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WO1994012667A1 - Inhibiteurs de metabolites d'acide arachidonique destine a la prevention de deteriorations neurologiques - Google Patents

Inhibiteurs de metabolites d'acide arachidonique destine a la prevention de deteriorations neurologiques Download PDF

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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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Edward W. Bernton
Marti Jett
Howard Gendelman
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The United States Department Of The Army
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
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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

L'invention concerne un procédé de traitement d'une encéphalite ou d'une encéphalopatie suivant une affection du système nerveux central, consistant à administrer des doses thérapeutiquement efficaces de compositions inhibant la libération du facteur d'activation plaquettaire et/ou de métabolite d'arachidonate. L'invention concerne également des procédés de triage de composés présentant une activité neuroprotectrice, lesdits procédés consistent à infecter des monocytes ou des lymphocytes avec un organisme infectieux dont on sait qu'il provoque une détérioration des neurones, à ajouter la culture infectée obtenue à une culture d'astrocytes, à ajouter un composé de test, à laisser s'écouler suffisamment de temps pour permettre la production du facteur de nécrose tumorale α, à soustraire des aliquotes du surnageant de la culture, à ajouter les aliquotes à des cultures de cellules de neurones et à identifier les surnageants présentant un effet neuroprotecteur.
PCT/US1993/011542 1992-11-27 1993-11-29 Inhibiteurs de metabolites d'acide arachidonique destine a la prevention de deteriorations neurologiques WO1994012667A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0876143A4 (fr) * 1995-09-11 2001-06-27 Yissum Res Dev Co Produits pharmaceutiques inhibiteurs du facteur de necrose tumorale alpha (tnf-alpha)
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
WO2007059507A3 (fr) * 2005-11-15 2007-10-18 Baxter Int Compositions comprenant des inhibiteurs de lipoxygenase et de la cyclodextrine
US20140066497A1 (en) * 1999-03-22 2014-03-06 Immugen Pharmaceuticals, Inc. Treatment of immune dysregulation using cannabinoid derivatives

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US5134150A (en) * 1985-12-12 1992-07-28 Smithkline Beecham Corporation Inhibition of the 5-lipoxygenase pathway
US5144030A (en) * 1987-02-17 1992-09-01 Abbott Laboratories Fluorescene polarization immunoassay for tetrahydrocannabinoids

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US5134150A (en) * 1985-12-12 1992-07-28 Smithkline Beecham Corporation Inhibition of the 5-lipoxygenase pathway
US5144030A (en) * 1987-02-17 1992-09-01 Abbott Laboratories Fluorescene polarization immunoassay for tetrahydrocannabinoids

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Title
AIDS RESEARCH AND HUMAN RETROVIRUS, Volume 8, No. 4, issued April 1992, E.W. BERNTON et al., "No Direct Neuronotoxicity by HIV-1 Virions or Culture Fluids from HIV-1-Infected T Cells or Monocytes", pages 495-503. *
ANNALS OF NEUROLOGY, Volume 32, No. 1, issued July 1992, M. TARDIEU et al., "Human Immunodeficiency Virus Type-1-Infected Monocytic Cells CAn Destroy Human Neural Cells After Cell-to-Cell Adhesion", pages 11-16. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0876143A4 (fr) * 1995-09-11 2001-06-27 Yissum Res Dev Co Produits pharmaceutiques inhibiteurs du facteur de necrose tumorale alpha (tnf-alpha)
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
WO2007059507A3 (fr) * 2005-11-15 2007-10-18 Baxter Int Compositions comprenant des inhibiteurs de lipoxygenase et de la cyclodextrine

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