8.0. The white solid is filteredwashed with hexane and dried to yield 39.5 g of product (71% yield). mp 113-4 °C, TLC Rf= 0.42 by 50% EtOAc/Hexane

^•H NMR (CDC13, ppm) 7.67 (d, 2H, ArH); 6.56 (d, 2H, ArH); 3.3 (m, IH) ; 2.9 (d, 3H,NCH3) 1.8 (m, 2H) ; 1.4 (m, 11H, cycloheptyl). ¹³C-NMR (CDC1₃): 129.6, 111.9, 55.1, 36.5, 30.73, 28.5, 24.12

MS: MH+ = 283

Synthesis of AN-32139 : N-cγcloheptyl-4- [N-methγl-N-bromoacetγlamino7 benzene sulfonamide A mixture of 1 g (3.2 mmol) of AN-20607 and 0.5 ml ( 3.5 mmol) of triethylamine in 10 mL of DCM was stirred at 0°C. Bromoacetyl bromide (1.3 g, 6.3 mmol) was added slowly to this mixture. Reaction allowed to stir at room temperature for 4 hrs. Worked up by partitioning between water and ethyl acetate, the organic layer is separated washed with brine and dried over sodium sulfate to give a crude brown oil ( 1.1 g) . TLC Rf= 0. 5 by 10 % MeOH in DCM.

Η NMR (CDC13, ppm) 8.0 (d, 2H, ArH); 7.4 (d, 2H, ArH), 4.5 (m, IH, NH) , 3.7 (bs, 2H, CH2) 3.4 (s, 3H, NCH3); 1.4 (m, 13H, cycloheptyl). ¹³C-NMR (CDC1₃): 129.6, 111.9, 55.1, 36.5, 30.73, 28.5, 24.12 MS: MH⁺ = 405

Synthesis of AN-31936: N-cycloheptyl-4-[N-methγl-N-diethylphosphonoacetylamino]benzen e sulfonamide

A mixture of 1.1 g (3.5 mmol) of AN-32139 and 1.0 ml ( 5.2 mmol) of triethylphosphite in 30 mLs of toluene was refluxed for 17 hrs. Worked up by partitioning between water and ethyl acetate, the organic layer is separated washed with brine and dried over sodium sulfate. Product crystallizes from DCM/hexane.

TLC Rf= 0. 45 by 10 % MeOH in DCM.

^!H NMR (CDC13, ppm) 8.0 (d, 2H, ArH); 7.4 (d, 2H, ArH), 4.5 (m, IH, NH) , 4.2 (g, 2H, 0CH2), 3.4 (s, 3H, NCH3); 1.4 (m, 13H, cycloheptyl).

¹³C-NMR (CDC1₃): 129.6, 63.3 55.61, 36.47, 28.47, 24.03, 16.97, 16.88 MS: MH⁺ = 461

Synthesis of AN-28588 : Methyl 3- (l-{4-[3- (4-Acetyl-3-hγdroxy-2-propylphenoxγ) pro poxy ] phenyl } -4-phenylthio) propionate

Prepared by procedure described by Abdullah et. al., (Bioorganic & Medicinal Chemistry Letters, Vol. 5. pp 519-522 (1995) and US Patent No: 5,453,443 ( Perrier et. al.,) Date of Patent: Sep. 26, 1995.

Compound obtained as an oil. TLC Rf= 0.6 by 50% EtOAc/Hexane IH NMR (CDC13, ppm) 12.75 (s, IH, OH); 7.6 (d. 2H, ArH) ; 7.3 ( , IH) ; 2.9 (d, 3H,5H, ArH) 7.15 (d, IH, ArH) , 6.8 (d, 2H, ArH) , 6.4(d, IH, ArH) , 4.2(m, 4H, OCH2) , 3.85(t, IH) , 3.8 (S, 3H, OCH3) , 2.65 (m, 3H) , 2.56 (S, 3H, CH3 ) , 2.48 (m, 3H) , 2.29 (t, 2H) , 1.92 (m, 2H) , 1.5 (m, 4H) , 0.91 (t, 3H, CH3) .

¹³C-NMR (CDC1₃) : 177.4, 163,3, 162.6, 158.4, 142.5, 134.8, 130.6, 129.4, 128.9, 128.8, 126.4, 118.8, 114.9, 114.8, 103.3, 66.3, 64.7, 52.3, 49.8, 36.6, 36.1, 34.7, 29.9, 26.9, 26.1, 24.5, 22.6, 14.9 MS: Negative Ion spectrum [M-H]^~ = 577

Synthesis of AN-28589:

3-(l-{4-[3-(4-Acetγl-3-hγdroxy-2-propγlphenoxγ) pro poxy ] phenyl J -4-phenylthio) propionic acid Prepared by procedure described by Abdullah et. al.,

(Bioorganic & Medicinal Chemistry Letters, Vol. 5. pp 519-522 (1995) and US Patent No: 5,453,443 ( Perrier et. al.,) Date of Patent: Sep. 26, 1995. Compound obtained as an oil. TLC Rf = 0.15 by 50% EtoAc/Hexane Η NMR (CDCI₃, ppm) 12.75 (s, IH, OH); 7.6 (d, 2H, ArH); 7.3 (m, 5H, ArH) 7.15 (d, IH, ArH), 6.8 (d, 2H, ArH), 6.4(d, IH, ArH), 4.2(m, 4H, OCH2), 3.8 (t, IH), 2.65 (m, 3H), 2.56 (s, 3H, CH3), 2.48 ( , 3H), 2.29 (t, 2H), 1.92 (m, 2H), 1.5 (m, 4H), 0.91 (t, 3H, CH3) . ¹³C-NMR (CDCI3): 177.4, 163,3, 162.6, 158.4, 142.5, 134.8, 130.6, 129.4, 128.9, 128.8, 126.4, 118.8, 114.9, 114.8, 103.3, 66.3, 64.7, 49.8, 36.6, 36.1, 34.7, 29.9, 26.9, 26.1, 24.5, 22.6, 14.9 MS: Negative Ion spectrum [M-H]^" = 564

Biological Activity of Compounds Fig. 13 shows the activities of the benzenesulfonamides of

Structural Formula II with respect to their activities in inhibiting PLA₂ activity (IC₃₀: concentration of benzenesulfonamide to produce 70% inhibition of enzyme activity) and inhibiting neuronal degeneration in human cortical neuron cultures treated with Aβ peptide (ICJ_Q; concentration of benzenesulfonamide to produce 50% inhibition of cell toxicity) .

Table 4 shows the effect of the various AN compounds on: (1) percent inhibition of cPLA2, (2) the ED50 for reduction of neurotoxicity Table 4

AN-Number CPLA2 % Ab-neurotox. IL-1 inhibition inhibition ED50 IC50

36653 29% @25uM 19uM 4.6 uM

36654 6% @25 uM NA NA

36655 30% @25 uM NA 5.3 uM

36657 12% @25 uM NA 2.2 uM

36690 7% @25 uM NA 14.8 uM

36721 7% @25 uM 25.9 uM

36722 4% @25 uM 5.0 uM

36724 11% @25 uM 6.3 uM

36831 5% @25 uM 14 uM 1.6 uM

36832 10% @25 uM 9 uM 13.4 uM

22669 34% @25 uM 50 uM

22831 30% @25 uM 5 uM

28589 80% @25 uM 10.5 uM 18.0 uM

28588 5% @25 uM NA NA

For chemical synthetic methods, the following are incorporated herein by reference: European Patent Publication EP 0 468 054 Al (1991) to Oinuma; Nace and Managle (1959) J. Org. Chem. 24; 1792; Oinuma et al.

(1991) J. Medicinal Chem. 34: 2260; Smiles and Stewart Organic Synthesis collective, vol. 1, 8-10; Stojanovic et al. Glas. Hem. Drus. , Beograd, 36(9-10): 393-400 (1971).

Summary of Experimental Examples

The Experimental Examples are indicative that agents which selectively inhibit PLA₂ activity protect human neurons from toxicity resulting from exposure to pathogenic Aβ peptide and other neurodegenerative stimu1i. Although the present invention has been described in some detail by way of illustration for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the claims.

Claims

1. A method for identifying active agents which inhibit neuronal degeneration induced by Aβ peptide, comprising administering an agent to a cell population comprising neurons, wherein said cell population is exposed to an amount of pathogenic Aβ capable of inducing neuronal degeneration in the cell population, and determining whether the presence of said agent produces inhibition of PLA₂ activity and also produces a detectable reduction in the amount and/or rate of neuronal degeneration in the cell population; if said agent produces PLA₂ inhibition in neurons and/or inhibits neuronal degeneration, the agent is thereby identified as an active agent.

2. The method according to claim 1, wherein determining whether the presence of said agent produces inhibition of PLA₂ activity is performed by in vitro assay using a predetermined quantity of PLA₂ enzyme.

3. The method according to claim 2, wherein the

PLA₂ is produced by expression of a recombinant expression vector encoding human cPLA₂ in a host cell.

4. The method according to claim 1, wherein the cell population comprises neurons or neuronal cell lines of human or rodent origin.

5. The method according to claim 1, wherein the detectable reduction in the amount and/or rate of neuronal degeneration is determined in a mammal.

6. The method of claim 1, wherein the pathogenic Aβ comprises an Aβ peptide having amino acids 25-35 of Aβl-40.

7. The method of claim 6, wherein the pathogenic βA is AjSl-40 or AjSl-42.

8. A method for reducing or retarding Aβ-mediated neurodegeneration in a cell population comprising cortical neurons exposed to an amount of pathogenic βA sufficient to produce neurodegeneration, comprising administering an efficacious dose of a PLA₂ inhibitor to retard or inhibit Aβ- mediated neuronal degeneration.

9. The method of claim 8, wherein the cell population comprises cultured human or rodent cortical neurons.

10. The method of claim 9, wherein the cell population further comprises cultured microglial cells, astrocytes, an astrocytoma cell line, or a microglial cell line.

11. The method of claim 8, wherein A/31-40 is present at 25 μM or 50 μM.

12. A method for inhibiting neuronal degeneration induced by Ajδ peptide, comprising administering an efficacious dose of a selective inhibitor of PLA₂.

13. The method of claim 12, wherein the selective inhibitor of PLA₂ is administered at a dose level predetermined to reduce A/3-induced or NGF withdrawal-induced neuronal cell death by 50% (IC₅₀) .

14. A composition for therapy of neurodegenerative disease, comprising an efficacious dosage of a selective PLA₂ inhibitor.

15. A composition of claim 14, wherein the selective PLA₂ inhibitor is able to cross a blood-brain barrier of a human to produce a therapeutically efficacious concentration in cerebrospinal fluid.

16. A composition of claim 14, wherein the selective PLA₂ inhibitor has a structure according to Structural Formula I or Structural Formula II.

17. A composition of claim 16, wherein the selective PLA₂ inhibitor is AN20606, AN22831, AN 20628, AN22757, AN22669, AN23019, AN22831, AN36653, AN36654, AN36655, AN36657, AN36690, AN36721, AN36722, AN36724, AN 36831, AN36832, AN20517, AN20607, AN32139, AN31936, AN28588, or AN28589.

18. A kit comprising a composition of claim 14 and instructions for administering an efficacious dosage to a patient having Alzheimer's disease.

19. A PLA₂ inhibitor having a structure according to Structural Formula I or Structural Formula II

20. The PLA₂ inhibitor of claim 19, which is selected from the group consisting of: AN20606, AN22831, AN 20628, AN22757, AN22669, AN23019, AN22831, AN36653, AN36654, AN36655, AN36657, AN36690, AN36721, AN36722, AN36724, AN 36831, AN36832, AN20517, AN20607, AN32139, AN31936, AN28588, or AN28589.