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WO1994012667A1 - Inhibitors of arachidonic acid metabolites for preventing neurological damage - Google Patents

Inhibitors of arachidonic acid metabolites for preventing neurological damage Download PDF

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Publication number
WO1994012667A1
WO1994012667A1 PCT/US1993/011542 US9311542W WO9412667A1 WO 1994012667 A1 WO1994012667 A1 WO 1994012667A1 US 9311542 W US9311542 W US 9311542W WO 9412667 A1 WO9412667 A1 WO 9412667A1
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hiv
infected
cells
culture
monocytes
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PCT/US1993/011542
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French (fr)
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Edward W. Bernton
Marti Jett
Howard Gendelman
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The United States Department Of The Army
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Publication of WO1994012667A1 publication Critical patent/WO1994012667A1/en

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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
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    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
    • C—CHEMISTRY; METALLURGY
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    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • This invention relates to the field of prevention of neuro- nal damage arising from diseases that cause increase in levels of platelet activation factor (PAF) and/or metab ⁇ olites of arachidonic acid. Some of the more deleterious effects of CNS infections can be mitigated by administra ⁇ tion of inhibitors of PAF and/or arachidonic acid metabo- lites. Compositions and methods disclosed herein also have application to treatment of retroviral infections, includ ⁇ ing human immunodeficiency virus. Background of the Invention;
  • Tissues of the cen ⁇ tral nervous system consist of several distinct cell types. Neurons are electrically active cells arranged in networks where cells are functionally and anatomically interconnect ⁇ ed by synapses. Release of specific molecules called neurotransmitters at synapses allow signalling between neurons.
  • the term glia or neuroglia applies to the stroma of non-neural supportive cells.
  • Astrocytes are the domi- nant glia, and function to control the constituents of the central nervous system (CNS) microenvironment within the limits optimal for neuronal viability and function.
  • oligodendrocyte is a specialized cell which produces and maintains myelin, an electrical insulator which surrounds fascicles of neuronal axons, and is re ⁇ quired for effective propagation of signals along these axons. Dysfunction of these cells can cause loss of my- elination and disease conditions such as multiple sclero ⁇ sis.
  • the microglia are the brain macrophages derived from the same bone-marrow precursors as tissue macrophages, but assuming specialized morphology and function in the CNS microenvironment.
  • the cells initiate the inflammatory response within these cells and represent the overwhelming majority of CNS cells harboring virus and allowing viral replication in many viral infection, including HIV.
  • th system described below which models the interactions between virus-infected microglia, astrocytes, and neurons
  • primary cultures of human monocyte/macrophages are infecte with virus as a practical surrogate for human microglia, which would otherwise need to be obtained from fresh human brain tissue.
  • the cellular and molecular mechanisms by which infection and inflammation lead to damage of neurons has been studied.
  • TNF tumor necrosis factor
  • LT lymphotoxin
  • astrocytes secrete TNF ⁇ in response to a variet of biologic stimuli, particularly to cytokines IL-1 and interferon- ⁇ (INF- ⁇ ) , which are known to be present in the central nervous system during neurologic diseases associat ed with inflammation (Journal of Immunology, Vol 144, No. (April 15, 1990) 2999-3007).
  • PAF platelet activating factor
  • Virus-infected brain macrophages may origi ⁇ nate from an expansion of latently infected monocytes that carry HIV into the brain (the "Trojan horse” hypothesis) and later produce virus.
  • virus may pene ⁇ trate the brain through a disrupted blood-brain barrier by infected T cells or as free viral particles.
  • results are identical, selective productive infection of brain macrophages and macroglia. Whether these HIV-infected brain macrophages induce disease through metabolic, immune and/or viral-induced mechanisms is criti ⁇ cal to our understanding of HIV neuropathogenesis. Several reports suggest that low-level infection of neurons and glia can produce neurologic impairment during HIV infec- tion.
  • CNS dysfunction in the case of HIV infec ⁇ tion include coexistence of opportunistic CNS infection, secretory toxic factors from infected monocytes, gpl20- mediated neuronal growth factor (NGF) blockade or killing, and neuronotoxicity by HIV tat or other viral regulatory components. All or many of these mechanisms may result in cytotoxic effects in neurons and/or oligodendrocytes.
  • gpl20 may antagonize normal vasoactive intestinal peptide (VIP) function in the brain or be directly toxic to neurons. Studies show that gpl20 can induce neurono ⁇ toxicity by increasing Ca ++ levels in cultured neurons and are prevented by Ca ++ channel antagonists.
  • VIP vasoactive intestinal peptide
  • Secretory products from HIV-infected cells may alter neuronal viability, damage myelin, or stimulate neurotransmitters resulting in neuronal dysfunc- tion.
  • Macrophages play important roles in steady-state immune and tissue function.
  • the regulatory role of macro ⁇ phages occurs through the release of numerous secretory molecules made under a variety of physiologic conditions. Changes in the secretion or release of certain of these mediators may lead to disease.
  • In support of this idea are recent studies demonstrating that disordered secretion of one or more cellular factors from HIV-infected macrophages produce neuronal death in vitro.
  • HlV-in- fected U937 cells a myelomonocytic cell line, released toxic factors that destroyed cultured chick and rat neu ⁇ rons.
  • the monocyte-produced neurotoxins were heat-stable and protease-resistant and acted by way of N-methyl-D- aspartate (NMDA) receptors.
  • NMDA N-methyl-D- aspartate
  • interferon alpha IFN ⁇
  • PBMC peripheral blood mononuclear cells
  • neuronotoxins could be produced from glia during cell-to-cell interactions with infected brain macrophages
  • HIV-infec ⁇ tion of human monocytes causes activation of phospholipase A2 and lipoxygenase, resulting in the production of potent, short-lived lipid biomediators such as leukotrines, and known collectively as 5- and 12-lipoxygenase metabolites.
  • These mediators appear to effect both the cell of origin, the infected macrophage) and the function of nearby unin- fected cells such as astrocytes and neurons.
  • a variety of inter-cellular and intra-cellular signal ⁇ ling pathways can lead to the activation of phospholipase A or C, which results in the release of arachidonic acid and diacyl glycerol from cell membrane phospholipid components.
  • Diacyl glycerol acts within the cell to increase intracel- lular Ca levels, leading to phosphorylation and activation of cellular enzymes which regulate cellular functions.
  • Arachidonic acid is then metabolized via either the cyclo- oxygenase enzyme, into bioactive lipids known as prosta- glandins, or via several lipoxygenase enzymes into a variety of derivatives including certain powerful pro- inflammatory lipid signalling molecules known as leuko- triennes.
  • PAF synthesis is believed to largely occur through the activation of phospholipase A2, which hydro- lyzes the sn-2 acyl fatty acid (predominantly arachidonic acid) of 1-0-alkyl phosphatidylcholine in membrane phospho- lipids, releasing lyso-PAF. Lyso-PAF is subsequently reacylated by arachidonyl-CoA acyltransferase, regenerating the parent phospholipid, or by lyso-PAF acetyl CoA acetyl- transferase, producing PAE.
  • This invention to provides a method of treating en- cephalitis or encephalopathy secondary to CNS infection by administration of therapeutically effective amounts of compositions containing agents that inhibit release of platelet activation factor (PAF) and/or arachodonate etab- olites.
  • Appropriate pharmaceutical agents include (1) inhibitors of the lipoxygenase enzyme pathway and/or PAF production, (2) receptor antagonists of a class of inflam ⁇ matory mediators known as leukotrienes, and (3) receptor antagonists of PAF which may be administered either alone or in combination.
  • the active agents used in accord with the teachings of this disclosure may also be given in combination with other therapeutic agents such as antibiot ⁇ ics, immune enhancers, and HIV-inhibitors.
  • methods described herein may be practiced in conjunction with administration of AZT, DDI and DDC and other known therapeutic agents used in treatment of retro- viral infections.
  • the manner of administration will depend on the course of the disease process, the susceptibility of the active agent to inactivation in the gut or upon exposure to en ⁇ zymes, and the duration of treatment required.
  • This disclosure also teaches a method for screening drugs for use for neuroprotective efficacy in CNS infection based on the inhibition of induction of neurotoxic factors using a unique cell culture system.
  • the methods of treatment also have general applicability for treatment of encephalitis due to parasitic or bacterial infection of the central nervous system (CNS) .
  • the method of screening pharmaceutical compounds for neuroprotective activity comprises infecting monocytotropic cells with an organism known to cause neuronal damage. After effectively infecting the cells astrocytes (prefera ⁇ bly astrocytoma cells) are added to the infected culture.
  • the number of astrocytes added is roughly equivalent to the number of cells in the origi ⁇ nally infected culture.
  • a test compound to be evaluated for neuroprotective activity To some of the cultures there is added a test compound to be evaluated for neuroprotective activity. The cultures to which no test compound is added serve as controls. The cultures are then incubated for a sufficient period of time to allow production of TNF ⁇ . Reduction of more than 70% TNF ⁇ in cultures containing test compounds was considered evidence that the test compound had inhibitory activity. The supernatant is then withdrawn and assayed for TNF ⁇ . The supernatants may then be frozen for later use or used immediately. Aliquots of supernatant are then added to cultures of neurons (preferably embryonic neurons) .
  • the neuron/culture mixture is evaluated for evidence of neurotoxic or neuronocytopathic effects.
  • the method mimics cell-to-cell interaction which, in vivo, allows or potentiates both the production of TNF ⁇ and of neuronotoxic factors.
  • the infective agent used to infect the cell culture may vary. Examples of useful infective agents are HIV, dengue virus, toxoplasmosis, cryptococcus, and listeria.
  • HIV-infected monocytes and macrophages are believed to represent the largest reservoir of virus in the infected patient. HIV remains latent and persistent in these cells for long periods of time. However, during acute HIV infec ⁇ tion and subsequent to a variety of poorly understood activation signals, activation of viral replication in the cells results in abundant release of infected virons. Additionally, cells of monocyte lineage represent the reservoir of infection within the central nervous system, and viral replication in these cells is most closely linked with the wide range of neuropathology associated with HIV disease.
  • Inhibitors of the lipoxygenases prevented induction of TNF- ⁇ and IL-l ⁇ in infected cells by HIV-infected monocytes in co-culture, as do PAF antagonists. Inhibitors of the lipoxygenase pathway also were shown to decrease the cyto- pathic effects of productive HIV infection on human mono ⁇ cytes. Inhibitors of the cyclooxygenase enzyme, which represents a parallel and competing pathway of arachodonate metabolism, have the opposite effect. The data suggest a cascade of events where PAF and lipoxygenase products of HIV-infected monocytes elicit abnormal secretion of TNF ⁇ and IL-1 from surrounding unin- fected cells.
  • HIV-infected macrophages evidence activation of phospholipase A2, resulting in formation of arachidonic acid from membrane phospholipids.
  • the phospholipase A2 is preferentially metabolized by the cell via the 5-lipoxy- genase pathway, resulting in production of a series of biologically active compounds, including 5-HPETE, 5-HETE and leukotrienes and results in release of PAF.
  • the com- pounds have the effect of .both acting on the HIV-infected cell (autocrine activity) and on neighboring uninfected cells (paracrine activity) to induce release of cytokines which include interleukin-1 and TNF.
  • PAF and lipoxygenase products act on the infected cell to mobilize intracellular calcium and diacylglycerol, leading to the activation of protein kinase-C (PKC) .
  • PKC protein kinase-C
  • PKC activates, via phosphorylation, specific cellular enzymes. This cascade of events appears to be required for both release of solu ⁇ ble neuronotoxins by HIV-infected monocytes co-cultured with normal astrocytes and active replication of HIV within the macrophage or monocyte.
  • 5-lipoxygenase inhibitors can be used to block the production of the 5- HETE, 5-HPETE, leukotriene, and other 5-lipoxygenase prod- ucts, shunting arachidonic acid into the cyclooxygenase pathway to produce prostaglandins (which may also impede HIV replication) .
  • Leukotriene receptor antagonists may be administered to block the actions of leukotrienes at their cellular receptors. Both the 5-lipoxygenase inhibitors and the leukotriene receptor antagonists appear to decrease release of PAF. Compositions containing PAF antagonists may be administered to obstruct the actions of PAF at its cellular receptor. Furthermore, the protein-kinase C inhibitors may block the penultimate result of the process on the cells by suppressing activation of enzymes by pro ⁇ tein-kinase C.
  • Examples of 5 lipoxygenase inhibitors include zi- leuton, nordihydroguaiaretic acid (NDGA) , (+)-2,2-dibutyl- 5(2-quinolylmethoxy)-1,2,3,4-tetrahydro-l-napthol and 5- [[3,5-bis(1,1,dimethylethyl)-4-hydroxyphenyl]methylene]-3- (dimethylamino)-4-thiazolidinone.
  • NDGA nordihydroguaiaretic acid
  • (+)-2,2-dibutyl- 5(2-quinolylmethoxy)-1,2,3,4-tetrahydro-l-napthol and 5- [[3,5-bis(1,1,dimethylethyl)-4-hydroxyphenyl]methylene]-3- (dimethylamino)-4-thiazolidinone.
  • leuko ⁇ triene receptor antagonist is 7[3(4-acetyl-3-methoxy-2- propylphenoxy)-propoxy]-3,4-dihydro-8-propyl-2H-l-benzo- pyrano-2-carboxylic acid.
  • PAF antagonists examples include ll-nor- ⁇ 8 -tetrahydrocannabinol-9-carboxylic acid (THC-7-oic acid) and derivatives thereof, the ginkolides (triazolothienodiazepine derivatives), trans-2,5-bis(3,4,5 trimethoxyphenyl)-1,3,-dioxolane (a diaryltetrahydrofuran) , the 1,4-dihydropyridines, and l-O-hexadecyl-2-O-acetyl-sn- glycero-3-phospho-(N,N,N-trimethyl) hexanolamine.
  • protein-kinase-C inhibitors include gossypol, sphingo- sine, and staurosporine. Materials and Methods
  • Monocytes Isolation and culture of monocytes and neural cells.
  • Monocytes were recovered from PBMC of HIV and hepatitis B- seronegative donors after leukoperesis and purified by countercurrent centrifugal elutitration. Cell suspensions were >98% monocytes by criteria of cell morphology on Wright-stained cytosmears, by granular peroxidase and by nonspecific esterase. Monocytes were cultured as adherent monolayers (1 x 10 6 cells/ml in 24 mm plastic culture wells) in Dublvecco's modified Eagle's medium (DMEM) (Signa, St.
  • DMEM Dublvecco's modified Eagle's medium
  • the cells were grown as adherent monolayers in DMEM with 10% heat inactivated fetal calf serum (FCS) and 50 ⁇ g/ml gentamicin.
  • FCS heat inactivated fetal calf serum
  • Human endothelial cells were a gift of P.I. Lelkes ffProc. Natl. Acad. Sci. USA 87:6161-6165).
  • Cell lines were fully characterized to their cell origins.
  • Primary human astrocytes were prepared from second trimester fetal brain tissue obtained from elective abortions performed in full compliance with both NIH and University of Rochester guidelines.
  • Brain tissue composed of telencephalon with both cortical and ventricu ⁇ lar surfaces were dissected in cold Hank's basic salt solution (HBSS) with HEPES and 50 ⁇ g/ml gentamicin, then transferred to 20 ml ice cold (4°C) DMEM/F12 (Gibco) with 10% heat inactivated FCS.
  • HBSS cold Hank's basic salt solution
  • DMEM/F12 Gibco
  • the cell suspension was centrifuged at 500 rp , washed 2X in media, then plated in DMEM containing 10% fetal calf serum (FCS) and 50 ⁇ g/ml gentamicin into 75 cm 2 tissue culture flasks at a cell density of 2 X 10 5 cells/ml. Media was exchanged every 3 days. Non-adherent microglia and oligo- dendrocytes were removed by gentle agitation and circular shaking of cultured cell preparations 10 days after plat ⁇ ing. The purity of the astrocyte cultures was > 95% by immuno staining of glial fibrillary acidic protein (GFAP) .
  • FCS fetal calf serum
  • Cells were cultured as adherent monolayers in DMEM with 10% heat-inactivated FCS, 10 ⁇ g/ml gentamicin and 1% glutamine. All culture reagents were screened and found negative for endotoxin contamination.
  • Reverse transcriptase activity was determined in replicate samples of culture fluids added to a reaction mixture of 0.05% NONIDENT P-40 (Sigma Chemical Co.), 10 ⁇ g/ml poly(A) , 1.25 U/ml oligo (dT) (Pharmacia, Piscataway, NJ) , 5 mM dithiothreitol (Pharmacia), 150 mM KC1, 15 mM MgCl, and 3 H- dTTP(2 Ci/mmol, Amersham Corp., Arlington Heights, Illi ⁇ nois) in pH 7.9 Tris-HCl buffer for 24 hours at 37° C. Radiolabeled nucleotides were precipitated with cold 10% trichloroacetic acid (TCA) and washed with 5% TCA and 95% ethanol in an automatic cell harvester (Skatron Inc. ,
  • VSV vascular stomatitis virus
  • TNF bioactivity was performed according to stan ⁇ dard procedures (J. Immunol.. 141:4196).
  • the murine L929 cell line was propagated in DMEM, 5% FCS, 1% glutamine and 20 ⁇ g/ml gentamicin. Cells were retrieved in log phase and placed (0,05 X 10 5 / ell) in 96-well plates (Costar) with actinomycin D. Culture fluids were inoculated into cell monolayers and degree of cell lysis determined by crystal violet staining after a 24 hour incubation.
  • cytokine RNAs were estimated after reverse transcription and antisense primers and PCR amplification of the cDNA transcripts.
  • the mRNA for the cellular enzyme, glyceraldehyde 3-phosphate dehy- drogenase (GAPDH) served as an internal control to allow analysis and comparison of RNA species between different samples.
  • GAPDH glyceraldehyde 3-phosphate dehy- drogenase
  • 2.0 ⁇ g total cellular RNA in 0.025 ml was mixed with 0.3 ⁇ g of the antisense primers GAPDH, TNF ⁇ , TNFB, IL-l ⁇ , IL-1B, IL-6, IFN ⁇ , IFN ⁇ AND IFNB.
  • reaction mixture was heated at 70° C for 5 minutes, cooled on ice, and treated with 500 U of Moloney murine leukemia virus RT (BRL, Be- thesda, Maryland) and 0.5mM each of all four deoxy- nucleotide triphosphates. Reverse transcription reactions were at 37°C for 15 minutes, then stopped by heating at 95°C for 10 minutes.
  • reaction mixtures were divided into equal ali- quotes and mixed with 0.5 ⁇ g sense and antisense primers, 0.5 mM deoxynucleotide triphosphates, and 2 U Amplitaq (Cetus Corp.).
  • oligonuc- leotides were synthesized on an Applied Biosystems DNA synthesizer (Applied Biosystems, Inc., Foster City, Cali- fornia) and checked for purity by polynucleotide kinase labeling and sequence gel analysis. Oligonucleotides were typically 95% pure.
  • Fetal rat cortical explant cultures Fetal Sprague- Dawley rat (15 day gestational age) forebrains were disso- ciated by trituration into a single cell suspension and adjusted to 1 x 10 6 viable cells/ml. Cells were plated in poly-L-lysine treated plastic culture wells in 1:1 mixture of Eagle and Ham's F12K medium with 50 U/ml penicillin, 50 ⁇ g /ml of streptomycin, 600 ⁇ g/ml glucose, 10% horse serum, and 10% FCS.
  • Ara-C cytosine arabinoside
  • the composition of these Ara-C treated cultures at day 10 was 70% to 85% neurons by neuron-specific enolase (NSE, Dako Corp., Carpinteria, California), 10% to 15% microglia by latex-bead phagocytosis and rat OX-6 staining, and less than 5% to 10% astrocytes by morphology and glial fibril- lary acidic protein (GAF, Dako Corp.) staining.
  • NSE neuron-specific enolase
  • GAF glial fibril- lary acidic protein
  • Morphologic changes in these neuron-enriched cell cultures correlated directly with MTT levels and were scored as 0 (no neuritic outgrowth) , + (dendritic outgrowth 2 to 4 perikaryons distance in > 50% of cells/field) , or ++ (dendritic outgrowth 4 to 8 perikaryons distance in > 50% of cells/field) .
  • 100 cells in 4 fields/culture were examined on successive days.
  • identi ⁇ cal microscopic fields were photographed at serial inter ⁇ vals to decrease sampling variability.
  • the reaction was stopped by the addition of 10 ⁇ l formic acid and 25 ⁇ l butylated hydroxytoluene in methanol and the samples placed on dry ice. Supernatant fluids were removed and the cells scraped following the addition of 500 ⁇ l of HPLC-grade water. The cell lysate was combined with the culture supernatants from each well and placed into 10 ml polypropylene centrifuge tubes. The fractured cell-super- natant mixtures were centrifuged at 400 X g for 5 minutes and the clarified supernatants stored under argon at -70°C in 4 ml dram vials.
  • a 2-ml thawed sample was adjusted to pH 4.0 with 22M formic acid and micro- centrifuged for 1 minute.
  • the internal standard mix was added to the cell lysate. It contained hydroxy- eicosadienoic acid (for spectrophotometric verification of elution position accuracy) and 14 C eicostrienoic acid (for quantitation of sample recovery and inter/intra sample comparisons) .
  • Disintegrations per minute (DPM's) of each sample were adjusted based on recovered 14 C eicostrienoic acid.
  • a C18 Sep-Pak cartridge (Waters Associate, Milford, Maine) was activated by placing 4 ml of HPLC-grade methanol through the cartridge.
  • the C18 Sep-Pak was washed with 10 ml of HPLC-grade water.
  • the sample was applied to the C18 Sep-Pack cartridge followed by a 2.5 ml wash and arachi ⁇ donic acid metabolites were quantitatively eluted with a mixture of 85% acetonitrile and 15% methanol.
  • the eluant was concentrated and dried with a Speed-Vac concentrator (Savant Instruments, Inc., Farmingdale, New York).
  • the arachidonic acid metabolites were then dissolved in metha ⁇ nol and transferred to HPLC vials (National Scientific Co., Lawrenceville, Georgia) for injection.
  • the arachidonic acid metabolites extracted were then injected into a Beck- man reverse phase C-18 column using a Beckman analytical HPLC system.
  • Spectrophotometric analysis were performed with a Beckman 166 UFV detector. One minute fractions were collected during a 96 minute chromatography run and total DPM determined in each fraction. A second separately collected elution was performed on the Sep-Pak that quanti ⁇ tatively removed platelet activating factor (PAF) . The eluate was dried and analyzed using a quantitative RIA assay kit (Amersham Corp.). Results:
  • composition of fetal rat brain cultures at the time of experimental inoculation was 70% to 85% neurons (neurofiliment and neuron-specific enolase-positive cells) , 10% to 15% microg ⁇ lia (OX-6-positive cells that ingest latex beads) , 5% to 10% astrocytes (GFAP positive cells) and 0 to 3% fibro- blasts (vimentin-positive cells) .
  • Additions of fluids from HIV-infected and control uninfected monocytes to the rat brain explants showed neuronotrophic activity.
  • NSE + neurons treated with HIV-infected and control monocyte fluids for 5 days were 2 to 3 fold higher than equal num ⁇ bers of neurons treated with culture medium alone.
  • Fetal rat brain cells inoculated with fluids from HIV-infected or control uninfected U251 MG, U373 MG or HTB 148 cells also showed no neuronotoxicity.
  • cytokines may produce neurotoxicity and thus contribute to the pathogenesis of CNS disease.
  • Two cytokines, IL-l ⁇ and TNF ⁇ are associated with glial prolif ⁇ eration, neurotoxicity and demyelination. Interestingly, these cellular effects are all prominent in neuropathic infections.
  • mRNAs for IL- l ⁇ , IL-l ⁇ , TNF ⁇ and TNF ⁇ mRNAs in cell lysates of uninfect ⁇ ed and HIV-infected monocyte cultures were absent. Howev ⁇ er, the predicted 237bp and 179bp amplification products of TNF ⁇ and IL-l ⁇ mRNAs were readily seen in cell lysates of uninfected and HIV-infected monocytes and glia (U251 MG and HTB 148 cell lines.
  • TNF ⁇ and IL-l ⁇ mRNAs were not detected in cell lysate and culture fluids of uninfected control or HIV-infected monocytes co-cultured with endothelial cells or neuroblastoma cells.
  • Control uninfected HIV-infected U251 MG cells also showed no TNF ⁇ and IL-l ⁇ mRNAs.
  • Replicate experiments with primer pairs for 3 other cytokines were below the limits of PCR detection) .
  • TNF ⁇ and IL-l ⁇ protein and biological activity were seen only in co-cul ⁇ ture fluids of HIV-1 infected monocytes and glia.
  • the TNF ⁇ and IL-l ⁇ proteins were observed in co-culture of HIV-1 infected monocytes and astroglial. Maximum levels were present 12 to 48 hours following co-cultivation. In a series of 4 replicate experiments, maximum levels of TNF ⁇ were 1,000 to 9,000 pg/ml (mean of 5,000) while IL-l ⁇ levels ranged from 400 to 5000 pg/ml mean 900) .
  • the re ⁇ sults were confirmed by assays of TNF activity.
  • Cytokines produced during the interactions between HIV-infected monocytes and glia required viable mixtures of both cell types.
  • U251 MG or HTB 148 were mixed with 4% paraformaldehyde-fixed or freeze-thawed HIV-infected monocytes, cytokines were not detected.
  • TNF ⁇ and IL-l ⁇ were further tested.
  • Toxicity of recombinant human rhTNF ⁇ (Amgen, Thousand Oaks, California) and rhIL-l ⁇ (Collaborative Research, Bedford, Maine) was tested on rat fetal neuronal cultures in the same manner.
  • the cytokine concentrations used in these assays were extrapolated from data from co-cultures of HIV-infected monocytes and astro ⁇ glia (1 to 10 ng/ml of recombinant protein) .
  • Inoculation of TNF ⁇ and IL-l ⁇ alone or in combination to fetal neuronal cultures produced no neuronotoxicity.
  • mycoplasma and endotoxin contamination were ruled out as the neurono- toxin in the cell and viral preparation.
  • Mycoplasma was not detected by hybridization assays in any of 10 randomly selected culture fluids.
  • the levels of endotoxin contami- nation as detected by the LIMULUS amebocyte lysate assay were ⁇ 50 pg/ml.
  • the addition of polymyxin B at 15 ⁇ g/ml, a concentration known to inhibit an LPS-induced cytokine production had no significant effect on cytokine levels observed in HIV-infected monocyte-astroglia co-cultures.
  • the induction of arachidonic acid metabolites during co-cultures of HIV-infected monocytes and astroglia implies a relationship between cytokine regulation and neurono ⁇ toxicity.
  • the use of the methods disclosed herein provides means for screening neuroprotective drugs.
  • the results indicate that arachidonic acid metabolites are upregulated in monocytes infected with HIV. These metabolic products can regulate TNF ⁇ and IL-l ⁇ production in macrophages.
  • TNF was implicated as a cause of amplification of arachidonic acid metabolites in response to IL-1, while PAF was shown to enhance TNF production, implying an autocrine/paracrine loop between arachidonic acid metabolites and cytokines and vice versa.
  • Arachidonic acid metabolic products released from uninfected control monocytes, HIV-infected monocytes, uninfected monocytes with U251 MG astroglial cells, and HIV-infected monocytes with U251 MG astroglial cells were separated by HPLC and measured. Cells were radiolableled with [ 3 H arachidonic acid for 18 hours before co-culture. The arachidonic metabolites were identified based on elu- tion position standards (Table 2) and use of increasing polar solvents. The elution profiles of uninfected control and HIV-infected monocytes were virtually indistinguish ⁇ able.
  • Uninfected mono ⁇ cytes co-cultured with astroglia produced 15-HETE, a metab ⁇ olite found in low quantities in HIV-infected cells.
  • the differences in the metabolic arachidonate profiles persist ⁇ ed through 90 minutes.
  • HIV-infected mono- cyte-astroglia co-cultures showed a >20 fold increase in LTD 4 and uninfected co-cultures showed a >14-fold increase in 5-HETE.
  • LTB 4 oxidation product levels were cyclic but were always greater in HIV-infected co-cultures and in ⁇ creased 4-fold from co-cultures of uninfected monocytes and astroglia.
  • Macrophage-ColonyStimulating Factor had been infected 7 days previously with a monocytotropic HIV strain.
  • Suspensions of glial cells were added to wells with mono ⁇ layers of HIV-infected or uninfected human monocytes at an approximately 1:1 ratio. .At various time intervals ali- quots of media were removed, filtered, and flash-frozen for PAF determination by RIA.
  • Media alone added to unin ⁇ fected or infected monocytes resulted in PAF levels of less than 20 pg/ml at all time-points. Minutes after Uninfected HIV-infected media exchange Human monocytes Human monocytes
  • the PAF levels were evaluated by quantitative RIA. Release of glutamic acid, a neuronal excitotoxin, from primary rat fetal cortex explants in tissue culture was also studied. Supernatants were removed 5 minutes after additions and glutamate levels determined by reverse-phase high pressure chromatography with derivitization and fluorescent detection.
  • PAF platelet-activating factor
  • IL-l ⁇ and TNF ⁇ response were cytokine-specific, and correlated with neuronotoxicity, occurring only during co-culture of infected monocytes and astroglia.
  • TNF ⁇ , IL-l ⁇ , IL-6, IFN ⁇ AND IFNT were not produced under these conditions.
  • Mixtures of neuronal and endothelial cells with infected monocytes failed to elicit cytokines or neuronotoxins.
  • the addition of TNF ⁇ and IL-l ⁇ alone or in combination to neurons at con ⁇ centrations found in infected monocyte astroglia culture fluids did not produce neuronotoxicity. Viable glial cell- to-cell interactions were required.
  • infected monocyte culture fluids sucrose-gradient concentrated viral particle, and paraformaldehyde-fixed or freeze-thawed HIV-infected monocyte cell membranes failed to produce cytokine or neuronotoxic activity when placed on astro ⁇ cytes.
  • Two factors were identified as required for re- sponse: lipidic compounds derived from membrane phospho- lipids, including products of 5-lipoxygenase, and PAF. These products, potent low molecular weight mediators of immune activation, were secreted within 90 seconds of the mixture of the infected monocytes with the astroglia.
  • pathology of the brain tissue includes neuronal loss, reactive astrogliosis and myelin damage.
  • the pathology may result from acute, over ⁇ whelming infection, such as in acute epidemic encephalitis or may result from chronic infection such as AIDS.
  • macrophages contribute to disease progression by several mechanisms. At sites of infection macrophages secrete scores of toxic effector molecules that damage tissue. Proliferation of autoreactive T-cells through indigenous antigen presentation also lead to tissue damage such as that seen in multiple sclerosis (MS) and rheumatoid arthritis (RA) . In MS, cytokines produced from macrophages and T-cells produce myelin damage.
  • MS multiple sclerosis
  • RA rheumatoid arthritis
  • Dexamethasone a potent inhibitor of phospholipase A was added to the infected monocyte/astroglia preparation, and was shown to decrease TNF ⁇ 60 fold at a concentration of 10 "5 M after 12 hours.
  • Nordihydroguaiaretic acid (NDGA) an inhibitor of the lipoxygenase pathway, at a concentra- tion of 5 x 10 "5 , decreased TNF ⁇ five fold at 12 hours.
  • Indomethacin a cyclooxygenase inhibitor, increased TNF ⁇ production. (See Table 4) .
  • compositions were also tested for their activity (Table 5) .
  • Other active agents include N- methyl-3-[4-(2,5-dimethylpyrrol-l-yl)phenyl]propene- hydroxamic acid, N-(3-phenoxycinnamyl)-acetohydroxamic acid, (BW A4C) and leukotriene receptor antagonist sold as LY203647, which is l-[2-hydoxy-3-propyl-4(4-lH-tetrazol-5- ly)butoxy]phenyl ethanone.
  • THC-7-oic acid 11-Nor- ⁇ 8 - tetrahydrocannabinol-9-carboxylic acid
  • THC-7-oic acid 11-Nor- ⁇ 8 - tetrahydrocannabinol-9-carboxylic acid
  • This compound is a non- psychoactive metabolite of tetrahydrocannabinol which has been known to display potent bronchodilatory, anti-inflam- matory and analgesic activity.
  • the compound is minimally soluble in water, but may be formulated in oils or be prepared in cyclodextrin. The preparation of such composi ⁇ tions is known.
  • U.S. Patent 4,727,064 to Pitha which is incorporated herein by reference in its entirety, disclose methods of preparation of .buccal compositions. Any pharma ⁇ ceutically safe lipophylic agent such as a pharmaceutically acceptable glycol may be used to solubilize the THC-7-oic acid.
  • the dosage will be administered in sufficient amounts to provide a neuroprotective concentration in the blood.
  • dexamethasone will be given in sufficient amounts to provide a concentration of 10 "4 to 10- "6 molar concentration in the blood with the preferred dosage being a dosage that will deliver a blood concentration of O.l ⁇ M to 50 ⁇ M.
  • Nordihydroguaiaretic acid would be given in sufficient amount to provide about the same concentration, with the preferred dosage being sufficient to provide a concentration of 10 "6 to 10 "5 molar concentration in the blood.
  • sphingosine .2 ⁇ M to 100 ⁇ M
  • gossypol .2 ⁇ M to 100 ⁇ M
  • THC-7-oic acid the blood concentration should range from .04 ⁇ M to 50 ⁇ M. More preferred methods of administration are provided below.
  • THC-7-oic acid given orally dissolved in peanut oil, at a dosage of 2 to 10/mg/kg per day, to maintain a effective serum concentration of 5-25 micromolar, or THC-7-oic acid which has been solubilized and stabilized by dissolution as a 10 % solution in absolute ethanol, followed by addition of 2 volumes of 30% pyrogen-free aqueous solution of beta cyclodextrin containing .5% wt./vol of cysteamine HC1 as an antioxidant, followed by sterile filtration and aliquoting into sterile vials such as to contain 10 to 100 mg THC each. Vials are then lyophilized under vacuum sealed under nitrogen.
  • Each vial can then be reconstituted with sterile saline at the time of use to provide a solubilized product suitable for intravenous administration, or THC-7-oic acid solubilized with either ethanol or polyethylene glycol, and packaged in a pressurized, metered-dose container, to deliver a dose of 0.05 to 0.2 mg, for nasal insufflation.
  • This delivery system would be utilized approximately every 3 hrs while awake to maintain a therapeutic blood level and to circumvent inactivation by hepatic first pass metabolism resulting after oral administration, or THC-7-oic acid compounded with beta-cyclodextrin and stearic acid and methoxycellulose or other appropriate binders to form a tablet suitable for sub-lingual administration and buccal absorption.
  • THC-7-oic acid compounded with beta-cyclodextrin and stearic acid and methoxycellulose or other appropriate binders to form a tablet suitable for sub-lingual administration and buccal absorption.
  • the ginkolides including BN52021, BN50726 , BN 50739, and BN50730 (.25-5 mg/kg/day PO,) are usually administered at dosage of .1-1 mg/kg/day IV, buccally, or intranasally, or by inhalation) .
  • CV-3988 trans-2,5-bis-(3,4,5,-tri ethoxy-phenyl)-1,- 3,dioxolane (a diaryltetrahydrofuran) may be administered IV, buccally, or intranasally, or by inhalation at dosage of about .02-2 mg/kg/day.
  • l-O-hexadecyl-2-O-acetyl- sn-glycero-3-phospho-(N,N,N- tri-methyl) hexanolamine may be administered, for example, IV, buccally, or intranasally, or by inhalation at dosage of about .01-1 mg/kg/day.
  • the 1,4,-dihydropyridines including PCA 4233 and PCA 4248 may be given ad dosage of about 1 - 5 mg/kg/day PO, IV, buccally, or intranasally, or by inhalation.
  • Triazolobenzodiazepine compounds such as alprazolam, also known as triazolam, are usually administered to attain a serum concentration of 0.5 to 5 uM, with a dosage of 0.5-5 mg/kg per day PO.
  • Lipoxygenase enzyme inhibitors including nordihydro- guaracetic acid (NDGA) , are administered to attain a serum concentration of 1 - 10 uM and may be given, PO, IV, buc ⁇ cally, or intranasally, or by aerosol inhalation.
  • the preferred dosage is about 2-10 mg/kg/day.
  • a preferred dosage of A-64077 is usually 10-25 mg/kg/day and is usually administered PO, IV, buccal ⁇ ly, or intranasally, or by aerosol inhalation.
  • MK 886 is usually administered at dosage of 1-25 mg/kg/day, PO, IV, buccally, or intranasally, or by aerosol inhalation.
  • LY221068, 5-[ [3 ,5-bis(1,1,-dimethylethyl)-4-hydroxy phenyl]methylene]-3-(dimethylamino)-4-thiazolidinone is preferably administered PO, IV, buccally, or intranasally, or by aerosol inhalation. The most common dosage is about .2 - 2 mg/kg/day .
  • 1,2,3,4,-TETRAHYDRO-1-NAPTHOL is given at a preferred dosage of 0.5-5 mg/kg, PO, IV buccally, or intranasally, or by aerosol inhalation.
  • CGS 22745 an N-methyl-3-[4-(2,5,-dimethylpyrrol-l- yl)phenyll]propenehydroxamic acid inhibitor is 5-lipoxy- genase, usually given orally at 0.2 - 4 mg/kg per day.
  • Phospholipase A2 inhibitors including gossypol, a plant natural product used in China as a male contracep ⁇ tive, is usually administered PO (about 1-10 mg/kg/day) or buccally, intranasally or IV at 0.1-2 mg/kg to obtain a serum level of about 2-10 uM (about 1 - 10 mg/kg/day) and Scalaradial, a marine product, is given to obtain a desired serum level of 0.5 to 5 uM are effective for purposes of the invention..
  • the leukotrienne receptor antagonists including SC 41930 (7[3(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,- 4,-dihydro 8-propyl-2H-l-benzopyran-2-carboxylic acid give at dosage of about .2-4 mg/kg/day PO and LY203647, l-[2-hydroxy-3-propyl-4-(4-l-OH-tetrazol-5-ly)butoxy]phenyl ethanone, given at dosage of about 0.5-5 mg/kg/day, PO, buccally, or by aerosol via nasal or oral inhalation may be used.
  • Agents that can be readily absorbed from the mucous membrane can be deliver as cyclodextrin inclusion complexes for buccal administration.
  • Powders may be snorted from vials for delivery to the nasal membranes or may be provid ⁇ ed as mists for inhalation.
  • HIV-infected monocytes co-cultured with: endothelial cells +
  • HTB 148 Neuroglia
  • HIV-1 ⁇ freeze-thawed HIV-infected monocytes + paraform-fixed HIV-infected monocytes +
  • Fetal rat brain expants were cultured for 10 days, then treated for 5 days with monocytes media, conditioned media containing 20% v/v fluids from uninfected monocytes, HIV- 5.
  • HIV-1 ⁇ virus stock > lxio 8 HIV particles/ml by grid count on transmission microscopy
  • PAF production in cultured cells Monocytes cutured 7 days as adherent monolayers were exposed to HIV at a moi of 0.1. One week after infection, virus-infected monocytes were co-cultured with equal numbers of U251 MG astroglial cells. At 60 and 120 minutes of co-culture, the cells were lysed and PAF levels determined by RIA.
  • THC-7-oic acid 25 41 32 55 46 10 56 58 45 68
  • NDGA could be used in combination with AZT to provide synergistic effects that would make it possible to effectively treat patients using lower dosages of AZT.

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Abstract

This invention provides a method of treating encephalitis or encephalopathy secondary to CNS infection by administration of therapeutically effective amounts of compositions which inhibit the release of platelet activation factor and/or arachidonate metabolites. The invention also provides methods for screening for compounds that have neuroprotective activity wherein the methods comprise infecting monocytes or lymphocytes with an infectious organism known to cause neural damage, adding the resulting infected culture to a culture of astrocyte cells, adding a test compound, allowing sufficient time to pass for the production of TNF-alpha, withdrawing aliquotes from the supernatant of the culture, adding the aliquotes to cultures of neural cells and identifying which supernatants impart a neuroprotective effect.

Description

INHIBITORS OF ARACHIDONIC ACID METABOLITES FOR PREVENTING NEUROLOGICAL DAMAGE Field of the Invention;
This invention relates to the field of prevention of neuro- nal damage arising from diseases that cause increase in levels of platelet activation factor (PAF) and/or metab¬ olites of arachidonic acid. Some of the more deleterious effects of CNS infections can be mitigated by administra¬ tion of inhibitors of PAF and/or arachidonic acid metabo- lites. Compositions and methods disclosed herein also have application to treatment of retroviral infections, includ¬ ing human immunodeficiency virus. Background of the Invention;
Infection of the central nervous system (CNS) by viral, bacterial or protozoal pathogens results in high morbidity and mortality, even in those cases where specific anti-microbial therapy is available. Tissues of the cen¬ tral nervous system consist of several distinct cell types. Neurons are electrically active cells arranged in networks where cells are functionally and anatomically interconnect¬ ed by synapses. Release of specific molecules called neurotransmitters at synapses allow signalling between neurons. The term glia or neuroglia applies to the stroma of non-neural supportive cells. Astrocytes are the domi- nant glia, and function to control the constituents of the central nervous system (CNS) microenvironment within the limits optimal for neuronal viability and function. They also have the capacity to take up and to release biomolecules which effect neuronal function. Normally, neither neurons nor astrocytes divide in the adult animal, but in the presence of inflammation or specific cytokines, astrocytes begin to divide and proliferate, as well as releasing a variety of products which effect neuronal function. The oligodendrocyte is a specialized cell which produces and maintains myelin, an electrical insulator which surrounds fascicles of neuronal axons, and is re¬ quired for effective propagation of signals along these axons. Dysfunction of these cells can cause loss of my- elination and disease conditions such as multiple sclero¬ sis. The microglia are the brain macrophages derived from the same bone-marrow precursors as tissue macrophages, but assuming specialized morphology and function in the CNS microenvironment. The cells initiate the inflammatory response within these cells and represent the overwhelming majority of CNS cells harboring virus and allowing viral replication in many viral infection, including HIV. In th system described below, which models the interactions between virus-infected microglia, astrocytes, and neurons, primary cultures of human monocyte/macrophages are infecte with virus as a practical surrogate for human microglia, which would otherwise need to be obtained from fresh human brain tissue. The cellular and molecular mechanisms by which infection and inflammation lead to damage of neurons has been studied. However, effective means for prevention and/or treatment of the damaging effects of these infec¬ tions has not been available. It has previously been know that both tumor necrosis factor (TNF) and lymphotoxin (LT induced injury to oligodendrocytes in a time and dose- dependent manner (Journal of -Immunology, Vol. 147, No. 5
(September 1, 1991) 1522-1529) . Chung and Benveniste have shown that astrocytes secrete TNFα in response to a variet of biologic stimuli, particularly to cytokines IL-1 and interferon-τ (INF-τ) , which are known to be present in the central nervous system during neurologic diseases associat ed with inflammation (Journal of Immunology, Vol 144, No. (April 15, 1990) 2999-3007). Poubelle, et al. taught that platelet activating factor (PAF) enhances concomitant production of TNFα. One of the major viruses which cause severe neuronal damage is human immunodeficiency virus (HIV) , the causativ agent of AIDS. Recent studies of HIV-infected cultures have cast new light on some of the mechanisms which give rise to neuronal damage. Several studies have demonstrated entry of virus unto the CNS early after infection with the HIV, either during the acute seroconversion reaction or during subclinical infection. Debate has centered around exactly how HIV enters the brain and preferentially infects macrophages. Virus-infected brain macrophages may origi¬ nate from an expansion of latently infected monocytes that carry HIV into the brain (the "Trojan horse" hypothesis) and later produce virus. Alternatively, virus may pene¬ trate the brain through a disrupted blood-brain barrier by infected T cells or as free viral particles. In either case, the results are identical, selective productive infection of brain macrophages and macroglia. Whether these HIV-infected brain macrophages induce disease through metabolic, immune and/or viral-induced mechanisms is criti¬ cal to our understanding of HIV neuropathogenesis. Several reports suggest that low-level infection of neurons and glia can produce neurologic impairment during HIV infec- tion.
Theories of CNS dysfunction in the case of HIV infec¬ tion include coexistence of opportunistic CNS infection, secretory toxic factors from infected monocytes, gpl20- mediated neuronal growth factor (NGF) blockade or killing, and neuronotoxicity by HIV tat or other viral regulatory components. All or many of these mechanisms may result in cytotoxic effects in neurons and/or oligodendrocytes. For example, gpl20 may antagonize normal vasoactive intestinal peptide (VIP) function in the brain or be directly toxic to neurons. Studies show that gpl20 can induce neurono¬ toxicity by increasing Ca++ levels in cultured neurons and are prevented by Ca++ channel antagonists.
Recent reports suggest that brain dysfunction may be related to cell-encoded toxins generated from virus-infec- ted macrophages. Secretory products from HIV-infected cells may alter neuronal viability, damage myelin, or stimulate neurotransmitters resulting in neuronal dysfunc- tion. Macrophages play important roles in steady-state immune and tissue function. The regulatory role of macro¬ phages occurs through the release of numerous secretory molecules made under a variety of physiologic conditions. Changes in the secretion or release of certain of these mediators may lead to disease. In support of this idea are recent studies demonstrating that disordered secretion of one or more cellular factors from HIV-infected macrophages produce neuronal death in vitro. In one report, HlV-in- fected U937 cells, a myelomonocytic cell line, released toxic factors that destroyed cultured chick and rat neu¬ rons. The monocyte-produced neurotoxins were heat-stable and protease-resistant and acted by way of N-methyl-D- aspartate (NMDA) receptors. The studies suggested that HIV-infected macrophages and macroglia in the brain contin¬ uously disrupted neurologic function leading to cognitive CNS dysfunction.
Two recent reports failed to confirm these observa¬ tions. One study demonstrated neurotoxicity only following cell-to-cell contact between HIV-infected monocytic and human neural cells. The inventors have found no morpho¬ logical alteration of neurons by HIV-1 infected monocyte fluids. Thus, if the macrophage plays a role in virus- induced neuropathology it may act through cell-to-cell interactions with neurons and glia to produce CNS tissue damage. It is postulated that this may occur through cytokine and/or other neuronotoxic factors released during cell-to-cell contact. It is known that similar mechanisms are operative for cytokine induction in peripheral blood. For example, interferon alpha (IFNα) is induced during cell-to-cell interaction between HIV-infected macrophages and uninfected peripheral blood mononuclear cells (PBMC) . Similarly, neuronotoxins could be produced from glia during cell-to-cell interactions with infected brain macrophages Unique to this invention is the discovery that HIV-infec¬ tion of human monocytes causes activation of phospholipase A2 and lipoxygenase, resulting in the production of potent, short-lived lipid biomediators such as leukotrines, and known collectively as 5- and 12-lipoxygenase metabolites. These mediators appear to effect both the cell of origin, the infected macrophage) and the function of nearby unin- fected cells such as astrocytes and neurons.
A variety of inter-cellular and intra-cellular signal¬ ling pathways can lead to the activation of phospholipase A or C, which results in the release of arachidonic acid and diacyl glycerol from cell membrane phospholipid components. Diacyl glycerol acts within the cell to increase intracel- lular Ca levels, leading to phosphorylation and activation of cellular enzymes which regulate cellular functions. Arachidonic acid is then metabolized via either the cyclo- oxygenase enzyme, into bioactive lipids known as prosta- glandins, or via several lipoxygenase enzymes into a variety of derivatives including certain powerful pro- inflammatory lipid signalling molecules known as leuko- triennes. PAF synthesis is believed to largely occur through the activation of phospholipase A2, which hydro- lyzes the sn-2 acyl fatty acid (predominantly arachidonic acid) of 1-0-alkyl phosphatidylcholine in membrane phospho- lipids, releasing lyso-PAF. Lyso-PAF is subsequently reacylated by arachidonyl-CoA acyltransferase, regenerating the parent phospholipid, or by lyso-PAF acetyl CoA acetyl- transferase, producing PAE. It has been shown that ara¬ chidonic acid metabolites of the lipoxygenase pathway are released when HIV-infected monocytes but not un-infected monocytes interact with surrounding uninfected cells. Also released is a phospholipid known as platelet activating factor or PAF, as shown by the referenced application and the data below. Both of these findings imply activation of the enzyme phospholipase A2. Due to mechanisms as yet undetermined, this cascade of events appears to be required for active replication (as opposed to latency) of HIV virus within the macrophage or monocyte or lymphocyte. Summary of the Invention;
This invention to provides a method of treating en- cephalitis or encephalopathy secondary to CNS infection by administration of therapeutically effective amounts of compositions containing agents that inhibit release of platelet activation factor (PAF) and/or arachodonate etab- olites. Appropriate pharmaceutical agents include (1) inhibitors of the lipoxygenase enzyme pathway and/or PAF production, (2) receptor antagonists of a class of inflam¬ matory mediators known as leukotrienes, and (3) receptor antagonists of PAF which may be administered either alone or in combination. The active agents used in accord with the teachings of this disclosure may also be given in combination with other therapeutic agents such as antibiot¬ ics, immune enhancers, and HIV-inhibitors. It is also a purpose of this invention to provide a method for inhibit- ing the replication and release of infective virus in cells using any of several classes of drugs disclosed herein to block the metabolic events transpiring after hydrolysis of membrane lipids by phospholipase enzyme activity. In treat¬ ment of HIV, methods described herein may be practiced in conjunction with administration of AZT, DDI and DDC and other known therapeutic agents used in treatment of retro- viral infections.
The manner of administration will depend on the course of the disease process, the susceptibility of the active agent to inactivation in the gut or upon exposure to en¬ zymes, and the duration of treatment required.
This disclosure also teaches a method for screening drugs for use for neuroprotective efficacy in CNS infection based on the inhibition of induction of neurotoxic factors using a unique cell culture system.
Detailed Description of the Invention;
It has been discovered, using a unique system to model intracellular interactions resulting in encephalopathy and encephalitis following viral infection, that specific arachidonate metabolites and platelet activating factor (PAF) mediate neuronal damage and that specific agents which inhibit their release or antagonize their action are useful for preventing or decreasing neural damage. The methods of treatment also have general applicability for treatment of encephalitis due to parasitic or bacterial infection of the central nervous system (CNS) . The method of screening pharmaceutical compounds for neuroprotective activity comprises infecting monocytotropic cells with an organism known to cause neuronal damage. After effectively infecting the cells astrocytes (prefera¬ bly astrocytoma cells) are added to the infected culture. In a preferred embodiment, the number of astrocytes added is roughly equivalent to the number of cells in the origi¬ nally infected culture. To some of the cultures there is added a test compound to be evaluated for neuroprotective activity. The cultures to which no test compound is added serve as controls. The cultures are then incubated for a sufficient period of time to allow production of TNFα. Reduction of more than 70% TNFα in cultures containing test compounds was considered evidence that the test compound had inhibitory activity. The supernatant is then withdrawn and assayed for TNFα. The supernatants may then be frozen for later use or used immediately. Aliquots of supernatant are then added to cultures of neurons (preferably embryonic neurons) . After sufficient time has been allowed for interaction (3 days was the period of time used in this instance) , the neuron/culture mixture is evaluated for evidence of neurotoxic or neuronocytopathic effects. The method mimics cell-to-cell interaction which, in vivo, allows or potentiates both the production of TNFα and of neuronotoxic factors. The infective agent used to infect the cell culture may vary. Examples of useful infective agents are HIV, dengue virus, toxoplasmosis, cryptococcus, and listeria.
It has been shown by the testing means that viral replication in monocytes or microglial cells in the pres- ence of astrocytes (the predominant non-neuronal cell type in the brain) results in release of specific arachidonate metabolites and PAF. It has been found that drugs which inhibit production of these factors can prevent the genera¬ tion of neurotoxic activity in infected cells. The active agents were found to inhibit the secretion of interleukin 1 (IL-1) and tumor necrosis factor-alpha (TNFα) in cultures of uninfected astrocytes in the presence of HIV-infected macrophages.
It has been shown herein that the direct addition of PAF to primary cultures of either rat or human neurons resulted in rapid and dramatic destruction of neurons without obvious toxicity to other cell types. This toxici¬ ty appears to be increased by the simultaneous presence of Lipoxin Af (LXA) and leukotrienes LTD4 and LTB4 or of TNF- α. The LXA, leukotrienes and TNF-α were also released in excess in the model system of infection. Addition of drugs known to inhibit the lipoxygenase pathway in the virus- infected cultures prevented the excess production of all three neurotoxic constituents, e.g. PAF, the leukotrienes and TNF-α., and prevented development of neurotoxic activi¬ ty in the supernatants. HIV-infected monocytes and macrophages are believed to represent the largest reservoir of virus in the infected patient. HIV remains latent and persistent in these cells for long periods of time. However, during acute HIV infec¬ tion and subsequent to a variety of poorly understood activation signals, activation of viral replication in the cells results in abundant release of infected virons. Additionally, cells of monocyte lineage represent the reservoir of infection within the central nervous system, and viral replication in these cells is most closely linked with the wide range of neuropathology associated with HIV disease. In setting up a model system for drug screen¬ ing, alteration in arachidonic acid metabolism in HIV- infected monocytes and other potent intracellular signal¬ ling molecules derived from membrane lipids which had been known to modulate inflammation and immunological responses were also studied. It was found thereby that infection of human monocytes with HIV is associated with dramatic alter- ation in the metabolism of membrane lipids into active soluble products. Arachidonic acid is preferentially metabolized by the lipoxygenase enzyme pathway into a variety of compounds including 5-HETE (5-hydroxyeicosa- tetraenoic acid), Lipoxin-A, Leukotriennes (LTB4, LTC4 and LTD4) . Release of PAF was also dramatically increased. Inhibitors of the lipoxygenases prevented induction of TNF- α and IL-lβ in infected cells by HIV-infected monocytes in co-culture, as do PAF antagonists. Inhibitors of the lipoxygenase pathway also were shown to decrease the cyto- pathic effects of productive HIV infection on human mono¬ cytes. Inhibitors of the cyclooxygenase enzyme, which represents a parallel and competing pathway of arachodonate metabolism, have the opposite effect. The data suggest a cascade of events where PAF and lipoxygenase products of HIV-infected monocytes elicit abnormal secretion of TNFα and IL-1 from surrounding unin- fected cells. HIV-infected macrophages evidence activation of phospholipase A2, resulting in formation of arachidonic acid from membrane phospholipids. The phospholipase A2 is preferentially metabolized by the cell via the 5-lipoxy- genase pathway, resulting in production of a series of biologically active compounds, including 5-HPETE, 5-HETE and leukotrienes and results in release of PAF. The com- pounds have the effect of .both acting on the HIV-infected cell (autocrine activity) and on neighboring uninfected cells (paracrine activity) to induce release of cytokines which include interleukin-1 and TNF. Furthermore, PAF and lipoxygenase products act on the infected cell to mobilize intracellular calcium and diacylglycerol, leading to the activation of protein kinase-C (PKC) . PKC activates, via phosphorylation, specific cellular enzymes. This cascade of events appears to be required for both release of solu¬ ble neuronotoxins by HIV-infected monocytes co-cultured with normal astrocytes and active replication of HIV within the macrophage or monocyte.
While TNFα and PAF have been shown in other systems to cause a cyclic stimulation of each other and PAF had been shown to prime cells for lytic activity of TNFα, the stud¬ ies had not been extended to provide a means for drug screening. The novel pharmacologic therapy to ameliorate damage arising from CNS infection can utilize, individually or conjointly, several classes of drugs. 5-lipoxygenase inhibitors can be used to block the production of the 5- HETE, 5-HPETE, leukotriene, and other 5-lipoxygenase prod- ucts, shunting arachidonic acid into the cyclooxygenase pathway to produce prostaglandins (which may also impede HIV replication) . Leukotriene receptor antagonists may be administered to block the actions of leukotrienes at their cellular receptors. Both the 5-lipoxygenase inhibitors and the leukotriene receptor antagonists appear to decrease release of PAF. Compositions containing PAF antagonists may be administered to obstruct the actions of PAF at its cellular receptor. Furthermore, the protein-kinase C inhibitors may block the penultimate result of the process on the cells by suppressing activation of enzymes by pro¬ tein-kinase C.
Examples of 5 lipoxygenase inhibitors include zi- leuton, nordihydroguaiaretic acid (NDGA) , (+)-2,2-dibutyl- 5(2-quinolylmethoxy)-1,2,3,4-tetrahydro-l-napthol and 5- [[3,5-bis(1,1,dimethylethyl)-4-hydroxyphenyl]methylene]-3- (dimethylamino)-4-thiazolidinone. An example of a leuko¬ triene receptor antagonist is 7[3(4-acetyl-3-methoxy-2- propylphenoxy)-propoxy]-3,4-dihydro-8-propyl-2H-l-benzo- pyrano-2-carboxylic acid. Examples of PAF antagonists include ll-nor-Δ8-tetrahydrocannabinol-9-carboxylic acid (THC-7-oic acid) and derivatives thereof, the ginkolides (triazolothienodiazepine derivatives), trans-2,5-bis(3,4,5 trimethoxyphenyl)-1,3,-dioxolane (a diaryltetrahydrofuran) , the 1,4-dihydropyridines, and l-O-hexadecyl-2-O-acetyl-sn- glycero-3-phospho-(N,N,N-trimethyl) hexanolamine. Examples of protein-kinase-C inhibitors include gossypol, sphingo- sine, and staurosporine. Materials and Methods
Isolation and culture of monocytes and neural cells. Monocytes were recovered from PBMC of HIV and hepatitis B- seronegative donors after leukoperesis and purified by countercurrent centrifugal elutitration. Cell suspensions were >98% monocytes by criteria of cell morphology on Wright-stained cytosmears, by granular peroxidase and by nonspecific esterase. Monocytes were cultured as adherent monolayers (1 x 106 cells/ml in 24 mm plastic culture wells) in Dublvecco's modified Eagle's medium (DMEM) (Signa, St. Louis, MO) with 10% heat-inactivated AB+ human serum, 50 μg/ml gentamicin, and 1000 U/ml highly purified (less than 0.01 ng/ml endotoxin) recombinant human macrophage colony stimulating factor (MCSF) (P-809, Cetus Corp., Emeryville, California) . Human brain tumor-derived cell lines were obtained for the following sources: U251 MG from D. Bigner (J. Neuropathol. EXP. Neurol. 40:201) . U373 MG from B. Westermark (Acta Pathol. Microbiol. Scand. 18:791. and SK- N-MC fin Vitro 8:410) and H4(HTB 148) (J . Natl. Cancer Inst. 52:71) from the American Type Culture Collection
(ATCC) , Rockville, MD. The cells were grown as adherent monolayers in DMEM with 10% heat inactivated fetal calf serum (FCS) and 50 μg/ml gentamicin. Human endothelial cells were a gift of P.I. Lelkes ffProc. Natl. Acad. Sci. USA 87:6161-6165). Cell lines were fully characterized to their cell origins. Primary human astrocytes were prepared from second trimester fetal brain tissue obtained from elective abortions performed in full compliance with both NIH and University of Rochester guidelines. Brain tissue composed of telencephalon with both cortical and ventricu¬ lar surfaces were dissected in cold Hank's basic salt solution (HBSS) with HEPES and 50 μg/ml gentamicin, then transferred to 20 ml ice cold (4°C) DMEM/F12 (Gibco) with 10% heat inactivated FCS. The tissue was mechanically dissociated by teasing through a Nitex bag with a glass pestle. Cells were resuspended in media and filtered through a 230 um sieve, then through a 140 urn sieve. The cell suspension was centrifuged at 500 rp , washed 2X in media, then plated in DMEM containing 10% fetal calf serum (FCS) and 50 μg/ml gentamicin into 75 cm2 tissue culture flasks at a cell density of 2 X 105 cells/ml. Media was exchanged every 3 days. Non-adherent microglia and oligo- dendrocytes were removed by gentle agitation and circular shaking of cultured cell preparations 10 days after plat¬ ing. The purity of the astrocyte cultures was > 95% by immuno staining of glial fibrillary acidic protein (GFAP) . Cells were cultured as adherent monolayers in DMEM with 10% heat-inactivated FCS, 10 μg/ml gentamicin and 1% glutamine. All culture reagents were screened and found negative for endotoxin contamination.
HIV infection of target cells. Adherent monocytes cultured for 7 days were exposed at a multiplicity of infection (MOI)=0.01 infectious virus/target cell to ADA, or 24 monocyte tropic HIV-1 strains. All viral stocks were tested and found free of mycoplasma contamination (Gene- probe II, Gene-probe Inc., San Diego, California). Culture medium was half-exchanged every 2 to 3 days. Reverse transcriptase (RT) activity was determined in replicate samples of culture fluids added to a reaction mixture of 0.05% NONIDENT P-40 (Sigma Chemical Co.), 10 μg/ml poly(A) , 1.25 U/ml oligo (dT) (Pharmacia, Piscataway, NJ) , 5 mM dithiothreitol (Pharmacia), 150 mM KC1, 15 mM MgCl, and 3H- dTTP(2 Ci/mmol, Amersham Corp., Arlington Heights, Illi¬ nois) in pH 7.9 Tris-HCl buffer for 24 hours at 37° C. Radiolabeled nucleotides were precipitated with cold 10% trichloroacetic acid (TCA) and washed with 5% TCA and 95% ethanol in an automatic cell harvester (Skatron Inc. ,
Sterling, VA) on glass filter discs. Radioactivity was estimated by liquid scintillation spectroscopy in accord with the teachings of Kalter, et al. (J . Immunol. 146:298). Chemical reagents. Dexamethasone (Sigma Chemical Co, St. Louis, Missouri) and indomethacin and nordihydro- guaiaretic acid (NDGA) (Cyman Chemical Co., Michigan) were used as described herein. All reagents were dissolved in ethanol and diluted with complete macrophage media. Final ethanol concentration in cell cultures was < 0.1%.
Ouantitation of cytokine activity. Culture fluids from control and HIV-infected monocytes were analyzed by ELISA for human cytokines TNFα, IL-2α, IL-1B, and IL-6
(Quantikine Immunoassay, Research and Diagnostics Systems, Minneapolis, Minnesota) . IFN activity in culture fluids was assayed by inhibition of cytopathic effects induced by vascular stomatitis virus (VSV) in MDBK cells (J . Virol. .37_:755) • TNF bioactivity was performed according to stan¬ dard procedures (J. Immunol.. 141:4196). The murine L929 cell line was propagated in DMEM, 5% FCS, 1% glutamine and 20 μg/ml gentamicin. Cells were retrieved in log phase and placed (0,05 X 105/ ell) in 96-well plates (Costar) with actinomycin D. Culture fluids were inoculated into cell monolayers and degree of cell lysis determined by crystal violet staining after a 24 hour incubation.
Coupled reverse transcription/PCR detection of cyto¬ kine and HIV-specific RNA. Levels of cytokine RNAs were estimated after reverse transcription and antisense primers and PCR amplification of the cDNA transcripts. The mRNA for the cellular enzyme, glyceraldehyde 3-phosphate dehy- drogenase (GAPDH) , served as an internal control to allow analysis and comparison of RNA species between different samples. 2.0 μg total cellular RNA in 0.025 ml was mixed with 0.3 μg of the antisense primers GAPDH, TNFα, TNFB, IL-lα, IL-1B, IL-6, IFNα, IFNτ AND IFNB. The mixture was heated at 70° C for 5 minutes, cooled on ice, and treated with 500 U of Moloney murine leukemia virus RT (BRL, Be- thesda, Maryland) and 0.5mM each of all four deoxy- nucleotide triphosphates. Reverse transcription reactions were at 37°C for 15 minutes, then stopped by heating at 95°C for 10 minutes. For PCR amplification of the cDNA products, reaction mixtures were divided into equal ali- quotes and mixed with 0.5 μg sense and antisense primers, 0.5 mM deoxynucleotide triphosphates, and 2 U Amplitaq (Cetus Corp.). The products of 25 cycles (1.5 minutes at 94'C, 1.5 minutes at 50°C, and 2.0 minutes at 72°C) were analyzed by Southern blot hybridization. The oligonuc- leotides were synthesized on an Applied Biosystems DNA synthesizer (Applied Biosystems, Inc., Foster City, Cali- fornia) and checked for purity by polynucleotide kinase labeling and sequence gel analysis. Oligonucleotides were typically 95% pure.
Fetal rat cortical explant cultures. Fetal Sprague- Dawley rat (15 day gestational age) forebrains were disso- ciated by trituration into a single cell suspension and adjusted to 1 x 106 viable cells/ml. Cells were plated in poly-L-lysine treated plastic culture wells in 1:1 mixture of Eagle and Ham's F12K medium with 50 U/ml penicillin, 50 μg /ml of streptomycin, 600 μg/ml glucose, 10% horse serum, and 10% FCS. After 5 days, cultures were treated with lOμM cytosine arabinoside (Ara-C) for 48 hours to deplete pro¬ liferating astrocytes, fibroblasts and microglial cells. The composition of these Ara-C treated cultures at day 10 was 70% to 85% neurons by neuron-specific enolase (NSE, Dako Corp., Carpinteria, California), 10% to 15% microglia by latex-bead phagocytosis and rat OX-6 staining, and less than 5% to 10% astrocytes by morphology and glial fibril- lary acidic protein (GAF, Dako Corp.) staining. Cells were treated with conditioned media from cell cultures for 1-7 days and analyzed for neuronotoxicity.
Ouantitations of neuronal cell growth and survival. The metabolic activity and number of viable cells/culture were assessed by conversion of 3-(4,5-dimethylthiazol-2yl)- 2,5-diphenyl tetrazolium (MTT) bromide and color intensity measured at OD490 m. Cell morphology in neuronal cultures depleted of glial cells was examined under phase-contrast microscopy or after fixation with 80% methanol and Wright- Giemsa stain. Morphologic changes in these neuron-enriched cell cultures correlated directly with MTT levels and were scored as 0 (no neuritic outgrowth) , + (dendritic outgrowth 2 to 4 perikaryons distance in > 50% of cells/field) , or ++ (dendritic outgrowth 4 to 8 perikaryons distance in > 50% of cells/field) . Generally, 100 cells in 4 fields/culture were examined on successive days. In some studies, identi¬ cal microscopic fields were photographed at serial inter¬ vals to decrease sampling variability. Analysis of f3H1 Arachidonic Acid metabolites released by HIV -infected monocytes and co-cultures of HIV-infected monocytes and astroglia. HIV-infected or controlled unin- fected monocytes (5 X 106 cells/ml) were cultured in 35-mm wells in media containing 10% AB+ human sera and [3H]AA (1.0 μCi/ml; 1 Ci=37 GBq/ American Radiolabeled Chemicals, Inc., St. Louis, Missouri) added to cultures for 18 hours. HPLC procedures followed previously published methods. A brief description of the protocol used in these experiments is outlined below: The monocytes were washed 3X with DMEM in 1% bovine serum albumin, then incubated for 20 seconds to 180 minutes without equal numbers of U251 MG astroglial cells. The reaction was stopped by the addition of 10 μl formic acid and 25 μl butylated hydroxytoluene in methanol and the samples placed on dry ice. Supernatant fluids were removed and the cells scraped following the addition of 500 μl of HPLC-grade water. The cell lysate was combined with the culture supernatants from each well and placed into 10 ml polypropylene centrifuge tubes. The fractured cell-super- natant mixtures were centrifuged at 400 X g for 5 minutes and the clarified supernatants stored under argon at -70°C in 4 ml dram vials. Before analysis, a 2-ml thawed sample was adjusted to pH 4.0 with 22M formic acid and micro- centrifuged for 1 minute. The internal standard mix was added to the cell lysate. It contained hydroxy- eicosadienoic acid (for spectrophotometric verification of elution position accuracy) and 14C eicostrienoic acid (for quantitation of sample recovery and inter/intra sample comparisons) . Disintegrations per minute (DPM's) of each sample were adjusted based on recovered 14C eicostrienoic acid. A C18 Sep-Pak cartridge (Waters Associate, Milford, Maine) was activated by placing 4 ml of HPLC-grade methanol through the cartridge. The C18 Sep-Pak was washed with 10 ml of HPLC-grade water. The sample was applied to the C18 Sep-Pack cartridge followed by a 2.5 ml wash and arachi¬ donic acid metabolites were quantitatively eluted with a mixture of 85% acetonitrile and 15% methanol. The eluant was concentrated and dried with a Speed-Vac concentrator (Savant Instruments, Inc., Farmingdale, New York). The arachidonic acid metabolites were then dissolved in metha¬ nol and transferred to HPLC vials (National Scientific Co., Lawrenceville, Georgia) for injection. The arachidonic acid metabolites extracted were then injected into a Beck- man reverse phase C-18 column using a Beckman analytical HPLC system. Spectrophotometric analysis were performed with a Beckman 166 UFV detector. One minute fractions were collected during a 96 minute chromatography run and total DPM determined in each fraction. A second separately collected elution was performed on the Sep-Pak that quanti¬ tatively removed platelet activating factor (PAF) . The eluate was dried and analyzed using a quantitative RIA assay kit (Amersham Corp.). Results:
Evidence of neuronotoxic activity. There was no evidence of neuronotoxic activity in culture fluids of HIV- infected monocytes or glial cells. Monocytes and glia (U251 MG, and H4 HTB 148) cells were infected with HIV-1^ at an M0I= 0.01. Culture fluids from the HIV-infected and control uninfected cells were half-exchanged at 2-3 day intervals, then added, 24 days after plating and 7 days following infection, to rat brain explant and/or SK-N-MC human neuroblastoma cells. The composition of fetal rat brain cultures at the time of experimental inoculation (day 10 of culture) was 70% to 85% neurons (neurofiliment and neuron-specific enolase-positive cells) , 10% to 15% microg¬ lia (OX-6-positive cells that ingest latex beads) , 5% to 10% astrocytes (GFAP positive cells) and 0 to 3% fibro- blasts (vimentin-positive cells) . Additions of fluids from HIV-infected and control uninfected monocytes to the rat brain explants showed neuronotrophic activity. Numbers of NSE+ neurons treated with HIV-infected and control monocyte fluids for 5 days were 2 to 3 fold higher than equal num¬ bers of neurons treated with culture medium alone. Fetal rat brain cells inoculated with fluids from HIV-infected or control uninfected U251 MG, U373 MG or HTB 148 cells also showed no neuronotoxicity.
The absence of neuronotoxic activity from HIV-infected monocytes or virus-infected neuroglial cells led to assay of cell mixtures for neuronotoxic activities. Monocytes were infected with HIV-1ADA for 7 days, harvested from Tef¬ lon flasks, then placed onto equal numbers of neural cells. Fluids were harvested at 24 and 48 hours after co-cultiva¬ tion, then placed onto rat brain explant for assay of neuronotoxicity. In contrast to previous results, fluids from co-cultures of HIV-infected monocytes with U251 MG, U373 MG or HTB 148 astroglial cells were profoundly neuro¬ notoxic (Table 1) . Within 2 days following fluid addition, neurons were swollen and vacuolated. Propodium iodide staining showed few viable neurons 2 days after fluid addition. These fluids were also toxic for SK-N-MC neuro- blastoma cells. The neural cell toxic activity was only produced in mixtures of HIV-infected monocytes and glial (U251 MG, U373 MG and HTB 148 cells) (Table 1) . Dose response analysis of culture fluids from HIV-infected onocyte-astroglia mixtures showed significant neurono¬ toxicity with dilutions of < 1/20 of the fluids. Indeed, a > 50% loss of viable neurons/well was evident when a 20- fold dilution of culture fluids of HIV-infected monocytes- astroglia was placed onto rat fetal neurons. The toxic effects were cell specific. Fluids obtained from mixtures of HIV-infected monocytes and SK-N-MC (neuroblastoma) or HIV-infected monocytes and endothelial cells showed no neuronotoxic activity (Table 1) . The HIV-infected mono- cytes and U251 glial cells fluids were not toxic for rat or human astrocyte and fibroblasts.
A variety of cytokines may produce neurotoxicity and thus contribute to the pathogenesis of CNS disease. Two cytokines, IL-lβ and TNFα are associated with glial prolif¬ eration, neurotoxicity and demyelination. Interestingly, these cellular effects are all prominent in neuropathic infections.
Initial experiments were performed to determine wheth¬ er the addition of HIV-infected monocytes to astroglia resulted in cytokine gene expression. The levels of TNFα and IL-1B were examined by coupled transverse transcrip- tion/PCR in cell co-cultures. The mRNA for the constitu¬ tive cellular enzyme, GAPDH, was examined as the reference cellular transcript. GAPDG mRNA amplification products were present at equivalent levels in al cell lysates. IFNT mRNA was not detected in any cell lysate, demonstrat- ing that a mixed T-cell reaction was not an explanation for any cytokine mRNA expression observed. The mRNAs for IL- lβ, IL-lα, TNFα and TNFβ mRNAs in cell lysates of uninfect¬ ed and HIV-infected monocyte cultures were absent. Howev¬ er, the predicted 237bp and 179bp amplification products of TNFα and IL-lβ mRNAs were readily seen in cell lysates of uninfected and HIV-infected monocytes and glia (U251 MG and HTB 148 cell lines. Interestingly, TNFα and IL-lβ mRNAs were not detected in cell lysate and culture fluids of uninfected control or HIV-infected monocytes co-cultured with endothelial cells or neuroblastoma cells. Control uninfected HIV-infected U251 MG cells also showed no TNFα and IL-lβ mRNAs. Replicate experiments with primer pairs for 3 other cytokines (IL-lα, TNFβ and IL-6 were below the limits of PCR detection) . These results, taken together, show a selective and cell-specific induction of TNFα and IL-lβ mRNA during interaction between HIV-infected or control uninfected monocytes and glia.
The high levels of mRNAs were not always associated with similarly high levels of proteins. TNFα and IL-lβ protein and biological activity were seen only in co-cul¬ ture fluids of HIV-1 infected monocytes and glia. The TNFα and IL-lβ proteins were observed in co-culture of HIV-1 infected monocytes and astroglial. Maximum levels were present 12 to 48 hours following co-cultivation. In a series of 4 replicate experiments, maximum levels of TNFα were 1,000 to 9,000 pg/ml (mean of 5,000) while IL-lβ levels ranged from 400 to 5000 pg/ml mean 900) . The re¬ sults were confirmed by assays of TNF activity. The under¬ lying basis for this 10-fold difference was related to levels of productive HIV infection. Peak cytokine levels occurred during the initial rise of reverse transcriptase (RT) activity, 3 to 5 days following virus infection and diminished to baseline by day 10. TNFα and IL-lβ proteins were also detected at low levels < 100 pg/ml in co-cultures of uninfected monocytes and astroglia. The latter results suggest that the interactions seen between HIV-infected monocytes and glia are an extension of a normal physiologi¬ cal response. In all experiments assayed, the cytokine response mirrored the neuronotoxic response. Testing of Inhibitory Agents:
Cytokines produced during the interactions between HIV-infected monocytes and glia required viable mixtures of both cell types. When U251 MG or HTB 148 were mixed with 4% paraformaldehyde-fixed or freeze-thawed HIV-infected monocytes, cytokines were not detected.
The role of TNFα and IL-lβ in the observed neurono- toxicity was further tested. Toxicity of recombinant human rhTNFα (Amgen, Thousand Oaks, California) and rhIL-lβ (Collaborative Research, Bedford, Maine) was tested on rat fetal neuronal cultures in the same manner. The cytokine concentrations used in these assays were extrapolated from data from co-cultures of HIV-infected monocytes and astro¬ glia (1 to 10 ng/ml of recombinant protein) . Inoculation of TNFα and IL-lβ alone or in combination to fetal neuronal cultures produced no neuronotoxicity. Moreover, mycoplasma and endotoxin contamination were ruled out as the neurono- toxin in the cell and viral preparation. Mycoplasma was not detected by hybridization assays in any of 10 randomly selected culture fluids. The levels of endotoxin contami- nation as detected by the LIMULUS amebocyte lysate assay were < 50 pg/ml. The addition of polymyxin B at 15 μg/ml, a concentration known to inhibit an LPS-induced cytokine production, had no significant effect on cytokine levels observed in HIV-infected monocyte-astroglia co-cultures. These results demonstrate a clear association of cytokine and neuronotoxicity in co-cultures of HIV-infected mono¬ cytes and astroglia.
The induction of arachidonic acid metabolites during co-cultures of HIV-infected monocytes and astroglia implies a relationship between cytokine regulation and neurono¬ toxicity. The use of the methods disclosed herein provides means for screening neuroprotective drugs. The results indicate that arachidonic acid metabolites are upregulated in monocytes infected with HIV. These metabolic products can regulate TNFα and IL-lβ production in macrophages. TNF was implicated as a cause of amplification of arachidonic acid metabolites in response to IL-1, while PAF was shown to enhance TNF production, implying an autocrine/paracrine loop between arachidonic acid metabolites and cytokines and vice versa.
Arachidonic acid metabolic products released from uninfected control monocytes, HIV-infected monocytes, uninfected monocytes with U251 MG astroglial cells, and HIV-infected monocytes with U251 MG astroglial cells were separated by HPLC and measured. Cells were radiolableled with [3H arachidonic acid for 18 hours before co-culture. The arachidonic metabolites were identified based on elu- tion position standards (Table 2) and use of increasing polar solvents. The elution profiles of uninfected control and HIV-infected monocytes were virtually indistinguish¬ able. Based on elution times of standard eicosanoids (Table 2), low levels of LTBA, LTD4 and lipoxin A4 were noted. Although increased amounts of 5-HETE methyl ester and lactone were in HIV-infected cells, the levels of these metabolites were indistinguishable between infected and uninfected cell co-cultures. Major increases were also seen in levels of 5-HETEs, methyl ester and 6-lactone. Interestingly, LTB4 levels were transient, produced at 90 seconds, but not sustained. This is in keeping with its known cyclical production. Moreover, cyclooxygenase prod- ucts were identified only at low levels. Uninfected mono¬ cytes co-cultured with astroglia produced 15-HETE, a metab¬ olite found in low quantities in HIV-infected cells. The differences in the metabolic arachidonate profiles persist¬ ed through 90 minutes. At that time, HIV-infected mono- cyte-astroglia co-cultures showed a >20 fold increase in LTD4 and uninfected co-cultures showed a >14-fold increase in 5-HETE. LTB4 oxidation product levels were cyclic but were always greater in HIV-infected co-cultures and in¬ creased 4-fold from co-cultures of uninfected monocytes and astroglia.
Induction of PAF release from HIV-infected mono¬ cytes by co- culture with uninfected human glial cell line U251 MG. (PAF, pg/ml.) U251 cells were grown as a sus¬ pension culture in Teflon coated flasks. Human monocytes cultured as monolayers in the presence of recombinant
Macrophage-ColonyStimulating Factor, and had been infected 7 days previously with a monocytotropic HIV strain. Suspensions of glial cells were added to wells with mono¬ layers of HIV-infected or uninfected human monocytes at an approximately 1:1 ratio. .At various time intervals ali- quots of media were removed, filtered, and flash-frozen for PAF determination by RIA. Media alone added to unin¬ fected or infected monocytes resulted in PAF levels of less than 20 pg/ml at all time-points. Minutes after Uninfected HIV-infected media exchange Human monocytes Human monocytes
1 55 56
3 135 255 6 <20 235
12 <20 134
20 <20 55
30 <20 <20 The PAF levels were evaluated by quantitative RIA. Release of glutamic acid, a neuronal excitotoxin, from primary rat fetal cortex explants in tissue culture was also studied. Supernatants were removed 5 minutes after additions and glutamate levels determined by reverse-phase high pressure chromatography with derivitization and fluorescent detection.
ADDITION Glutamate (uM)
Control (media) .07 + .02 Bacterial LPS + gamma interferon .68 + .07
TNF-alpha 10 ng/ml .12 ± .05
PAF 1 ug/ml .11 + .03
TNF + PAF .45 ± .06
Neuronocytopathic effects of supernatants from co- incubated HIV-infected human monocytes and U-235 astro¬ glial cells added at 10% vol/vol to 14 day cultures of rat cortical neurons studied. Neuronotoxicity assessed photo- microscopically at 48 hours following additions. Culture Components Neuronocytopathicity U235 astroglial cells 0 un-infected monocytes 0
HIV-infected monocytes 0
Uninfected monos + U235 0
HIV-monos + U235 3+ HIV-monos + U235 + 20 UM NDGA 0
HIV-monos + U235 + 2 UM MK801 0
Determinations of platelet-activating factor (PAF) in cerebrospinal fluid from patients with HIV and other conditions. Patient ID DIAGNOSIS PAF
(pg/ml) 01 Multiple sclerosis in crisis 343
02 Bacterial Meningitis 249
03 Rule out meningitis -negative 64.7 04 Rule out meningitis -negative 60.5
05 Rule out meningitis -negative 73.3
1A AIDs, no CNS opportunist infection 335
IB same, later sample 230
2A HIV infection 198 2B same, later sample 172
Increased levels of PAF in co-culture of HIV-infected monocytes and astroglia were strongly associated with TNFα production (Table 3) . To investigate possible temporal relationship between arachidonic acid metabolites and TNFα, dexamethasone, an inhibitor of phospholipase A, indometha- cin, a cyclooxygenase inhibitor, or NDGA, a lipoxygenase inhibitor, to co-cultures of HIV-infected monocytes and astroglia and measured TNFα production (Table 4) .
Monocytes were infected with HIV at an MOI = 0.01 for 7 days before addition of U251 MG astroglial cells. The compounds were added together with equal numbers of astro¬ glia. Both dexamethasone and NDGA markedly reduced the levels of TNFα in supernatant fluids of these co-cultured cells. Indomethacin increased TNFα levels. This increase likely reflected shunting of the arachidonate metabolites into the lipoxygenase pathway. The failure of NDGA to completely abrogate the TNFα response suggests that PAF also participates in this cytokine response. Indeed, TNFα is reproducably detected following addition of PAF to co- cultures of uninfected monocytes and astroglia.
As indicated above, IL-lβ and TNFα response were cytokine-specific, and correlated with neuronotoxicity, occurring only during co-culture of infected monocytes and astroglia. TNFβ, IL-lα, IL-6, IFNα AND IFNT were not produced under these conditions. Mixtures of neuronal and endothelial cells with infected monocytes failed to elicit cytokines or neuronotoxins. Interestingly, the addition of TNFα and IL-lβ alone or in combination to neurons at con¬ centrations found in infected monocyte astroglia culture fluids did not produce neuronotoxicity. Viable glial cell- to-cell interactions were required. Moreover, infected monocyte culture fluids, sucrose-gradient concentrated viral particle, and paraformaldehyde-fixed or freeze-thawed HIV-infected monocyte cell membranes failed to produce cytokine or neuronotoxic activity when placed on astro¬ cytes. Two factors were identified as required for re- sponse: lipidic compounds derived from membrane phospho- lipids, including products of 5-lipoxygenase, and PAF. These products, potent low molecular weight mediators of immune activation, were secreted within 90 seconds of the mixture of the infected monocytes with the astroglia. In instances of encephalitis, pathology of the brain tissue includes neuronal loss, reactive astrogliosis and myelin damage. The pathology may result from acute, over¬ whelming infection, such as in acute epidemic encephalitis or may result from chronic infection such as AIDS. It is known that macrophages contribute to disease progression by several mechanisms. At sites of infection macrophages secrete scores of toxic effector molecules that damage tissue. Proliferation of autoreactive T-cells through indigenous antigen presentation also lead to tissue damage such as that seen in multiple sclerosis (MS) and rheumatoid arthritis (RA) . In MS, cytokines produced from macrophages and T-cells produce myelin damage. In rheumatoid arthri¬ tis, the secretion of monokines leads to alteration in endothelial cell adhesion and neutrophil influx. Ultimate- ly, alterations in macrophage secretory and antigen pre¬ senting functions lead to brain inflammation in MS.
Several compounds were tested by the methods described above. While some of the compounds have been previously used for treatment of disease conditions, some of the active agents have not been previously shown to have thera¬ peutic use.
Dexamethasone, a potent inhibitor of phospholipase A was added to the infected monocyte/astroglia preparation, and was shown to decrease TNFα 60 fold at a concentration of 10"5M after 12 hours. Nordihydroguaiaretic acid (NDGA) , an inhibitor of the lipoxygenase pathway, at a concentra- tion of 5 x 10"5, decreased TNFα five fold at 12 hours.
Indomethacin, a cyclooxygenase inhibitor, increased TNFα production. (See Table 4) .
Several additional compositions were also tested for their activity (Table 5) . Other active agents include N- methyl-3-[4-(2,5-dimethylpyrrol-l-yl)phenyl]propene- hydroxamic acid, N-(3-phenoxycinnamyl)-acetohydroxamic acid, (BW A4C) and leukotriene receptor antagonist sold as LY203647, which is l-[2-hydoxy-3-propyl-4(4-lH-tetrazol-5- ly)butoxy]phenyl ethanone. One of the most effective inhibitors was 11-Nor-Δ8- tetrahydrocannabinol-9-carboxylic acid (THC-7-oic acid) , which acts as a PAF antagonist. This compound is a non- psychoactive metabolite of tetrahydrocannabinol which has been known to display potent bronchodilatory, anti-inflam- matory and analgesic activity. The compound is minimally soluble in water, but may be formulated in oils or be prepared in cyclodextrin. The preparation of such composi¬ tions is known. U.S. Patent 4,727,064 to Pitha, which is incorporated herein by reference in its entirety, disclose methods of preparation of .buccal compositions. Any pharma¬ ceutically safe lipophylic agent such as a pharmaceutically acceptable glycol may be used to solubilize the THC-7-oic acid.
The method of administration, the carrier effect and the patient's condition and size must be considered in determining dosage of any particular active agent. Usual¬ ly, the dosage will be administered in sufficient amounts to provide a neuroprotective concentration in the blood. For example, dexamethasone will be given in sufficient amounts to provide a concentration of 10"4 to 10-"6 molar concentration in the blood with the preferred dosage being a dosage that will deliver a blood concentration of O.lμM to 50μM. Nordihydroguaiaretic acid would be given in sufficient amount to provide about the same concentration, with the preferred dosage being sufficient to provide a concentration of 10"6 to 10"5 molar concentration in the blood. Suggested concentrations of other active agent are, for sphingosine, .2 μM to 100 μM, for gossypol, .2 μM to 100 μM, for THC-7-oic acid, the blood concentration should range from .04 μM to 50 μM. More preferred methods of administration are provided below. ll-nor-delta-8-tetrahydrocannabinol-9-carboxylic acid
(THC-7-oic acid), given orally dissolved in peanut oil, at a dosage of 2 to 10/mg/kg per day, to maintain a effective serum concentration of 5-25 micromolar, or THC-7-oic acid which has been solubilized and stabilized by dissolution as a 10 % solution in absolute ethanol, followed by addition of 2 volumes of 30% pyrogen-free aqueous solution of beta cyclodextrin containing .5% wt./vol of cysteamine HC1 as an antioxidant, followed by sterile filtration and aliquoting into sterile vials such as to contain 10 to 100 mg THC each. Vials are then lyophilized under vacuum sealed under nitrogen. Each vial can then be reconstituted with sterile saline at the time of use to provide a solubilized product suitable for intravenous administration, or THC-7-oic acid solubilized with either ethanol or polyethylene glycol, and packaged in a pressurized, metered-dose container, to deliver a dose of 0.05 to 0.2 mg, for nasal insufflation. This delivery system would be utilized approximately every 3 hrs while awake to maintain a therapeutic blood level and to circumvent inactivation by hepatic first pass metabolism resulting after oral administration, or THC-7-oic acid compounded with beta-cyclodextrin and stearic acid and methoxycellulose or other appropriate binders to form a tablet suitable for sub-lingual administration and buccal absorption. The following are suggested means and dosages for delivery of other compounds:
The ginkolides (triazolothienodiazepine derivatives) including BN52021, BN50726 , BN 50739, and BN50730 (.25-5 mg/kg/day PO,) are usually administered at dosage of .1-1 mg/kg/day IV, buccally, or intranasally, or by inhalation) . CV-3988 (trans-2,5-bis-(3,4,5,-tri ethoxy-phenyl)-1,- 3,dioxolane (a diaryltetrahydrofuran) may be administered IV, buccally, or intranasally, or by inhalation at dosage of about .02-2 mg/kg/day. l-O-hexadecyl-2-O-acetyl- sn-glycero-3-phospho-(N,N,N- tri-methyl) hexanolamine may be administered, for example, IV, buccally, or intranasally, or by inhalation at dosage of about .01-1 mg/kg/day.
The 1,4,-dihydropyridines, including PCA 4233 and PCA 4248 may be given ad dosage of about 1 - 5 mg/kg/day PO, IV, buccally, or intranasally, or by inhalation. Triazolobenzodiazepine compounds such as alprazolam, also known as triazolam, are usually administered to attain a serum concentration of 0.5 to 5 uM, with a dosage of 0.5-5 mg/kg per day PO.
Lipoxygenase enzyme inhibitors, including nordihydro- guaracetic acid (NDGA) , are administered to attain a serum concentration of 1 - 10 uM and may be given, PO, IV, buc¬ cally, or intranasally, or by aerosol inhalation. The preferred dosage is about 2-10 mg/kg/day.
A preferred dosage of A-64077 (Zileuton) is usually 10-25 mg/kg/day and is usually administered PO, IV, buccal¬ ly, or intranasally, or by aerosol inhalation.
MK 886 is usually administered at dosage of 1-25 mg/kg/day, PO, IV, buccally, or intranasally, or by aerosol inhalation. LY221068, 5-[ [3 ,5-bis(1,1,-dimethylethyl)-4-hydroxy phenyl]methylene]-3-(dimethylamino)-4-thiazolidinone is preferably administered PO, IV, buccally, or intranasally, or by aerosol inhalation. The most common dosage is about .2 - 2 mg/kg/day . FR110302, ( (+)-2,2,-DIBUTYL-5-(2-QUINOLYLMETHOXY)-
1,2,3,4,-TETRAHYDRO-1-NAPTHOL) , is given at a preferred dosage of 0.5-5 mg/kg, PO, IV buccally, or intranasally, or by aerosol inhalation.
CGS 22745, an N-methyl-3-[4-(2,5,-dimethylpyrrol-l- yl)phenyll]propenehydroxamic acid inhibitor is 5-lipoxy- genase, usually given orally at 0.2 - 4 mg/kg per day. Phospholipase A2 inhibitors, including gossypol, a plant natural product used in China as a male contracep¬ tive, is usually administered PO (about 1-10 mg/kg/day) or buccally, intranasally or IV at 0.1-2 mg/kg to obtain a serum level of about 2-10 uM (about 1 - 10 mg/kg/day) and Scalaradial, a marine product, is given to obtain a desired serum level of 0.5 to 5 uM are effective for purposes of the invention..
The leukotrienne receptor antagonists, including SC 41930 (7[3(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,- 4,-dihydro 8-propyl-2H-l-benzopyran-2-carboxylic acid give at dosage of about .2-4 mg/kg/day PO and LY203647, l-[2-hydroxy-3-propyl-4-(4-l-OH-tetrazol-5-ly)butoxy]phenyl ethanone, given at dosage of about 0.5-5 mg/kg/day, PO, buccally, or by aerosol via nasal or oral inhalation may be used.
Usually choice of route of administration is deter¬ mined by consideration of a method that will cause the active agent to reach to site where damage is likely to occur. Agents that can be readily absorbed from the mucous membrane can be deliver as cyclodextrin inclusion complexes for buccal administration. Powders may be snorted from vials for delivery to the nasal membranes or may be provid¬ ed as mists for inhalation.
TABLE 1: NEURON SURVIVAL AND DIFFERENTIATION OF RAT BRAIN EX¬ PLANTS TREATED WITH CULTURE FLUIDS FROM UNINFECTED AND HIV-1 INFECTED MONOCYTES AND ASTROGLIA CULTURED FOR 5 DAYS
Medium alone Neuronal survival and differentiation
Tissue Culture medium medium alone + medium with MCSF +
Monocyte culture fluids control monocytes ++
HIV-1ADA-infected monocytes ++
HIV and its products
"IV- 1HTLVI I IB +
HIV-124 +
HIV gpl20 +
HIV-infected monocytes co-cultured with: endothelial cells +
SK-N-MC (neuroblastoma) +
U251 MG (astroglia) 0
U373 MG (astroglia) 0
HTB 148 (neuroglia) 0
U251 MG astroglia co-cultured with: uninfected monocytes +
HIV-1^ + freeze-thawed HIV-infected monocytes + paraform-fixed HIV-infected monocytes +
Fetal rat brain expants were cultured for 10 days, then treated for 5 days with monocytes media, conditioned media containing 20% v/v fluids from uninfected monocytes, HIV- 5. lADA and HIV-l24-infected monocytes or with HIV-lHTLVmB or
HIV-1^ virus stock (> lxio8 HIV particles/ml by grid count on transmission microscopy) , and 500 ng/ml recombinant HIV- HTLVIIIB GP 12°- CONDITIONED MEDIA CONTAINING 20% V/V FLUIDS FROM hiv-l^-infected monocytes co-cultured with endotheli- 0 al cells, SK-N-MC (neuroblastoma) , and U251 glial cells and U251 MG astroglial cells incubated with HIV-1ADA, freeze- thawed HIV-infected monocytes, paraformaldehyde-fixed HIV- infected monocytes and uninfected monocytes were harvested after 24 hours and assayed for neurotoxicity. Neuron survival and extent of differentiation was estimated by cell morphology on phase contrast microscopy or on metha- nol-fixed, Wright-Gies a stained slides.
TABLE 2 ELUTION TIME OF SELECTED EICOSANOIDS
Metabolite Time (min) Pathway
6-kPGF, 6.0 cyclooxygenase
LTB4 oxidation prod. 8.3 5-lipoxygenase
TSB2 11.2 cyclooxygenase
PGF2α 14.6 cyclooxygenase
PGE2 18.5 cyclooxygenase
Lipoxin A4 21.8 5,15 lipoxygenase
LTC4 23.5 5 lipoxygenase
LTE4 26.4 5-lipoxygenase
LTB4 30.1 5-lipoxygenase
LTD4 41.0 5-lipoxygenase
15-HETE 44.0 15-lipoxygenase
15-HPETE 49.1 15-lipoxygenase
12-HETE 52.1 12-lipoxygenase
5-HETE 56.5 5-lipoxygenase
5- HPETE 60.4 5-lipoxygenase
15-HEPE *** 65.0 internal standard
15-HETE methyl ester 71.5 15-lipoxygenase
5-HETE methyl < ester 75.9 5-lipoxygenase
5-HETE σ lactone 79.5 5 lipoxygenase
Arachidonic acid 83.0
Eicosatrienoic acid 92.0 internal standard TABLE 3: PAF LEVELS FOLLOWING HIV INFECTION AND CO-CULTURE OF HIV-INFECTED MONOCYTES AND ASTROGLIA
PAF (pg/106 cells) Minutes 60 120
Cell cultures: Monocytes cultured with:
U251 MG astroglial cells 260 100
HIV-infected monocytes cutured with: medium 360 100
U251 MG astroglial cells 990 670
U251 MG astroglial cells cultured with: medium 130 200
PAF production in cultured cells: Monocytes cutured 7 days as adherent monolayers were exposed to HIV at a moi of 0.1. One week after infection, virus-infected monocytes were co-cultured with equal numbers of U251 MG astroglial cells. At 60 and 120 minutes of co-culture, the cells were lysed and PAF levels determined by RIA.
TABLE 4: EFFECT OF ARACHIDONIC ACID INHIBITORS ON THE TNFα LEVELS FOLLOWING CO-CULTURE OF HIV-INFECTED MONO¬ CYTES AND ASTROGLIA
TNFα protein (pg/ml) hours after co-culture: 12 48
Cell Treatments: medium 3100 1760 dexamethasone (1200"5M) 50 30 indomethacin (0.4 μg/ml) 5030 4040 nordihydroguaiaretic Acid 602 550
(NDGA) (5X10"5M)
Effect of arachidonic acid inhibitors on TNF produc- tion: Monocytes cultured 7 days as adherent monolayers were exposed to HIV at an MOI of 0.01. One week after infection, virus-infected monocytes were co-cultured with equal numbers of U251 MG astroglial cells. At the time of co-culture, the cells were incubated with medium, dexameth¬ asone (10"5M) , indomethacin (0.4 μg/ml) or NDGA (5xlO"5M) . TNFα production was measured by ELISa. Data represent means of duplicate determination of 1 of 3 experiments performed.
TABLE 5 SUGGESTED CONCENTRATION FOR INHIBITORS
INHIBITOR 41 02 *3 #4
NDGA 25 μM 10 μM 4 μM 1 μM
Sphingosine 50 μM 25 μM
SPH/NDGA 50/10 μM 25/10 μM 10/10 2/10 gossypol 50 μM 20 μM 4 μM 1 μM
THC-7-oic 25 μM 10 μM 2 μM 0.2μM
The above are suggested concentrations at which bene¬ ficial effects would be expected.
When several of the drugs were tested to determine whether or not the active agents could be used to prevent HIV viral replication as well as to prevent neuronal dam¬ age. It was found that several of the active agents inhib¬ ited HIV replication and release of reverse transcriptase. It can be seen, therefore, that NDGA is particularly use- ful, alone or in the presence of AZT, for inhibiting HIV replication. Used in combination with AZT, it was possible to inhibit replication using decreased doses of both the AZT and the NDGA. Other agents useful for inhibition of HIV replication include THC-7-oic acid, Sphingosine. (See tables 6 and 7.) TABLE 6: RETROVIRAL REVERSE TRANSCRIPTASE ACTIVITY INHIBITION
Agent Cone. Viral Rev. Transcriptas :e, % con- trol (μM) Day 4 Day 8 Day 14
NDGA 30 8 4 3
10 44 80 38
5 75 82 54
1 60 83 66
Sphingosine 50 10 47 73 25 26 55 66 10 42 103 89
THC-7-oiσ acid 50 17 17 6
25 22 20 14
10 31 46 62
2 53 52 61
CV-3988 20 72 126 120 5 73 89 120 1 48 54 108 0.2 56 94 106
Hex-PAF-16 50 26 36 100
25 30 37 95
10 75 82 110
2 72 83 118
After 7 days in culture, recombinant M-CSF, human monocytes in confluent monolayers were infected with HIV-1 at a multiplicity of infection of 10. Every other day media was half exchanged with fresh media containing vehicle or drug and the media removed was frozen for RT assay.
TABLE 7: EFFECT OF TREATMENT ON REVERSE TRANSCRIPTASE PRODUC¬ TION
RT as % of no drug control
Agent Cone. (μM) Day 4 Day 8 Day 11 Day 13
NDGA 30 8 2 1 1 10 17 14 34 56
THC-7-oic acid 25 41 32 55 46 10 56 58 45 68
Indomethacin 2 151 106 105 124
AZT 0.5 μg/ml 8 13 4 1 5.0 μg/ml 9 1 1 1
AZT 0.5 μg/ml with NDGA 10 μM 8 1 1 1
The data would indicate that NDGA could be used in combination with AZT to provide synergistic effects that would make it possible to effectively treat patients using lower dosages of AZT.

Claims

Claims :
1. A method of screening compounds for neuroprotective activity comprising the steps of:
1) infecting cultured monocytes or lymphocytes with an infectious organism known to cause neuronal damage;
2) adding to the infected cell culture astrocyte cells;
3) adding aliquotes of test compounds to cultures obtained in step 2; 4) allowing sufficient period of time for production of TNFα;
5) withdrawing aliquotes of supernatant from the cultures obtained in step 4;
6) adding aliquotes of supernatants obtained in step 5 to cultures containing neurons;
7) allowing sufficient time for the supernatants and cultures prepared in step 6 to interact; and
8) inspecting the interacting supernatant/culture product of step 7 to determine whether neurotoxic or neuronocytopathic effects are present.
2. A method of claim 1 wherein the number of astrocytes added to the infected culture is approximately equal to the number of monocytes or lymphocytes originally in the infected culture.
3. A method of claim 1 wherein the supernatants obtained in step 5 are stored in the frozen state before addi¬ tion to the cultures containing neurons.
4. A method of claim 2 wherein the astrocytes are astro- cytoma cells.
5. A method of claim 3 wherein the neurons are embryonic neurons.
6. A method of claim 1 wherein the infective organism is immunodeficiency virus.
7. A composition of matter comprising supernatant from a culture containing infected monocytes or lymphocytes and astrocytes, said supernatant being in a culture of neurons.
8. A composition of claim 7 wherein the neurons are em¬ bryonic neurons.
9. A composition of claim 7 wherein the supernatant also contains a compound having neuroprotective properties.
10. A composition of matter comprising a neuroprotective amount of ll-nor-Δ8-tetrahydrocannabinol-9-carboxylic acid in a pharmaceutically acceptable carrier.
11. A composition of claim 10 wherein the carrier contains a cyclodextrin.
12. A composition of claim 10 containing a pharmaceutical¬ ly acceptable glycol.
13. A composition of matter comprising a neuroprotective amount of NDGA in a pharmaceutically acceptable carri¬ er for administration to and absorption through the mucosa.
14. A composition of claim 13 which is a cyclodextrin inclusion complex.
PCT/US1993/011542 1992-11-27 1993-11-29 Inhibitors of arachidonic acid metabolites for preventing neurological damage WO1994012667A1 (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0876143A2 (en) * 1995-09-11 1998-11-11 Yissum Research Development Company Of The Hebrew University Of Jerusalem TUMOR NECROSIS FACTOR ALPHA (TNF-$g(a)) INHIBITING PHARMACEUTICALS
EP0876143A4 (en) * 1995-09-11 2001-06-27 Yissum Res Dev Co Tumor necrosis factor alpha (tnf-alpha) inhibiting pharmaceuticals
US6630507B1 (en) 1998-04-21 2003-10-07 The United States Of America As Represented By The Department Of Health And Human Services Cannabinoids as antioxidants and neuroprotectants
US20140066497A1 (en) * 1999-03-22 2014-03-06 Immugen Pharmaceuticals, Inc. Treatment of immune dysregulation using cannabinoid derivatives
US9173867B2 (en) * 1999-03-22 2015-11-03 Immugen Pharmaceuticals, Inc. Treatment of immune dysregulation using cannabinoid derivatives
WO2007059507A2 (en) * 2005-11-15 2007-05-24 Baxter International, Inc. Compositions comprising lipoxygenase inhibitors and cyclodextrin
WO2007059507A3 (en) * 2005-11-15 2007-10-18 Baxter Int Compositions comprising lipoxygenase inhibitors and cyclodextrin

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