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WO2006101920A2 - Compositions et procedes lies a la manipulation de la signalisation du recepteur 2a de l'adenosine pour traiter des lesions neuronales associees au vih - Google Patents

Compositions et procedes lies a la manipulation de la signalisation du recepteur 2a de l'adenosine pour traiter des lesions neuronales associees au vih Download PDF

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Publication number
WO2006101920A2
WO2006101920A2 PCT/US2006/009390 US2006009390W WO2006101920A2 WO 2006101920 A2 WO2006101920 A2 WO 2006101920A2 US 2006009390 W US2006009390 W US 2006009390W WO 2006101920 A2 WO2006101920 A2 WO 2006101920A2
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Prior art keywords
adenosine
tat
hiv
receptor
mitochondrial
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WO2006101920A3 (fr
Inventor
Stephen Dewhurst
Servio Ramirez
Shao-Ming Lu
Harris A. Gelbard
Sanjay B. Maggirwar
Shongshan Fan
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University of Rochester
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University of Rochester
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir

Definitions

  • HIV-I highly active antiretroviral therapy
  • CNS central nervous system CNS
  • intra-CNS viral burden correlates with both the severity of virally-induced neurologic disease, and with the generation of neurotoxic metabolites.
  • Many of these molecules are capable of inducing neuronal apoptosis in vitro, but neuronal apoptosis in vivo does not correlate with CNS dysfunction.
  • the mechanism of virally-induced neurologic disease is not known in the literature.
  • HIV neurotoxin comprising contacting the cell with a modulator of adenosine receptor signaling.
  • HIV-I associated dementia HAD
  • a method of treating or preventing HIV-I associated dementia (HAD) in a subject in need of such treatment or prevention comprising administering to the subject a therapeutically effective dose of a modulator of adenosine receptor signaling.
  • composition comprising a modulator of adenosine receptor signaling and a molecule that inhibits mitochondrial hyperpolarization in a neural cell.
  • FIG. 1 shows that an adenosine receptor 2 A (A 2A R) antagonist protects neurons against Tat-induced apoptosis.
  • CGNs were exposed to HIV-I Tat (Tat, 500 nM), or vehicle alone (NT), in the presence or absence of the A2A antagonist, ZM241385. After 48h, cultures were analyzed for the percentage of apoptotic cells using the TUNEL assay. Data represent mean + SEM for one experiment that was performed in triplicate; data are representative of two independent experiments.
  • FIG. 2 shows that ATL455, an A 2A R antagonist protects neurons against Tat- induced apoptosis.
  • CGNs were exposed to HTV-I Tat (Tat, 500 nM), or vehicle alone (No Tat), in the presence or absence of the A 2A R antagonists, ATL455 and ZM241385, or the A 2A R agonists ATL313 and CGS21680. After 48h, cultures were analyzed for the percentage of apoptotic cells using the TUNEL assay. Data represent mean + SEM for one experiment that was performed in triplicate; data are representative of two independent experiments.
  • Figure 3 shows that adenosine receptors control nitric oxide (NO) secretion induced by Tat.
  • Human primary monocytes were treated with Tat in the presence or absence of the indicated adenosine receptor agonists for 8h, and nitric oxide levels in the culture medium of the cells were then quantitated using the Griess reaction (Active Motif). Data represent mean+SEM of three experiments.
  • FIG 4 shows that Tat-induced monocyte activation is opposed by the A 2A R agonist, CGS21680.
  • Human primary monocytes were treated with Tat (100 nM) in the presence or absence of the A 2A R agonist, CGS21680 (CGS; l ⁇ M) or the A 2A R antagonist, ZM241385 (100 nM) for 4h.
  • TNF ⁇ levels in culture supernatants were then quantitated by ELISA. Data represent mean+SEM of three replicates, from a single representative experiment
  • Figure 5 shows Tat induces inflammatory gene expression by primary human monocytes.
  • FIG. 6 shows that Tat-induced monocyte activation is opposed by the A 2A R agonist, ATL313.
  • Human primary monocytes were treated with Tat (100 nM) in the presence or absence of the A 2A R agonist, ATL313 (1-4 nM) or the A 2A R antagonist, ATL455 (1-4 nM) for 4h.
  • TNF ⁇ levels in culture supernatants were then quantitated by ELISA. Data represent mean+SEM of three replicates, from a single representative experiment.
  • Figure 7 shows a dose response analysis for inhibition of Tat-induced monocyte activation by the A 2A R agonist, ATL313.
  • Human primary monocytes were treated with Tat (100 nM) in the presence or absence of the A 2A R agonist, ATL313 (at the indicated doses) for 4h.
  • TNF ⁇ levels in culture supernatants were then quantitated by ELISA. Data represent mean+SEM of three replicates, from a single representative experiment.
  • FIG. 8 shows that Tat and PAF increase synaptic vesicular activity. Images of (A) Brightf ⁇ eld and (B) fluorescent FMl -43 labeling show that FMl -43 uptake results in labeling that is punctate and oriented primarily along neuronal processes. (C) Moreover, activity dependent FM 1-43 release by KCl depolarization preferentially reduces the punctate vesicular labeling versus background signal. (D) In ⁇ 14 and >14 day old primary neurons, 2.5 ⁇ g/ml Tat and 4.25 ⁇ g/ml Tat induces an increase in spontaneous activity dependent vesicular uptake, an effect that is both dose and culture age dependent. (E) However, no effect is seen with mutated Tat protein, suggesting that Tat's effect is due to biologically specific activities of its functional region. (F) 464 nM PAF had an even greater effect on FMl -43 uptake at 24 hours.
  • FIG. 9 shows that Tat and PAF increase ROS in cortical neuronal cultures.
  • Treatment of rat cortical neurons for 24 hours with Tat or PAF induced elevated levels of ROS versus control, as measured with oxidizable dye indicator 5-(and-6)-chloromethyl- 2',7'dichlorodihydrofluorescem diacetate, acetyl ester (CM-H2DCFDA) (DCF).
  • CM-H2DCFDA oxidizable dye indicator 5-(and-6)-chloromethyl- 2',7'dichlorodihydrofluorescem diacetate, acetyl ester (CM-H2DCFDA) (DCF).
  • CM-H2DCFDA oxidizable dye indicator 5-(and-6)-chloromethyl- 2',7'dichlorodihydrofluorescem diacetate, acetyl ester
  • Figure 10 shows that antioxidants ameliorate the Tat-induced rise in vesicular uptake of neurotransmitter.
  • the antioxidant TUDCA completely eliminated Tat's effects on FM1-43 uptake for all doses of Tat.
  • FIG. 11 shows that Tat and PAF cause a dose-dependent mitochondrial hyperpolarization in cortical neurons.
  • A Treatment of rat cortical neurons with 100 ng/ml or 2.5 ⁇ g/ml Tat for 1, 4, 10, 24, 26, 36, or 48 hours resulted in a dose-dependent biphasic increase in mitochondrial membrane potential. At each dose, initial peaks were followed by periods of apparent mitochondrial membrane potential ( ⁇ m ) stabilization, followed by increased ⁇ m again at later time points, and the later increase in ⁇ m persisted until the end of the analysis (48 hours).
  • a biologically inactive mutated Tat peptide green produced no effect. Curve fits are non-formulaic software interpolations of the data points.
  • Figure 12 shows that neuronal ATP/ADP ratio increases then declines with chronic Tat exposure.
  • Primary cortical neurons were treated for 1, 24, or 48 hours with 0 (control), 0.1, or 2.5 ⁇ g/ml Tat.
  • ATP levels, as well as ATP/ADP ratios were sharply increased by both doses of Tat at 1 hour, and by 2.5 ⁇ g/ml Tat at 24 hours.
  • ATP levels, while still elevated had declined from peak levels at 24 hours, and ATP/ADP ratio had fallen to control level or below (i.e. ⁇ 1.0).
  • P-values shown are versus time matched control.
  • FIG. 13 shows that the KATP channel antogonist Tolbutamide attenuates Tat- induced increases in ⁇ m and neuronal apoptosis.
  • Co-treatment of 0.1 ⁇ g/ml or 2.5 ⁇ g/ml Tat treated cortical cultures with the mitochondrial KATP channel antagonist tolbutamide (100 ⁇ M) (A) completely prevented the Tat mediated rise in ⁇ m at 1 and 24 hours, and (B) partially attenuated Tat's dose-dependent induction of apoptotic cell death over 24 a «,.11 1! disturb- '!,_» >,,,. ⁇ l Ul 'I...I1 .•' 'UIl ..;f ..,..!'• hours. 100 ⁇ M tolbutamide alone had no significant effect on ⁇ m, and showed no toxicity.
  • Figure 14 shows that sublethal cPAF exposure leads to dendrite beading and disruption of spines.
  • cPAF causes dendrite beading and loss of dendritic spines.
  • A' 5 Low-magnification images show minimal changes in dendrite morphology and total spine number in control cultures (left). In cPAF-treated cultures (right), dendrite beading (red arrows) is accompanied by spine loss and sprouting of filopodia (green arrowheads) but gross aspects of dendrite structure are preserved.
  • B No cells developed dendritic swellings in control cultures, while 55 ⁇ 3% of cPAF-exposed cells beaded.
  • C 43 ⁇ 5% of 0 dendritic spines were lost during cPAF exposure, while total spine number was maintained in control cultures.
  • FIG. 15 shows that cPAF promotes dendrite beading and failure of long-term potentiation in acute hippocampal slices.
  • CAl pyramidal neurons were exposed to l ⁇ M cPAF or vehicle for 30-60min and simultaneously imaged and recorded by whole-cell 5 patch clamp before and after delivery of a high-frequency stimulus (HFS: three Is, 100Hz trains, every 20s) applied to Schaffer collateral axons via a bipolar stimulating electrode planted in the stratum radiatum.
  • HFS high-frequency stimulus
  • each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • any subset or combination of these is also specifically contemplated and disclosed.
  • the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise.
  • HIV neurotoxin comprising contacting the cell with a modulator of adenosine receptor signaling.
  • neuronal dysfunction include alterations in mitochondrial function, neurotransmitter uptake and release, neuronal architecture, synaptic transmission, and neuronal cell death.
  • an HIV neurotoxin can result in neuronal cell death.
  • neuronal cell death includes either apoptosis or necrosis of neurons that can occur as a result of exposure to neurotoxins associated with HIV.
  • apoptosis refers to programmed cell death that is signaled by the nuclei when age or state of cell health and condition dictates. Apoptosis is an active process requiring metabolic activity by the dying cell, often characterized by cleavage of the DNA into fragments that give a so called laddering pattern on gels. Cells that die by apoptosis do not usually elicit the inflammatory responses that are associated with necrosis. As used herein, necrosis refers to cell death in response to a major insult, resulting in a loss of membrane integrity, swelling and rupture of the cell. During necrosis, the cellular contents are released uncontrolled into the cell's environment which results in damage of surrounding cells and a strong inflammatory response in the corresponding tissue.
  • HIV-I associated dementia is comprised of a spectrum of conditions from the mild HIV-I minor cognitive-motor disorder (MCMD) to severe and debilitating AIDS dementia complex. Symptoms begin with motor slowing and may progress to severe loss of cognitive function, loss of bladder and bowel control, and paraparesis.
  • a classification system has been formulated for HIV associated dementia, wherein subjects are classified as being Stage 0 (Normal), Stage 0.5 (Subclinical or Equivocal), Stage 1 (Mild), Stage 2 (Moderate), Stage 3 (Severe), or Stage 4 (End-Stage).Thus, the subject of the provided method can therefore be classified as Stage 0, Stage 0.5, Stage 1, Stage 2, Stage 3, or Stage 4.
  • treat or “treatment” is meant a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the disease or condition itself rather than just the symptoms.
  • the treatment can be any reduction from native levels and can be but is not limited to the complete ablation of the disease, condition, or the symptoms of the disease or condition.
  • a disclosed method for treatment of HAD is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject with the disease when compared to native levels in the same subject or control subjects.
  • the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • to treat HAD in a subject can comprise improving the disease classification, (e.g. from stage 3 to stage 2, from stage 2 to stage 1, from stage 1 to 0.5 or from stage 0.5 to 0).
  • "preventing" means to preclude, avert, obviate, forestall, stop, or hinder something from happening, especially by advance planning or action.
  • to prevent HAD in a subject is to stop or hinder the subject from advancing in disease classification (e.g. from stage 0 to stage 0.5, from stage 0.5 to stage 1, from stage 1 to stage 2, from stage 2 to stage 3, or from stage 3 to stage 4).
  • HIV-I infected and/or functionally activated mononuclear cells and astrocytes can produce a number of soluble mediators, including the structural and regulatory proteins gpl20, Tat, and platelet activating factor (PAF), which can exert damaging effects on both developing and mature neural tissues.
  • soluble mediators including the structural and regulatory proteins gpl20, Tat, and platelet activating factor (PAF), which can exert damaging effects on both developing and mature neural tissues.
  • PAF platelet activating facotr
  • the neurotoxin of the present method can be a virally-derived toxin such as Tat or gpl20 or other viral protein (alone or in combination) or it may be a cellular pro- infllammatory mediator such as PAF, tumor necrosis factor (TNF)-alpha, interleukin (IL)- 1 beta, IL-6, nitric oxide or other cell-derived neurotoxin (alone or in combination).
  • a virally-derived toxin such as Tat or gpl20 or other viral protein (alone or in combination) or it may be a cellular pro- infllammatory mediator such as PAF, tumor necrosis factor (TNF)-alpha, interleukin (IL)- 1 beta, IL-6, nitric oxide or other cell-derived neurotoxin (alone or in combination).
  • TNF tumor necrosis factor
  • IL interleukin
  • nitric oxide cell-derived neurotoxin
  • Endogenous adenosine plays a pivotal role in the regulation of neural cell fate.
  • the actions of adenosine are mediated by specific receptors located on cell membranes, which belong to the family of G protein-coupled receptors.
  • adenosine receptors have been cloned: A 1 , A 2A , A 2B , and A 3 .
  • the disclosed modulator of adenosine receptor signaling can comprise any composition that will alter a biological property of either adenosine or adenosine receptors in a cell, such as for example their synthesis, degredation, translocation, binding, or phosphorylation, such that the alteration results in a net increase or decrease in adenosine receptor signaling in the cell.
  • the provided modulator can be a nucleic acid that alters expression of either adenosine or adenosine receptor in a cell, such as for example RNAi or antisense nucleic acids.
  • the provided modulator can be a polypeptide that alters the binding of adenosine to adenosine receptors, such as for example soluble adenosine receptors, mutant adenosine ligands or antibodies specific for adenosine or adenosine receptors.
  • the provided modulator can comprise informational molecules that modulate adenosine receptor expression (such as short-interfering RNAs or peptide nucleic acids) or molecules that may regulate downstream signaling events that may occur as a result of adenosine receptor stimulation.
  • the provided modulator of adenosine receptor signaling can be a small molecule comprising a modified adenosine (6-amino-9-beta-D-ribofuranosyl-9-H-purine).
  • Modifications that can be made to adenosine are well known in the art. These modifications include those that result in adenosine receptor agonists and antagonists. These agonists and antagonists can be either receptor selective or non-selective.
  • the modulator of the present method can be an adenosine 1 receptor (A 1 R) antagonist.
  • the modulator can be an adenosine 2A receptor (A 2 AR) antagonist.
  • the modulator can be an adenosine 2B receptor (A 2 ⁇ R) antagonist.
  • the modulator can be an adenosine 3 receptor (A 3 R) antagonist.
  • the modulator can be any adenosine receptor selective antagonist, whether known in the art or later developed.
  • a 2A R selective antagonists include ATL455, ZM241385, KW-6002 (istradefylline), SCH 58261, and the pharmaceutically acceptable salts thereof.
  • ZM241385 is 4(2-[7-Amino-2-(2-furyl)[l ,2,4]triazolo[2,3-a] [ 1 ,3,5]triazin-5- ylamino]ethyl)phenol (Poucher et al. (1995) The in vitro pharmacology of ZM 241385, a potent, non-xanthine, A 2a selective adenosine receptor antagonist. Br. J.Pharmacol. 115 1096; Poucher et al (1996) Pharmacodynamics of ZM 241385, a potent A 2a adenosine receptor antagonist, after enteric administration in rat, cat and dog. J.Pharm.Pharmacol. 48 601; Keddie et al (1996) In vivo characterisation of ZM 241385, a selective adenosine A 2A receptor antagonist. Eur. J.Pharmacol. 301 107.)
  • KW-6002 (istradefylline) is (E)-l,3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7- dhydro-lH-purine-2,6-dione. KW-6002 has been evaluated humans as a treatment for
  • SCH 58261 is 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-l,2,4- triazolo[l,5-c]pyrimidine.
  • the provided modulator can be an adenosine 1 receptor (A 1 R) agonist.
  • the modulator can be an adenosine 2A receptor (A 2 AR) agonist.
  • the modulator can be an adenosine 2B receptor (A 2 ⁇ R) agonist.
  • the modulator can be an adenosine 3 receptor (A 3 R) agonist, such as for example CFlOl (Aderis Pharmaceuticals).
  • the provided modulator can be any adenosine receptor selective agonist, whether known in the art or later developed.
  • Non-limiting examples OfA 2 AR selective agonist include ATL146e,
  • ATL146e is 4- ⁇ 3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydrofuran-2- yl)-9H-purin-2-yl]prop-2-ynyl ⁇ cyclohexanecarboxylic acid methyl ester. (Lappas CM, et al. A2A adenosine receptor induction inhibits IFN-gamma production in murine CD4+ T cells. J Immunol. 2005 Jan 15;174(2):1073-80.)
  • ATL313 is 4- ⁇ 3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4- dihydroxytetrahydrofuraii-2-yl)-9H-purin-2-yl]prop-2-ynyl ⁇ piperidine- 1 -carboxylic acid methyl ester (Lappas CM, et al. A2A adenosine receptor induction inhibits IFN-gamma production in murine CD4+ T cells. J Immunol. 2005 Jan 15;174(2):1073-80.)
  • CGS21680 is 4-[2-[[6-Amino-9-(N-ethyl-&-D-ribofuranuronamidosyl)-9H-purin-
  • any of the compounds described herein can be the pharmaceutically-acceptable salt thereof.
  • pharmaceutically-acceptable salts are prepared by treating the free acid with an appropriate amount of a pharmaceutically-acceptable base. For example, one or more hydrogen atoms of the SO3 ⁇ group can be removed with a base.
  • Representative pharmaceutically-acceptable bases are ammonium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, and the like.
  • the compound if it possesses a basic group, it can be protonated with an acid such as, for example, HCl or H2SO4, to produce the cationic salt.
  • an acid such as, for example, HCl or H2SO4
  • the reaction of the compound with the acid or base is conducted in water, alone or in combination with an inert, water-miscible organic solvent, at a temperature of from about 0 C to about 100 C such as at room temperature.
  • the molar ratio of the compounds described herein to base used are chosen to provide the ratio desired for any particular salts.
  • the starting material can be treated with approximately one equivalent of pharmaceutically-acceptable base to yield a neutral salt.
  • the pharmaceutically-acceptable salts of the compounds described herein can be used as prodrugs or precursors to the active compound prior to the administration.
  • the active compound if it is unstable, it can be prepared as its salt form in order to increase stability in dry form (e.g., powder).
  • dry form e.g., powder.
  • the severity of dementia in persons with HIV-I associated neurologic disease is strongly correlated with the number of macrophages and microglia within the basal ganglia and frontal lobes [Glass, J. D., et al. 1995. Ann Neurol 38:755-762].
  • the activation of microglia and brain macrophages plays a crucial role in the induction of neuronal dysfunction and damage.
  • the herein disclosed agonists of adenosine receptor signaling can inhibit HAD in a subject in part by inhibiting the recruitment of monocytes to the CNS.
  • the disclosed method can be further combined with other therapeutic approaches for the treatment of HTV-I infection or HAD.
  • the disclosed method can further comprise administering to the subject an antiretro viral compound.
  • Antiretro viral drugs inhibit the reproduction of retroviruses such as HIV.
  • Antiretro viral agents are virustatic agents which block steps in the replication of the virus. The drugs are not curative; however continued use of drugs, particularly in multi-drug regimens, can significantly slow disease progression. There are three main types of antiretro viral drugs, although only two steps in the viral replication process are blocked. Nucleoside analogs, or nucleoside reverse transcriptase inhibitors (NRTIs), act by inhibiting the enzyme reverse transcriptase.
  • NRTIs nucleoside reverse transcriptase inhibitors
  • Reverse transcriptase is an enzyme that is essential to making the DNA copy.
  • the nucleoside reverse transcriptase inhibitors are incorporated into the DNA strand. This is a faulty DNA molecule that is incapable of reproducing.
  • the non-nucleoside reverse transcriptase inhibitors act by binding directly to the reverse transcriptase molecule, inhibiting its activity.
  • Protease inhibitors act on the enzyme protease, which is essential for the virus to break down the proteins in infected cells. Without this essential step, the virus produces immature copies of itself, which are noninfectious.
  • a fourth class of drugs called fusion inhibitors block HIV from fusing with healthy cells.
  • the antiretroviral compound can comprise one or more molecules selected from the group consisting of protease inhibitors [PI], fusion inhibitors, nucleoside reverse transcriptase inhibitors [NRTI], and non-nucleoside reverse transcriptase inhibitors [NNRTI].
  • the antiretroviral compound of the provided method can be a PI, such as a PI selected from the group consisting of Indinavir, Amprenavir, Nelfmavir, Saquinavir, Fosamprenavir, Lopinavir, Ritonavir, and Atazanavir, or any combinations thereof.
  • the antiretroviral compound of the provided method can be a fusion inhibitor, such as for example Enfuvirtide.
  • the antiretroviral compound of the provided method can be a NRTI, such as a NRTI selected from the group consisting of Abacavir, Stavudine, Didanosine, Lamivudine, Zidovudine, Zalcitabine, Tenofovir, and Emtricitabine, or any combinations thereof.
  • a NRTI selected from the group consisting of Abacavir, Stavudine, Didanosine, Lamivudine, Zidovudine, Zalcitabine, Tenofovir, and Emtricitabine, or any combinations thereof.
  • the antiretroviral compound of the provided method can be a NNRTI, such as a NNRTI selected from the group consisting of Efavirenz, Nevirapine, and Delavirdine.
  • the disclosed method can further comprise administering to the subject an inhibitor of mitochondrial hyperpolarization.
  • mitochondrial hyperpolarization refers to an elevation in the mitochondrial transmembrane potential, ⁇ m (delta psi), i.e., negative inside and positive outside).
  • ⁇ m is the result of an electrochemical gradient maintained by two transport systems — the electron transport chain and the F 0 F 1 -ATPaSe complex.
  • the electron transport chain catalyzes the flow of electrons from NADH to molecular oxygen and the translocation of protons across the inner mitochondrial membrane, thus creating a voltage gradient with negative charges inside the mitochondrial matrix.
  • F 0 F 1 -ATPaSe utilizes the extruded proton to synthesize ATP.
  • MHP leads to uncoupling of oxidative phosphorylation, which disrupts ⁇ m and damages integrity of the inner mitochondrial membrane.
  • Disruption of ⁇ m has been proposed as the point of no return in cell death signaling. This releases cytochrome c and other cell- death-inducing factors from mitochondria into the cytosol.
  • the inhibitor of the present method can be a F 0 F 1 -ATPaSe agonists.
  • the inhibitor of the present method can be a KATP channel antagonist.
  • the KATP channel antagonist can be selected from the group consisting of Tolbutamide, hydroxydecanoic acid (5-HD), glibenclamide (glyburide), and meglitinide analog (e.g. Repaglinide, A-4166).
  • the inhibitor of the present method can be an electron transport inhibitor.
  • the electron transport chain (ETC) is the biomolecular machinery present in mitochnodria that couples the flow of electrons to proton pumps in order to convert energy from sugar to ATP.
  • the electron transport chain couples the transfer of an electron from NADH (nicotinamide adenine dinucleotide) to molecular oxygen (O 2 ) with the pumping of protons (H + ) across a membrane.
  • NADH nicotinamide adenine dinucleotide
  • O 2 molecular oxygen
  • the charge gradient that results across the membrane serves as a battery to drive ATP Synthase.
  • the electron transport chain is made up of several integral membrane complexes: NADH dehydrogenase (complex I), Coenzyme Q - cytochrome c reductase (complex III), and Cytochrome c oxidase (complex IV).
  • NADH dehydrogenase complex I
  • Coenzyme Q - cytochrome c reductase complex III
  • Cytochrome c oxidase complex IV
  • Succinate - Coenzyme Q reductase (Complex II) connects the Krebs cycle directly to the electron transport chain.
  • the inhibitor of the provided method can be an inhibitor of any component of the ETC.
  • the inhibitor can be an inhibitor of complex I, II, III, or IV.
  • diphenylene iodonium (DPI) and rotenone are specific inhibitors of complex I
  • succinate-q reductase (TTFA) is an inhibitor of complex II
  • antimycin A and myxothiazole are inhibitors of complex III
  • potassium cyanide (KCN) is an inhibitor of complex IV.
  • the inhibitor of the provided method can be selected from the group consisting of diphenylene iodonium (DPI), rotenone, antimycin, myxothiazole, succinate-q reductase (TTFA) 5 and potassium cyanide (KCN).
  • the inhibitor of the present method can be an uncoupler.
  • an "uncoupler” is a substance that allows oxidation in mitochondria to proceed without the usual concomitant phosphorylation to produce ATP; these substances thus “uncouple” oxidation and phosphorylation.
  • Trifluorocarbonylcyanide Phenylhydrazone FCCP is a chemical uncoupler of electron transport and oxidative phosphorylation. FCCP permeabilizes the inner mitochondrial membrane to protons, destroying the proton gradient and, in doing so, uncouples the electron transport system from the oxidative phosphorylation system. In this situation, electrons continue to pass through the electron transport system and reduce oxygen to water, but ATP is not synthesized in the process.
  • the uncoupler of the present method can agonize, antagonize or modulate the expression of endogenous mitochondrial uncoupling proteins (UCPs).
  • UCPs endogenous mitochondrial uncoupling proteins
  • the uncoupler of the present method can be the beta-adrenergic agonist CL-
  • the uncoupler of the present method can be a protonophore.
  • the inhibitor of the present method can be a protonophore.
  • a "protonophore” is a molecule that allows protons to cross lipid bilayers.
  • the protonophore can be FCCP.
  • the protonophore can also be 2,4,-dinitrophenol (DNP).
  • the protonophore can be also m- chlorophenylhydrazone (CCCP).
  • the protonophore can also be pentachlorophenol (PCP).
  • the disclosed method can further comprise contacting the cell with an antioxidant.
  • antioxidants are compounds that react with, and typically get consumed by, oxygen.
  • antioxidants typically react with oxygen, antioxidants also typically react with the free radical generators, and free radicals.
  • the herein disclosed antioxidant can be any antioxidant, and a non-limiting list would included but not be limited to, non-flavonoid antioxidants and nutrients that can directly scavenge free radicals including multi-carotenes, beta-carotenes, alpha-carotenes, gamma-carotenes, lycopene, lutein and zeanthins, selenium, Vitamin E, including alpha-, beta- and gamma- (tocopherol, particularly ⁇ -tocopherol, etc., vitamin E succinate, and trolox (a soluble Vitamin E analog) Vitamin C (ascoribic acid) and Niacin (Vitamin B3, nicotinic acid and nicotinamide), Vitamin A, 13-cis retinoic acid, , N-acetyl-L-cysteine (NAC), sodium ascorbate, pyrrolidin-edithio-carbamate, and coenzyme QlO; enzymes which catalyze the destruction of free
  • the disclosed method can further comprise contacting the cell with an antioxidant selected from the group consisting of tauroursodeoxycholic acid (TUDCA), N- acetylcysteine (NAC) (600-800 mg/day), Mito-Coenzyme QlO (Mito-CoQ) (300-400 mg/day), Mito-VitaminE (Mito-E) (100 - 1000 mg/day), Coenzyme QlO (300-400 mg/day), and idebenone (60 - 120 mg/day).
  • an antioxidant selected from the group consisting of tauroursodeoxycholic acid (TUDCA), N- acetylcysteine (NAC) (600-800 mg/day), Mito-Coenzyme QlO (Mito-CoQ) (300-400 mg/day), Mito-VitaminE (Mito-E) (100 - 1000 mg/day), Coenzyme QlO (300-400 mg/day), and idebenone
  • NAC N-acetylcysteine
  • Glutathione Glutathione
  • De Rosa SC Zaretsky MD, Dubs JG, Roederer M, Anderson M, Green A, Mitra D, Watanabe N, Nakamura H, Tjioe L Deresinski SC, Moore WA, EIa SW, Parks D, Miberg LA, Immunberg LA.
  • NAC is not used to replenish Glutathione (GSH) in HIV-infected subjects.
  • NAC is not used to treat HAD.
  • Coenzyme QlO has been used to treat patients having the AIDS related complex.
  • Coenzyme QlO increases T4/T8 ratios of lymphocytes in ordinary subjects and relevance to patients having the AIDS related complex. Biochem Biophys Res Commun. 1991 Apr 30;176(2):786-91.
  • Bile acids such as TUDCA lead to a significant improvement in serum transaminase activities in subjects with hepatitis B and C.
  • Coenzyme QlO is not used to treat patients having the AIDS related complex.
  • Coenzyme QlO is not used to treat HAD.
  • Idebenone has been used to treat subjects with senile cognitive decline (Bergamasco B, Villardita C, Coppi R. Effects of idebenone in elderly subjects with cognitive decline. Results of a multicentre clinical trial. Arch Gerontol Geriatr. 1992 Nov- Dec;15(3):279-86.) .) Thus, in one embodiment of the provided invention, Idebenone not used to treat subjects with senile cognitive decline, hi another embodiment of the method Idebenone is not used to treat HAD.
  • the disclosed method can further comprise administering to the subject a neurotoxin inhibitor.
  • the inhibitor can be a TNF ⁇ inhibitor, including TNF ⁇ -inhibitory monoclonal antibodies (e.g., etanercept), phosphodiesterase (PDE)-4 inhibitors (such as IC485, which can reduce TNF ⁇ production), thalidomide and other agents.
  • Etanercept is a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgGl.
  • the Fc component of etanercept contains the C H 2 domain, the C H 3 domain and hinge region, but not the C H I domain of IgGl.
  • Etanercept is produced by recombinant DNA technology in a Chinese hamster ovary (CHO) mammalian cell expression system. It consists of 934 amino acids and has an apparent molecular weight of approximately 150 kilodaltons.
  • Etanercept has been evaluated in HIV-infected subjects receiving highly active antiretroviral therapy (HAART) (Sha BE, Valdez H, Gelman RS, Landay AL, Agosti J, Mitsuyasu R, Pollard RB, Mildvan D, Namkung A, Ogata- Arakaki DM, Fox L, Estep S, Erice A, Kilgo P, Walker RE, Bancroft L, Lederman MM. Effect of etanercept (Enbrel) on interleukin 6, tumor necrosis factor alpha, and markers of immune activation in HIV-infected subjects receiving interleukin 2. AIDS Res Hum Retroviruses. 2002 Jun l0;18(9):661-5).
  • HAART highly active antiretroviral therapy
  • IC485 is an orally administered, small molecule inhibitor of PDE4. Inhibition of PDE4 leads to an increase in the second messenger, cAMP, within cells. This inhibition may in turn reduce the cell's production of tumor necrosis factor alpha (TNF-alpha) and a variety of other inflammatory mediators. IC485 is being evaluated in patients with chronic obstructive pulmonary disease.
  • the inhibitor can be a PAF receptor antagonist (such as lexipafant, WEB2086, WEB2170, BN-52021 or PMS-601), a PAF degrading-enzyme such as PAF- acetylhydrolase (PAF-AH), or a molecule that regulates the expression of PAF-AH (such as pioglitazone and other PPAR-gamma inhibitors).
  • a PAF receptor antagonist such as lexipafant, WEB2086, WEB2170, BN-52021 or PMS-601
  • PAF-AH PAF degrading-enzyme
  • PAF-AH PAF- acetylhydrolase
  • a molecule that regulates the expression of PAF-AH such as pioglitazone and other PPAR-gamma inhibitors.
  • Lexipafant has been used improve cognitive dysfunction in HIV-infected people (Schifitto G, Sacktor N, Marder K, McDermott MP, McArthur JC, Kieburtz K, Small S, Epstein LG. Randomized trial of the platelet-activating factor antagonist lexipafant in HTV-associated cognitive impairment. Neurological AIDS Research Consortium. Neurology. 1999 JuI 22;53(2):391-6). Lexipafant can be administered at for example 500 mg/day.
  • PMS-601 inhibits proinflammatory cytokine synthesis and HIV replication (Martin M, Serradji N, Dereuddre-Bosquet N, Le Pavec G, Fichet G, Lamouri A, Heymans F, Godfroid JJ, Clayette P, Dormont D. PMS-601, a new platelet-activating factor receptor antagonist that inhibits human immunodeficiency virus replication and potentiates zidovudine activity in macrophages. Antimicrob Agents Chemother. 2000 Nov;44(ll):3150-4.) TNF-alpha-mediated neuronal apoptosis can also be blocked by co-incubation with
  • PAF acetylhydrolase (PAF-AH) (Perry SW, Hamilton JA, Tjoelker LW, Dbaibo G, Dzenko KA, Epstein LG, Hannun Y, Whittaker JS, Dewhurst S, Gelbard HA. Platelet- activating factor receptor activation. An initiator step in HIV-I neuropathogenesis. J Biol Chem. 1998 JuI 10;273 (28): 17660-4). Pioglitazone can inhibit PAF-induced morphological changes through PAF-AH
  • Phosphatidylcholines (l-O-alcoxy-2-amino-2-desoxy-phosphocholines and 1- pyrene-labeled analogs) were synthesized and used to examine interactions with recombinant human PAF-AH (Deigner HP, Kinscherf R, Claus R, Fyrnys B, Blencowe C, Hermetter A. Novel reversible, irreversible and fluorescent inhibitors of platelet-activating factor acetylhydrolase as mechanistic probes. Atherosclerosis. 1999 May;144(l):79-90).
  • the disclosed method can further comprise administering to the subject an inhibitor of GSK-3 ⁇ .
  • the inhibitor can be valproate or lithium.
  • Valproate has been administered to HIV-infected patients receiving efavirenz or lopinavir (DiCenzo R, Peterson D, Cruttenden K, Morse G, Riggs G, Gelbard H, Schifitto G. Effects of valproic acid coadministration on plasma efavirenz and lopinavir concentrations in human immunodeficiency virus-infected adults. Antimicrob Agents Chemother. 2004 Nov;48(l 1):4328-31).
  • a typical dose of valproate comprises 250 mg twice daily.
  • the disclosed method can further comprise administering to the subject a compound that enhances CNS uptake.
  • Ritonavir influences levels of coadministered drugs in the CNS, due to effects on the activity of drug transporters located at the BBB (Haas DW, Johnson B, Nicotera J, Bailey VL, Harris VL, Bowles FB, Raffanti S, Schranz J, Finn TS, Saah AJ, Stone J Effects of ritonavir on indinavir pharmacokinetics in cerebrospinal fluid and plasma Antimicrob Agents Chemother. 2003 Jul;47(7):2131-7).
  • the disclosed methods can further comprise administering a drug that inhibits the P-glycoprotein drug efflux pump, or multidrug resistance-associated proteins at the blood- brain-barrier (BBB).
  • BBB blood- brain-barrier
  • LY-335979 Cho EF, Leake B, Wandel C, Jmamura H, Wood AJ, Wilkinson GR, Kim RB.
  • Pharmacological inhibition of P-glycoprotein transport enhances the distribution of HIV-I protease inhibitors into brain and testes.
  • Drug Metab Dispos include LY-335979 (Choo EF, Leake B, Wandel C, Jmamura H, Wood AJ, Wilkinson GR, Kim RB.
  • the disclosed method can further comprise administering to the subject a microglial deactivator.
  • Minocyclin is a potent microglial deactivator (Wu DC, Jackson- Lewis V, Vila M, Tieu K, Teismann P, Vadseth C, Choi DK, Ischiropoulos H, Przedborski S. Blockade of microglial activation is neuroprotective in the l-methyl-4-phenyl-l,2,3,6- tetrahydropyridine mouse model of Parkinson disease. J Neurosci. 2002 Mar 1;22(5):1763-71; Yrjanheikki J, Keinanen R, Pellikka M, Hokfelt T, Koistinaho J.
  • Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc Natl Acad Sci U S A. 1998 Dec 22;95(26): 15769-74). Futher, minocycline can potently inhibit HJV-I viral production from microglia (Si Q, Cosenza M, Kim MO, Zhao ML, Brownlee M, Goldstein H, Lee S. A novel action of minocycline: inhibition of human immunodeficiency virus type 1 infection in microglia. J Neurovirol. 2004 Oct;10(5):284-92). Thus, the microglial deactivator can be minocycline. A typical dosage of minocyclin comprises 200 mg/day.
  • the disclosed method can further comprise administering to the subject an inhibitor of glutamate damage.
  • the inhibitor can be a beta-lactam antibiotic such as for example ceftriaxone, which can have direct effects on glutamate transporter expression.
  • beta-lactam ceftriaxone When delivered to animals, the beta-lactam ceftriaxone increases both brain expression of GLTl that inactivates synaptic glutamate (Rothstein JD, Patel S, Regan MR, Haenggeli C, Huang YH, Bergles DE, Jin L, Dykes Hoberg M, Vidensky S, Chung DS, Toan SV, Bruijn LI, Su ZZ, Gupta P, Fisher PB. Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature. 2005 Jan 6;433(7021):73-7) A typical dosage of cephtriaxone is 50 mg/kg/day.
  • the inhibitor of glutamate damage can be a TNF ⁇ inhibitor or a microglial deactivator (descrived above), which can have indirect effects on glutamate transporters.
  • compositions comprising a modulator of adenosine receptor signaling and a molecule that inhibits mitochondrial hyperpolarization in a neural cell.
  • the modulator of the provided composition can be any adenosine receptor signaling modulator provided herein.
  • the composition can comprise the A 2A R antagonist ATL455 or ZM241685.
  • the molecule of the disclosed composition that inhibits mitochondrial hyperpolarization in a neural cell can be any such molecule provided herein.
  • the provided composition can comprise one or more of a KATP antagonist, an inhibitor of electron transport, a protonophore, or an antioxidant.
  • the provided composition can further comprise an antiretro viral compound.
  • the antiretro viral compound can be any antiretroviral compound as disclosed herein, such as one or more molecules selected from the group consisting of protease inhibitors [PI], nucleoside reverse transcriptase inhibitors [NRTI], and non-nucleoside reverse transcriptase inhibitors [NNRTI].
  • the provided composition can further comprise a neurotoxin inhibitor.
  • the provided composition can further comprise an inhibitor of GSK-3 ⁇ .
  • the provided composition can further comprise a compound that enhances CNS uptake.
  • the provided composition can further comprise a microglial deactivator.
  • the provided composition can further comprise an inhibitor of glutamate damage.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose.
  • the dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, the disclosed A 2A R antagonists can be administered at published dosages, such as those approved for human use.
  • a typical daily dosage of the disclosed modulators of adenosine receptor signaling used alone can range from about 0.05 to 5 mg/kg of body weight or more per day, depending on the factors mentioned above.
  • the disclosed A 2A R antagonists e.g. ATL455, KW6002 and ZM241685
  • the disclosed A 2 AR agonists e.g. ATL146e, ATL313 and CGS21680
  • a typical daily dosage of the disclosed inhibitors of hyperpolarization used alone can range from about .001 mg/kg to up to 50 mg/kg of body weight or more per day, depending on the factors mentioned above.
  • the disclosed KATP channel antagonists can be administered at from .02 mg/kg to about 30 mg/kg of body weight per day.
  • Tolbutamide can be administered at from about 0.25 to 3 g/day; glibenclamide (glyburide) can be administered at from about 1.25 to 20 mg/day; and meglitinide analog (e.g. Repaglinide, A-4166) can be administered at from about 0.5 to 4 mg/day.
  • the disclosed inhibitors of the ECC can be administered at from .001 mg/kg to 1 mg/kg of body weight per day.
  • the disclosed protonophore e.g., FCCP, DNP, CCCP, PCP
  • the disclosed beta-adrenergic agonist CL-316,243 can be administered at 0.01 to up to 1 mg/kg, including 0.1 mg/kg, of body weight or more per day.
  • the disclosed antioxidants can be administered at from 1 mg/day to 1000 mg/day.
  • N-acetylcysteine NAC
  • Mito-Coenzyme QlO Mito-CoQ
  • Mito-VitaminE Mito-E
  • Coenzyme QlO can be administered from about 300 mg/day to 400 mg/day
  • idebenone can be administered at from about 60 mg/day to 120 mg/day.
  • compositions can also be administered in vivo in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
  • an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
  • compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions can include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions can also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
  • the pharmaceutical composition can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration can be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the disclosed compositions can be administered intracranially intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • compositions are recognized to cross the blood-brain- barrier.
  • CGS21680 (Agnati LF, Leo G, Vergoni AV, Martinez E, Hockemeyer J, Lluis C, Franco R, Fuxe K, Ferre S. Neuroprotective effect of L-DOPA co- administered with the adenosine A2A receptor agonist CGS 21680 in an animal model of Parkinson's disease. Brain Res Bull. 2004 Aug 30;64(2):155-64); Istradefylline (Weiss SM, Benwell K, Cliffe IA, Gillespie RJ, Knight AR, Lerpiniere J, Misra A, Pratt RM, Revell D, Upton R, Dourish CT.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsif ⁇ ers, dispersing aids or binders may be desirable.
  • compositions can be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glycolic
  • compositions may be administered orally or parenterally (e.g., intravenously, intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, intracranially, topically or the like, including topical intranasal administration or administration by inhalant.
  • intracranial administration means the direct delivery of substances to the brain including, for example, intrathecal, intracisternal , intraventricular or transsphenoidal delivery via catheter or needle.
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.
  • compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
  • the exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • Parenteral administration of the composition is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These can be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al.,
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)).
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced.
  • receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration.
  • Example 1 Adenosine 2A (A 2 A) receptor antagonist protects against Tat- induced neuronal apoptosis.
  • CGNs Primary rat cerebellar granule neurons (CGNs) were exposed to a neurotoxic concentration of HIV-I Tat, in the presence or absence of an A 2A R antagonist (ZM241385). The A 2A R antagonist was able to protect neurons against the otherwise lethal effects of exposure to HIV-I Tat ( Figure 1). These results demonstrate that A 2A R antagonists can have therapeutic potential in the context of HAD. It is important to note that CGNs express adenosine A 2A receptors [Vacas, J., et al. 2003. Brain Res 992:272- 280]. Primary cultures of rat cerebellar granule neurons (CGNs) were exposed to HIV-
  • Tat 50OmM alone or together with increasing doses of the A 2A R antagonist ATL-455 or A 2A agonists ATL313 or CGS21680.
  • Cells are treated with the test compounds for defined time intervals of up to 48 hours (1, 4, 8, 24, and 48 hours).
  • the A 2 AR antagonist ATL455 protected neurons against Tat-induced apoptosis (Fig. 2). Further, exposure of neurons to an A 2A R agonists (ATL313 and CGS21680) did not result in an increase in cell death in the presence of Tat (Fig. 2).
  • RNAi can also be used to suppress expression OfA 2 AR and mimic the effects of A 2A R antagonist treatment.
  • a 2A R expression is inhibited using lentivirus vectors that encode A 2A R-specific shRNA.
  • CGN cultures are transduced with viral vectors encoding shRNA targeted to A 2 AR, or scrambled shRNA sequences, or nothing (GFP only).
  • siRNA has been used to modify the levels OfA 2A R and A 2B R in cardiac fibroblasts (Chen Y, Epperson S, Makhsudova L, Ito B, Suarez J, Dillmann W, Villarreal F. Functional effects of enhancing or silencing adenosine A2b receptors in cardiac fibroblasts. Am J Physiol Heart Circ Physiol. 2004 Dec;287(6):H2478-86. Epub 2004 JuI 29).
  • a 2A R-specific antibodies (Alpha Diagnostics International) are used to perform both immunoblot and immunofluorescence assays to examine knockdown OfA 2A R. A knockdown of 80% or greater is followed by the treatment of the transduced neurons with candidate HIV-I neurotoxins (or mock-treated, for controls).
  • a 2A receptors are generally accepted to couple to the Gs- adenylate cyclase (AC)-protein kinase A (PKA) pathway [Fredholm, B. B., et al. 2001. Pharmacol Rev 53:527-552], but can also couple to pathways involving G-proteins other than Gs (Go, Gal 5/16, Gi/o) or to cAMP PKA independent signal transduction pathways.
  • AC Gs- adenylate cyclase
  • PKA protein kinase A
  • neurons are treated as described above, and the activity of cellular kinases such as PKA, ERK, AKT, JNK, and GSK-3 ⁇ are evaluated.
  • the activation of transcription factors e.g., AP-I, CREB, and NF-kB, is evaluated, as these factors represent potential endpoint targets of the signaling pathways initiated by A 2A R).
  • Transcription factor activity is assessed using assays of protein nuclear translocation, nuclear DNA binding activity and transient transcriptional reporter assays.
  • Antagonism OfA 2A R signaling is expected to have a substantial effect on the activation of these transcription factors.
  • Example 2 A 2A R agonists prevent HIV-I induced monocyte activation. Microglial and macrophage activation contributes to neuronal damage in HIV-I infected individuals [Poluektova, L., et al. 2004. J Immunol 172:7610-7617], and is associated with increased expression of inducible nitric oxide synthase (iNOS) and production of NO.
  • iNOS inducible nitric oxide synthase
  • HIV-I Tat produced a dose-dependent increase in iNOS expression in human primary monocytes, which was maximal at 100 nM.
  • the effect of adenosine receptor activation on Tat-induced upregulation of iNOS expression and NO release was then determined. Tat-treatment resulted in a 4-fold increase in iNOS expression by primary monocytes and a similar elevation in NO release (Fig. 3).
  • a 2A R agonist on TNF ⁇ release by primary human monocytes was also determined.
  • Monocytes were stimulated with HIV-I Tat, in the presence or absence of the A 2A R agonist CGS21680 or the A 2A R antagonist ZM251385 and TNF ⁇ release was then measured in cell culture supernatants using an ELISA assay.
  • the results show that the A 2A R agonist abrogated the Tat-mediated increase in monocyte-derived TNF ⁇ production.
  • Quantitative real-time PCR analysis was used to measure TNF ⁇ message levels in Tat-treated monocytes.
  • Primary human monocytes were prepared using CD14 + immunomagnetic selection, and then exposed to LPS or to HTV-I Tat for 4 hours, in the presence or absence of the A 2A R agonist CGS21680.
  • Culture supernatants were tested for TNF ⁇ levels by ELISA assay (Fig. 5A), and RNA was extracted from cell pellets for quantitative reverse-transcription PCR (qRTPCR) analysis (Fig. 5B).
  • the A 2A R agonist CGS21680 strongly suppressed TNF ⁇ release in Tat-exposed monocytes (Fig. 5B). Moreover, a strong concordance was observed between the TNF ⁇ ELISA (Fig. 5A) and the qRTPCR analysis (Fig. 5B). Notably, the A 2A R agonist CGS21680 strongly suppressed TNF ⁇ release (and TNF ⁇ transcription) in Tat-exposed monocytes, but had little effect on ILl ⁇ mRNA levels in these cells (Fig. 5B).
  • a second A 2A R agonist ATL313 demonstrated very similar results to those shown in Figure 4 (see Fig 6 & 7). It was determined that exposure of monocytes to an A 2A R antagonist did not result in an increase in cellular activation in the presence of Tat (see data for ATL455; Fig. 6) and that the approximate IC50 for ATL313's inhibitory effect on
  • Tat-stimulated TNF ⁇ release in monocytes is ⁇ InM. This is consistent with the known receptor-binding properties of this compound (see Table 1).
  • Table 1 the results shown in Figures 6 and 7 confirm and extend the findings in Figures 4 and 5, using a commercially relevant molecule (ATL313).
  • Cortical neuronal cultures were treated with Tat or cPAF for varying periods of time as an in vitro model of pre-synaptic nerve terminal function, using the lipophilic, fluorescent styryl dye iV-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM 1-43), which binds to synaptic vesicle membranes and is taken up into nerve terminals during normal activity (i.e., synaptic vesicle recycling).
  • the lipophilic, fluorescent styryl dye iV-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide FM 1-43
  • Fluorescent signal can be quantified to number of neurons, and after chemical depolarization with high concentrations of KCl, fluorescent signal is abolished, giving an index of nerve terminal activity in real-time.
  • This model was used to demonstrate that both Tat and PAF increased vesicular uptake (of neurotransmitter) in rodent cortical cultures (Figure 8).
  • the staining seen with FM 1-43 was punctate and largely oriented along neuronal processes (Figure 8A, B), and released in a quantal fashion with a depolarizing concentration of KCl, all suggestive of synaptic vesicle recycling (Figure 8C).
  • Tat caused a dose-dependent increase in FMl -43 uptake that was even more pronounced in aged cultures ( Figure 8D), perhaps due to incomplete maturation of synaptic receptors in the younger cultures [Gelbard, H.A., et al., J Virol, 1994. 68(7): p. 4628-35].
  • an inactive mutant Tat protein had no effect on FM1-43 uptake, indicating the effect was a biologically specific effect of Tat (Figure 8E).
  • PAF caused an even more robust increase in FM 1-43 uptake (Figure 8F), in keeping with its role as a mediator of pre-synaptic glutamate release [Clark, G.D., et al., Neuron, 1992. 9(6): p. 1211-6].
  • ROS reactive oxygen species
  • Mitochondria generate the energy for synaptic transmission in the form of ATP, and as a byproduct of this oxidative phosphorylation, they are also one of the primary producers of intracellular ROS.
  • Two key mitochondrial parameters were examined in response to Tat and PAF: 1) mitochondrial membrane potential ( ⁇ m ) [as assessed by mitochondrial uptake of the lipophilic cationic dyes tetramethylrhodamine ethyl or methyl ester (TMRE or TMRM respectively)], and 2) ATP production (as measured by luciferase assay).
  • the electronegative mitochondrial membrane potential provides the driving force for calcium buffering and ATP production by the mitochondria, and as such, ⁇ m is frequently used as an indicator of mitochondrial health and energetic capacity. Moreover, fluctuations in ⁇ m can trigger mitochondrial release of pro-apoptotic factors, an event most frequently associated with a loss of ⁇ m . To the contrary, however, Tat treatment of rodent cortical neurons resulted in a dose-dependent, biphasic increase in ⁇ m over 48 hours (Figure 1 IA).
  • Tat 100 ng/ml
  • a low dose of Tat caused a gradual increase in ⁇ m , peaking with a 17% increase in mitochondrial TMRE uptake at 4 hours (p ⁇ .004), then declining to baseline by 14 hours, before rising again (6% increase vs. control vehicle) 26 hours after application, followed by a plateau at 36 hours that persists until the end of the analysis (48 hours) (19% increase vs.
  • Example 4 Approaches to normalize synaptic transmission in models of postsynaptic injury
  • An additional pathologic consequence of exposure to HIV-I neurotoxins is an increase in oxidized cellular phospholipids that can bind to and activate the PAF-R [Marathe, G.K., et al., Vascul Pharmacol, 2002. 38(4): p. 193-200].
  • Both energetic stress and PAF-R signaling have the potential to impair post-synaptic neurotransmission.
  • a morphologic correlate of dendrite damage in HAD was identified, which is activity- induced dendritic swelling or "beading" in the presence of the HIV neurotoxin cPAF. This beading is also accompanied by impairment of synaptic activity.
  • Figure 14 shows that exposing hippocampal neurons in vitro to sublethal (130 nM) concentrations of cPAF for 60 hours results in loss of dendritic spines (but not neurites) and dendrite beading, without cell death. Additionally, in an acute hippocampal slice model, shorter cPAF exposures (l ⁇ M for 30-60 minutes) increased neuronal susceptibility to beading in response to synaptic activity, and results in failure of long-term potentiation in the beaded dendrites (Figure 15). Increased neuronal susceptibility to beading in response to synaptic activity was also observed in hippocampal cultures following brief (1 hour) cPAF exposures.
  • PAF can lower the threshold for synaptic injury, and that by impairing synaptic function, beading can serve as an important functional marker of dendritic injury, and can underlie the reversible impairments of neuronal function seen in HAD.
  • the in vitro and in vivo models of dendritic injury disclosed herein are sensitive and reproducible, and have great utility to determine the ability of adjunctive therapies to restore function (i.e., synaptic transmission) during exposure to HIV-I neurotoxins.
  • Example 5 Bioenergetic defects in neurons exposed to HIV-I neurotoxins HIV neurotoxins can increase vesicle recycling, mitochondrial membrane potential, ATP/ADP ratios, and reactive oxygen species. Furthermore, blocking the Tat- induced rise in ⁇ m protects the neurons against apoptotic cell death. Thus, the observed increases in ⁇ m are not simply a compensatory response to increased metabolic demand, since in that case blocking the rise in ⁇ m would likely lead to energetic failure and exacerbate cell death.
  • HIV neurotoxins could be a pathogenic mechanism leading to excessive production of ROS and ATP, resulting in synaptic stress, excitotoxicity, and eventual demise of synaptic contacts and dendritic arbor, i.e., "synaptic apoptosis" [Mattson, M.P., J.N. Keller, and J.G. Begley, Exp Neurol, 1998. 153(1): p. 35- 48].
  • These pathogenic mechanisms could contribute to the reversible component of HAD, but if left unchecked, could ultimately result in permanent neurologic deficit with or without cell loss.
  • HIV-neurotoxin-induced mitochondrial hyperpolarization is associated with a rise in NADH/NAD + ratio and increased oxygen consumption by the electron transport chain (ETC) and to further clarify Tat and PAF 's effects on oxidative phosphorylation
  • ETC electron transport chain
  • Tat and PAF 's effects on oxidative phosphorylation primary rodent cortical or hippocampal neurons are treated with a range of doses of Tat (10 ng/ml, 100 ng/ml, and 2.5 ⁇ g/ml) and cPAF (2 nM, 20 nM, and 500 nM) over 1, 4, 8, 12, 24, 36, 48, and 72 hours, and NADH/NAD + ratio and rates of O 2 consumption at these time points assessed.
  • ATP/ADP ratios and ⁇ m are also assessed at these time points to verify previous findings.
  • a mitochondrial hyperpolarization is associated not only with increased ATP production as disclosed herein, but also with increased O 2 consumption and increased NADHTNAD + ratio.
  • the doses of Tat and PAF described herein have been determined by previous studies to represent sub-threshold (i.e., no measurable effects), sub-lethal (measurable effects on intracellular parameters but induces little cell death at 18-24 hours), and toxic (strong effects on intracellular parameters and induces apparent cell death at 18-24 hours or greater) in neuronal culture systems. These doses have been selected to cover the range of responses expected for the cellular functions being assessed.
  • Tat or cPAF doses altered, if warranted.
  • specificity of effects is confirmed by substituting Tat with an equimolar amount of a biologically inactive mutant Tat protein ( ⁇ Tat31-61) [Gurwell, J.A., et al., Neuroscience, 2001. 102(3): p. 555-63] or applying cPAF with the PAF receptor antagonists WEB 2086 (10 ⁇ M) or BN52021 (10 ⁇ M).
  • oligomycin partially or fully blocks the Tat or PAF-induced rise in ⁇ m , then reversal of the F 1 F 0 -ATPaSe is at least in part responsible for the mitochondrial hyperpolarization.
  • Tat or PAF + oligomycin induces a rise in ATP/ADP ratios versus Tat or PAF alone, due to reduced consumption of ATP by the reversed F 1 Fo- ATPase.
  • Mitochondrial complexes I and III are the chief ROS producers of the ETC [Nicholls, D.G., Iht J Biochem Cell Biol, 2002. 34(11): p. 1372-81]. ROS levels are assessed by with the oxidizable dye indicator 5-(and-6)-chloromethyl-2',7'- dichlorodihydrofluorescein diacetate, acetyl ester (CMH 2 DCFDA, abbreviated as "DCF”) (Molecular Probes, Eugene, OR) as described herein. IfROS levels are higher in Tat/PAF treated cultures, and this effect is blocked by rotenone and/or myxothiazole, then the hyperpolarization is contributing to enhanced ROS production from the ETC in Tat/PAF treated neurons.
  • CHC 2 DCFDA acetyl ester
  • Complex II and IV inhibitors succinate-q reductase (TTFA) and potassium cyanide (KCN) respectively can also be used herein.
  • Complexes II and IV contribute to ROS production, but generally not as much as complexes I and III [Nicholls, D.G., Int J Biochem Cell Biol, 2002. 34(11): p. 1372-81].
  • Caspase 9 activation of Apafl, another key component of the CytC/Apafl/Caspase9 apoptosome, and Caspase 3 are also examined.
  • Tat or PAF ⁇ glucose or NADH are co-incubated, and ⁇ m , O 2 consumption, NADH levels, ATP/ADP ratios, and apoptotic cell death assessed at the 24, 48, and 72 hour time points.
  • NADH and glucose are titrated to select doses that are not toxic to the neuronal cultures.
  • KATP channel antagonists As disclosed herein, the KATP channel antagonist and hypoglycemic agent Tolbutamide block the rise in ⁇ m from the HIV-I virotoxin Tat and protect against cell death. Tolbutamide's principal action is thought to occur via inhibitions of KATP channels [Liss, B. and J. Roeper, MoI Membr Biol, 2001. 18(2): p. 117-27], although it activates glycolysis as well [Kaku, K., Y. Inoue, and T. Kaneko,
  • the first and third mechanisms are the most likely candidates for tolbutamide's protective and stabilizing effects based on the data disclosed herein, as enhancing glycolysis would only further increase ⁇ m , ATP, and ROS production.
  • Controlled uncoupling, or regulation of mitochondrial permeability to H+ or other ions could underlie protective actions of several drugs disclosed herein, notably those that link pro-apoptotic mitochondrial proteins with mitochondrial membrane permeability (see rationale for "protease and enzyme inhibitors” below).
  • aberrant control of mitochondrial polarity represents a link between mitochondrial status and dendrite beading, via volume- regulated anion channels (VRACs).
  • Antioxidant compounds As disclosed herein, Tat and PAF induce ROS production in neurons, which could stem from increased oxidative phosphorylation and ATP production. Mitochondrial production of ROS leads to enhanced excitotoxic synaptic activity, leading to synaptic damage, or activates cell death pathways directly. Synapses are particularly vulnerable to oxidative stress. As disclosed herein, the antioxidant compound Tauroursodeoxycholic acid (TUDCA) protects against Tat-induced increases in synaptic activity. Together these data indicate that antioxidant compounds protect against synaptic damage and dysfunction in HAD.
  • TDCA Tauroursodeoxycholic acid
  • Candidate antioxidants are evaluated with the herein disclosed biological models of HAD, including the mitochondria-targeted antioxidants Mito-Coenzyme QlO (Mito- CoQ) and Mito-VitaminE (Mito-E) [Green, K., M.D. Brand, and M.P. Murphy, Diabetes, 2004. 53 Suppl 1: p. S110-8] ⁇ as well as regular antioxidants such as Coenzyme QlO and ibedenone [Smith, R.A., et al., Proc Natl Acad Sci U S A, 2003. 100(9): p. 5407-12] [Green, K., M.D. Brand, and M.P. Murphy, Diabetes, 2004. 53 Suppl 1 : p. S 110-8] .
  • Candidate compounds are tested for their efficacy in preventing pathologic outcomes against Tat and PAF treatment in primary assay systems that provide physiological and morphologic correlates of neuronal dysfunction HAD, i.e. whether they block mitochondrial hyperpolarization and cell death, prevent aberrant synaptic transmission and dendrite loss, and/or prevent dendrite swelling or "beading".
  • the compounds identified herein are tested for their ability to block Tat and PAF-induced mitochondrial hyperpolarization, as assessed by the TMRM assay (below), and cell death, as assessed by a combined assay for both necrosis and apoptosis [Perry, S.W., L.G. Epstein, and H.A. Gelbard, Biotechniques, 1997. 22(6): p. 1102-6].
  • AU potential therapeutic compounds are first be titrated in full dose response curves to determine non-toxic doses for neurons. Dose ranges of Tat and PAF are extended if necessary to clearly determine whether protective effects exist.
  • HAART particularly protease inhibitors [PIs] and nucleoside reverse transcriptase inhibitors [NRTIs]
  • adjunctive neuroprotective agents disclosed herein is of great value in terms of defining future regimens of HAART that are tailored specifically to the amelioration of CNS disease and/or use in combination with specific adjunctive regimens that target mitochondrial bioenergetics, synaptic dysfunction, and neuronal apoptosis.
  • pro- and anti- apoptotic proteins could participate not only in pathologic mechanisms at the synapse, but also physiologic processes of synaptic regulation, such as growth cone rearrangement or synaptic strengthening [Jonas, E., J Bioenerg Biomembr, 2004. 36(4): p. 357-61].
  • PIs that alter the processing of mitochondrial apoptotic regulatory proteins such as BCL-xL could both impact mitochondrial and cellular/synaptic function - as well as cell fate.
  • PIs e.g. Ritonavir
  • NRTIs e.g. Tenofovir
  • Pi-using kaletra [lopinavir/ritonavir] + efavirenz
  • Pi-sparing efavirenz + AZT + 3TC
  • PI Indinavir penetrates the CNS
  • Adjunctive neuroprotective agents provided herein are combined with different antiretrovirals, starting with individual drugs alone (PIs, NRTIs) and then progressing to the three defined HAART regimes (see Table 2, above). Selection of dose ranges is based on physiologically achievable drug levels and/or the EC50 and EC99 for each drug. Thus, as an example, Atazanvir is used at doses between 1-100 nM [Colonno, R.J., et al., Antimicrob Agents Chemother, 2003. 47(4): p.
  • Carbamyl-PAF (c-PAF; abbreviated "PAF” herein), a non-hydrolyzable form of PAF, is obtained from Biomol (Plymouth Landing, PA).
  • Cells that are used in the compositions and methods disclosed herein include:
  • CGNs Primary rat cerebellar granule neurons
  • CGNs represent a highly homogenous population of primary neurons which are susceptible to Tat and PAF- mediated cell killing and have been used in many of previous studies.
  • CGN cells are isolated according to published procedures [Maggirwar, S. B., et al. 1999. J Neurochem 73:578-86] [Tong, N., et al. 2001. Eur J Neurosci 13:1913-22].
  • CN Primary rat cortical neurons
  • CN are readily established, and available in significant cell numbers; they have the particular advantage that they fully reprise all the neuronal circuitry necessary to drive striatal projection neurons and are therefore used to identify electrophysiologic parameters of synaptic transmission to develop a functional bioassay for measuring the neuroprotective efficacy of potential therapeutic agents.
  • CN are susceptible to Tat and PAF-mediated cell killing as well as Tat and PAF- mediated mitochondrial dysfunction and synaptic apoptosis. These cells are isolated and maintained using methods described herein and in [Perry, S. W., et al. 2004. J Neurosci Res], incorporated by reference herein for its teaching of these methods.
  • HN Primary rat hippocampal neurons
  • the hippocampus plays a central role in learning and memory — processes which are adversely affected in HXV-associated neurologic disease.
  • HN are susceptible to Tat and PAF-mediated cell killing [Kruman, II, et al. 1998. Exp Neurol 154:276-88] [Nath, A., et al. 1996. J Virol 70:1475- 80] as well as Tat-mediated excitotoxicity [Song, L., et al 2003. J Neurovirol 9:399-403].
  • Tat-mediated excitotoxicity Long, L., et al 2003. J Neurovirol 9:399-403].
  • These cells also undergo reductions in dendritic arborization in response to Tat [Maragos, W. F., et al. 2002.
  • HNs are be prepared from embryonic day 18 rats by modification of the protocol by Brewer [Brewer, G. J., et al. 1993. J Neurosci Res 35:567-76].
  • hippocampi are dissected from a litter of E18 embryonic rats, dissected free of meninges and other tissue, and incubated in 2.0 ml of Ca + /Mg + -free Hanks balanced salt solution (HBSS) (with 1 OmM HEPES, pH7.3) with PSN antibiotics (penicillin 50 mg/L; streptomycin 50 mg/L; neomycin 100 mg/L) plus 0.5 ml for 2.5% trypsin (for 0.25% final) for 15 min at 37°C per brain.
  • HBSS Hanks balanced salt solution
  • TMRM mitochondrial membrane potential
  • primary rodent neurons are treated with reagent under normal culture conditions for the indicated time period, followed by removal of the media, 1x2 minute wash in pre-warmed 37°C HBSS plus 10 niM glucose and 10 mM HEPES
  • HBSS+ HBSS+
  • TMRE/M 1 nM TMRE/M
  • Random field images are taken using an Olympus IX-70 microscope and 4Ox objective (fluorescent excitation: 545; emission: 610) and Apogee KX32ME CCD camera, and the mean relative fluorescent unit (RFU) value of the neuronal soma and processes (excluding mitochondria deficient regions) is quantified using Scanalytics IPLab software.
  • Measuring production of intracellular ROS Generation of ROS in response to Tat and PAF is assessed with the oxidizable dye indicator 5-(and- 6)-chloromethyl-2', T- dichlorodihydrofluorescein diacetate, acetyl ester (CM-H 2 DCFDA, abbreviated as "DCF”) (Molecular Probes, Eugene, OR).
  • DCF dichlorodihydrofluorescein diacetate, acetyl ester
  • DCF acetyl ester
  • Oxidation by ROS including hydrogen peroxide (H 2 O 2 ), hydroxyl radical (HO ' ), peroxyl radical (HOO " ), or peroxynitrite anion (ONOO " )
  • H 2 O 2 hydrogen peroxide
  • HO ' hydroxyl radical
  • HOO " peroxyl radical
  • ONOO " peroxynitrite anion
  • Adenosine di- and tri-phosphate (ADP and ATP respectively) levels in cortical cultures were measured after treatment using a kit from Cambrex (Rockland, ME). Briefly, cortical neurons were plated in white poly-d-lysine coated 96 well plates at a density of 32,000 cells/well. Each treatment group contained five replicate wells. After treatment, ATP and ADP levels were then measured using an ATP/ADP assay kit (Cambrex, Rockland, ME).
  • This kit assays ATP levels via the luciferase reaction, then assays ADP levels by a proprietary method of ADP to ATP conversion; the difference in luminescence signal pre-and post- ADP conversion represents the ADP levels in the culture.
  • Luminescent signal was integrated over 1 second and read on a Packard LumiCount microplate luminometer (Meriden, CT). For each condition, data represents the mean ⁇ SEM ATP or ADP signal of five replicates per condition, expressed as percent increase over corresponding untreated, time-matched control. Background luminescence was negligible, but nonetheless was subtracted from all readings before data analysis. Experimental conditions were run two or more times with similar results.
  • FM 1-43 uptake assay Total spontaneous activity dependent vesicular uptake was assessed using the lipophilic styryl dye N- (3- triethylammoniumpropyl)-4-(4- (dibutylamino)styryl) pyridinium dibromide (FM1-43).
  • FM1-43 is an amphipathic molecule with a +2 charge that prevents it from passively crossing membranes [Ryan,
  • FM1-43 was loaded in the absence of a depolarizing stimulus to neurons. This method loads FMl -43 into vesicles undergoing both spontaneous miniature synaptic release and spontaneous action-potential (AP) dependent release. FM1-43 was loaded for 15 minutes, a length of time that has been determined to label spontaneous activity without saturating the vesicle pool [Prange, O. and T.H. Murphy, J Neurosci, 1999. 19(15): p. 6427-38].
  • This absolute FM 1-43 uptake value reflects the total activity of the culture, and is equivalent to the synaptic or vesicular release probability of the culture [Prange, O. and T.H. Murphy, J Neurosci, 1999. 19(15): p. 6427-38].
  • Cell death is assessed by visualizing fragmented DNA per the TUNEL method as described previously as well [Perry, S.W., et al., J Neurosci Res, 2004. 78(4): p. 485-92], and where necessary, a dual stain with Trypan blue (to identify necrotic cells)/ApopTag reagent [Perry, S. W., L.G. Epstein, and H. A. Gelbard, Biotechniques, 1997. 22(6): p. 1102-6]. Briefly, after treatment in 24 well plates, cells are fixed with Histochoice MB Tissue Fixative (Amresco) then TUNEL-labeled with the ApopTag kit (Chemicon) according to kit instructions.
  • HIV neurotoxins including Tat and PAF induce a reversible synaptic dysfunction that is morphologically characterized by dendrite beading, and eventually lead to permanent synaptic deficit (i.e. synaptic apoptosis) if the local concentration of HIV-I neurotoxins increases to irreversibly toxic levels.
  • Tat and PAF increase pre-synaptic terminal activity and induce mitochondrial hyperpolarization, contemporaneously with increased ROS and ATP production. ROS and ATP both induce synaptic activity directly [Cheng, J., et al., Neuroscience, 1998. 82(1): p. 97-106] [Kamsler, A. and M. Segal, MoI Neurobiol, 2004. 29(2): p.
  • Explant hippocampal slices are exposed to HIV-I neurotoxins ⁇ agents that reverse mitochondrial hyperpolarization. Beading and LTP are then be measured:
  • FCCP normalizes ⁇ m back to baseline levels but not below; here it is used simply to determine the effects of normalizing ⁇ m on beading and LTP in this model; it is not being considered as an adjunctive therapeutic agent.
  • Other candidate therapeutics that normalize ⁇ m e.g. glibenclamide are tested in place of FCCP, with a view to use in future animal or human studies.
  • Hippocampal slice preparation Brains from anesthetized P 14-28 rodents are rapidly removed and cooled in sucrose based artificial cerebrospinal fluid (ACSF) containing in niM: 110 sucrose, 60 NaCl, 3 KCl, 1.25 NaH 2 PO 4 , 28 NaHCO 3 , 10 D- glucose, 0.5 CaCl 2 , 7 MgSO 4 , 0.6 ascorbate.
  • the brains are blocked and fixed to a specimen stage with cyanoacrylate. Coronal slices (250-400 ⁇ m in thickness) are cut with a vibroslicer (World Precision Instruments, Fl, USA) equipped with a Peltier cooling system. The middle four to six slices of each hippocampus (with the entorhinal cortex removed) are placed into a holding chamber prior to use.
  • Dendritic arbors of individual CAl pyramidal cells are imaged while simultaneously recording excitatory postsynaptic potentials in the same cell by patch clamp recordings in whole-cell configuration. Recording pipettes (3-6 MO resistance) are pulled from 1.5mm borosilicate glass using a horizontal Flaming/Brown micropipette puller (Sutter Instrument Co, CA), fire-polished, and filled with intracellular recording solution (in mM: KCl 20, potassium gluconate 130, EGTA 0.5, HEPES 10, MgSO 4 2, ATP 2.5, GTP 0.5, pH 7.3).
  • Alexa 568 hydrazide (30 ⁇ M) is included in the recording pipette and is injected by small negative current pulses for 15-20 min prior to recording in order to fill the dendritic arbor.
  • Hippocampal slices are bathed in artificial CSF (containing, in mM: NaCl 125, KCl 2.5, NaH 2 PO 4 1.25, NaHCO 3 25, CaCl 2 2, MgCl 2 1, D-glucose 25, oxygenated and buffered with 5% CO 2 ).
  • Membrane potentials of CAl pyramidal cells are recorded in current clamp mode, and postsynaptic potentials are evoked by test pulses of constant-current stimulation applied via a bipolar stimulating electrode placed 50-200 ⁇ m away in the stratum radiatum.
  • Bicuculline (10 ⁇ M) is included in the bath to isolate excitatory postsynaptic potentials.
  • HFS high-frequency LTP induction stimulus
  • EPSPs are recorded for 60 min following HFS.
  • dendrites are imaged at 4Ox magnification by fluorescence microscopy, using a shutter to limit fluorescent light exposure and prevent phototoxic injury to the slice.
  • Serial images of dendrites are compared to detect development of focal swellings along dendritic shafts following HFS. Cells are scored as beaded if swellings develop along any of their dendrites.
  • Field potential recording and induction of LTP in hippocampal slices Single-cell recordings are complemented by extracellular field potential recordings: while single cell recordings allow correlation of electrophysiologic data with dendritic beading in the same cell, extracellular recordings sample responses from populations of dendrites and are less invasive.
  • Extracellular recording electrodes filled with 2 M NaCl and 2% pontamine blue (electrode impedance 2-4 MO), are placed within the stratum radiatum layer of area CAl.
  • Field excitatory post-synaptic potentials (EPSPs) are evoked by stimulation of the Schaffer collateral-commissural afferents once every 30 sec and the initial (1-2 ms) slope is measured.
  • Baseline responses are recorded for at least 20 min prior to induction of LTP by tetanic stimulation (four individual 100 Hz trains delivered for 1 sec each at the test intensity with inter-train intervals of 15 sec). Field responses are measured for 1 hr after applying tetanic stimulation; % baseline values are determined from the final 10 min interval recorded. Field potentials are recorded using an Axoclamp-2B amplifier (Axon Instruments, CA) and amplified further by an EXl differential amplifier (Dagan Corporation, MN, USA). Data acquisition and analyses are performed using pClamp 8 software (Axon Instruments, CA, USA) on a Pentium IV PC computer (Dell, DSf, USA).
  • Giniatullin A.R. and R. A. Giniatullin, Dual action of hydrogen peroxide on synaptic transmission at the frog neuromuscular junction. J Physiol, 2003. 552(Pt 1): p. 283-93. Glass, J. D., H. Fedor, S. L. Wesselingh, and J. C. McArthur. 1995. Immunocytochemical quantitation of human immunodeficiency virus in the brain: correlations with dementia. Ann Neurol 38:755-762.
  • HIV-I protein Tat induces apoptosis of hippocampal neurons by a mechanism involving caspase activation, calcium overload, and oxidative stress.
  • Masliah, E., et al. Changes in pathological findings at autopsy in AIDS cases for the last 15 years. Aids, 2000. 14(1): p. 69-74.
  • HIV-I Tat induces neuronal death via tumor necrosis factor-alpha and activation of non-N-methyl-D-aspartate receptors by a NFkappaB-independent mechanism. J Biol Chem, 1998. 273(28): p. 17852-8.
  • Gadd45 beta mediates the NF-kappa B suppression of JNK signalling by targeting MKK7/JNKK2. Nat Cell Biol 6:146-153.
  • Glycogen synthase kinase 3beta-mediated apoptosis of primary cortical astrocytes involves inhibition of nuclear factor kappaB signaling. MoI Cell Biol 23:4649-4662. Schwanstecher, C. and D. Bassen, KATP-channel on the somata of spiny neurones in rat caudate nucleus: regulation by drugs and nucleotides. Br J Pharmacol, 1997. 121(2): p. 193-8.
  • Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc Natl Acad Sci U S A 95:15769-15774.

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L'invention concerne des compositions et des procédés liés au traitement et à la prévention de la démence liée au VIH-1, qui utilisent un modulateur de la signalisation d'un récepteur de l'adénosine.
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WO2024168320A1 (fr) * 2023-02-09 2024-08-15 Anand Rene Procédés et compositions pharmaceutiques pour le traitement de la démence
WO2024215814A3 (fr) * 2023-04-10 2024-11-21 Anand Rene Procédés et compositions pharmaceutiques pour le traitement du vieillissement

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US5356894A (en) * 1990-05-29 1994-10-18 Rodney Peter W Morpholinyl substituted [1,2,4]-triazolo[1,5-a]triazine as antagonist
US5877180A (en) * 1994-07-11 1999-03-02 University Of Virginia Patent Foundation Method for treating inflammatory diseases with A2a adenosine receptor agonists
US6063775A (en) * 1997-04-29 2000-05-16 Berman; Charles L. Retardation of metalloproteinase incidental to HIV and/or AIDS
US20030162695A1 (en) * 2002-02-27 2003-08-28 Schatzberg Alan F. Glucocorticoid blocking agents for increasing blood-brain barrier permeability

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WO2024168320A1 (fr) * 2023-02-09 2024-08-15 Anand Rene Procédés et compositions pharmaceutiques pour le traitement de la démence
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