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WO2006019841A2 - S - Google Patents

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Publication number
WO2006019841A2
WO2006019841A2 PCT/US2005/024893 US2005024893W WO2006019841A2 WO 2006019841 A2 WO2006019841 A2 WO 2006019841A2 US 2005024893 W US2005024893 W US 2005024893W WO 2006019841 A2 WO2006019841 A2 WO 2006019841A2
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WO
WIPO (PCT)
Prior art keywords
hiv
cells
subject
cell
egr
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PCT/US2005/024893
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English (en)
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WO2006019841A3 (fr
Inventor
Steven Zeichner
Vyjayanthi Krishnan
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Government Of The United States Of America
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Publication of WO2006019841A2 publication Critical patent/WO2006019841A2/fr
Publication of WO2006019841A3 publication Critical patent/WO2006019841A3/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • 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
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • the human immunodeficiency virus type 1 (HIV-I, also referred to as HTLV- III LAV or HTLV-III/LAV) and, to a lesser extent, human immunodeficiency virus type 2 (HIV-2) is the etiological agent of the acquired immune deficiency syndrome (AIDS) and related disorders.
  • AIDS acquired immune deficiency syndrome
  • AIDS While AIDS, itself, does not necessarily cause death, in many individuals the immune system is so severely depressed that various other diseases (secondary infections or unusual tumors) such as herpes, cytomegalovirus, Kaposi's sarcoma and Epstein-Barr virus related lymphomas among others occur, which ultimately results in death. These secondary infections may be treated using other medications. However, such treatment can be adversely affected by the weakened immune system.
  • Some humans infected with the AIDS virus seem to live many years with little or no symptoms, but appear to have persistent infections.
  • Another group of humans suffers mild immune system depression with various symptoms such as weight loss, malaise, fever and swollen lymph nodes. These syndromes have been called persistent generalized lymphadenopathy syndrome (PGL) and AIDS related complex (ARC) and may or may not develop into AIDS. In all cases, those infected with the HIV are believed to be persistently infective to others.
  • PDL persistent generalized lymphadenopathy syndrome
  • ARC AIDS related complex
  • LTR long terminal repeat
  • TNF ⁇ is present in markedly enhanced levels in HIV infected individuals, suggesting that the cytokine plays an important role in the pathogenesis of AIDS.
  • Lahdevirta J., Am. J. Med., 85:289-291 (1988).
  • the compounds are those of formula I (trans-resveratrol), formula II (cis-resveratrol) or formula III (taxifolin):
  • aspects of the invention are methods of treating or preventing HIV infection in a subject comprising administration of an effective amount of an Egr 1 activator, including those having a structure represented by the formulae herein.
  • compounds e.g., compounds represented by the formulae herein
  • the compounds of the present invention can induce lytic replication in cells latently infected with an immunodeficiency virus such as HIV by targeting a product of a gene, which is differentially expressed in latently infected cells and lytic replicating cells.
  • the compounds of the present invention can treat cells infected acutely and chronically by immunodeficiency viruses, for example, HIV, preferably HIV-I, and thus can be used to treat humans infected by HIV.
  • immunodeficiency viruses for example, HIV, preferably HIV-I
  • the cell is preferably a lymphocytic cell or a monocytic cell. In preferred embodiments, the cell is a human cell.
  • Other aspects are a method of reducing latent HIV reservoirs in an HIV- infected subject comprising administration to the subject of an effective amount of an Egr 1 activator (e.g., a compound of the formulae herein); a method of increasing (e.g., 2-20 fold relative to cells treated with AZT) expression of p24 in a latently HIV- infected cell comprising administration to the cell of an effective amount of an Egr 1 activator (e.g., a compound of the formulae herein); a method of activating latent HIV-provirus in a cell in a subject comprising administration to the subject of an effective amount of an Egr 1 activator (e.g., a compound of the formulae herein); and a method of activating latent HIV-provirus in a cell in a subject comprising administration to the subject of an effective amount of an Egr 1 activator (e.g., a compound of the formulae herein).
  • an Egr 1 activator e.g.,
  • the methods delineated herein can further include administration of one or more additional anti-HIV therapeutic agents.
  • the additional agent(s) can be one or more reverse transcriptase inhibitors, one or more protease inhibitors r, or combination thereof.
  • the Egr 1 activator in the methods herein is resveratrol.
  • Another aspect is a method of screening for a compound capable of activating latent HIV-infected cells comprising contacting an ACH-2 cell or J 1.1 cell or Ul cell with a test compound and determining the level of p24 expression; the method can be wherein increased expression of p24 in cells treated with a test compound relative to non-treated cells indicates a compound capable of activating or reactivating latent HIV-infected cells.
  • the test compound can include those of any of the formulae delineated herein.
  • Another aspect is a method of reducing latent HIV-reservoirs in a subject by controlled activation of viral replication including administration of an effective amount of a Egr 1 activator.
  • Controlled activation is that activation initiated by administration of an effective amount of Egr 1 activator such that the Egr 1 activator reactivates (directly or indirectly) replication processes in latent HIV-infected cells.
  • the latent HIV-reservoirs are collections of latent HIV-infected cells, that is cells in which the HIV-replication is considered to be in a latent state.
  • the method can further include administration with one or more antiviral agents, thus both depleting the latent cell reservoir and inducing their lytic replication whereupon the cells in that state are subjected to and susceptible to the antiretroviral therapy, which controls their proliferation.
  • the compounds of the present invention can treat cells infected acutely and chronically by immunodeficiency viruses, for example, HIV, preferably HIV-I, and thus can be used to treat humans infected by HIV.
  • immunodeficiency viruses for example, HIV, preferably HIV-I
  • Other aspects are a method of inhibiting HIV replication in a cell comprising administration to the cell of an effective amount of an Ergl activator; a method of treating latently HIV-infected cells in a subject comprising administration to the subject of an effective amount of an Erg 1 inhibitor; and a method of modulating lytic replication in an HIV-infected cell in a subject comprising administration to the subject of an effective amount of a Ergl inhibitor.
  • the cell is a lymphocytic cell or a monocytic cell.
  • the invention provides a method of treating or preventing HIV infection in a subject.
  • the method comprises identifying the subject as in need of Egr 1 activation, and administration of an effective amount of an Egr 1 activator.
  • the method comprises administration of an effective amount of an Egr 1 activator having the structure of formula (I), (II), or (III):
  • the method further comprises administration to the subject of an effective amount of an HIV protease inhibitor.
  • the invention provides a method of treating latently HIV- infected cells in a subject comprising administration to the subject of an effective amount of an Egr 1 activator that is taxifolin.
  • the invention provides a method of modulating lytic replication in an HIV-infected cell in a subject comprising administration to the subject of an effective amount of an Egr 1 activator that is taxifolin.
  • the cell is a lymphocytic cell. In other preferred embodiments, the cell is a monocytic cell. In certain preferred embodiments, the cell is a human cell.
  • the invention provides a method of reducing latent HIV reservoirs in an HIV-infected subject comprising administration to the subject of an effective amount of an Egr 1 activator that is taxifolin.
  • the invention provides a method of increasing (e.g., fold increase relative to cells treated with AZT) expression of p24 in a latently HIV- infected cell comprising administration to the cell of an effective amount of an Egr 1 activator that is taxifolin.
  • the invention provides a method of activating latent HIV-provirus in a cell in a subject comprising administration to the subject of an effective amount of an Egr 1 activator that is taxifolin.
  • the method further comprises administration of one or more additional anti-HIV therapeutic agents to the subject.
  • the additional agent(s) are one or more reverse transcriptase inhibitors, one or more protease inhibitors, or combination thereof.
  • the invention provides a method of treating HIV infection in a subject.
  • the method comprises the steps of identifying a subject as in need of reactivation of a replication process in latent HIV-infected cells; administering to the subject an effective amount of an Egrl activator to reactivate the replication process; and administering to the subject one or more HIV antiviral agents to inhibit the induced lytic replication in the subject and/or the cells.
  • the method provides a means for decreasing or eliminating the pool of latently infected cells and also decreasing the viral population.
  • the invention provides a method of screening for a compound capable of activating latent HIV-infected cells comprising contacting an ACH-2 cell or Jl.1 cell or Ul cell with a test compound and determining the level of p24 expression in the cell or cells compared to a control.
  • the test compound comprises an Egr 1 activator.
  • the invention provides a method of treating HIV infection in a subject comprising identifying the subject as in need of Egr 1 activation, administering an effective amount of an Egr 1 activator to reactivate HIV replication in infected cells, and further administering an effective amount of an additional antiviral agent that is a non-nucleoside reverse transcriptase inhibitor, a protease inhibitor, or other non-nucleoside antiviral agent.
  • the methods delineated herein include administering to a subject (e.g., a human or an animal) in need thereof an effective amount of one or more Egrl activators, e.g., compounds as delineated herein.
  • the methods can also include the step of identifying that the subject is in need of treatment of diseases or disorders described herein, e.g., identifying that the subject is in need of reactivation of a replication process or processes in latent HIV-infected cells.
  • the identification can be in the judgment of a subject or a health professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or a diagnostic method).
  • Tests for HIV infection include polymerase chain reaction-based (PCR-based) amplification and detection of viral RNA; Western blot detection of anti-HIV antibodies; agglutination assays for anti-HIV antibodies; ELISA-based detection of HIV-specific antigens (e.g., p24); line immunoassay (LIA); and other methods known to one of ordinary skill in the art.
  • PCR-based polymerase chain reaction-based
  • Western blot detection of anti-HIV antibodies e.g., agglutination assays for anti-HIV antibodies
  • ELISA-based detection of HIV-specific antigens e.g., p24
  • line immunoassay line immunoassay
  • the methods of the invention can include the step of obtaining a sample of biological material (such as a bodily fluid) from a subject; testing the sample to determine the presence or absence of detectable HIV infection, HIV particles, or HIV nucleic acids; and determining whether the subject is in need of treatment according to the invention, i.e., identifying whether the subject is in need of reactivation of a replication process or processes in latent HIV-infected cells.
  • a sample of biological material such as a bodily fluid
  • the methods delineated herein can further include the step of assessing or identifying the effectiveness of the treatment or prevention regimen in the subject by assessing the presence, absence, increase, or decrease of a marker, including a marker or diagnostic measure of HIV infection, HIV replication, viral load, or expression of an HIV infection marker; preferably this assessment is made relative to a measurement made prior to beginning the therapy.
  • a marker including a marker or diagnostic measure of HIV infection, HIV replication, viral load, or expression of an HIV infection marker; preferably this assessment is made relative to a measurement made prior to beginning the therapy.
  • Such assessment methodologies are known in the art and can be performed by commercial diagnostic or medical organizations, laboratories, clinics, hospitals and the like.
  • the methods can further include the step of taking a sample from the subject and analyzing that sample.
  • the sample can be a sampling of cells, genetic material, tissue, or fluid (e.g., blood, plasma, sputum, etc.) sample.
  • the methods can further include the step of reporting the results of such analyzing to the subject or other health care
  • the terms “subject” and “patient” are used interchangeably.
  • the terms “subject” and “subjects” refer to an animal, preferably a mammal including a non-primate (e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey, ape, monkey, or human), and more preferably a human.
  • the subject is an immunocompromised or immunosuppressed mammal, preferably a human (e.g., an HIV infected patient).
  • the subject is a farm animal (e.g., a horse, a cow, a pig, etc.) or a pet (e.g., a dog or a cat).
  • the subject is a human.
  • the invention also provides pharmaceutical compositions comprising a compound of any of the formulae herein and a suitable carrier therefore for use in the methods and conditions referred to above.
  • Figure 1 shows the flow cytometric analysis of chronically infected ACH-2 cells before and after induction. Uninduced cells and cells from serial time points were fixed and permeabilized for intracellular p24 labeling. As an isotype control, cell samples were labeled with mouse IgGl. For each sample, 100,000 events were collected. In the figure, each sample histogram labeled for p24 (darker color) is overlaid with the control histogram labeled for the isotype control (lighter color).
  • A Uninduced ACH-2 cells, showing minimal p24 accumulation with 8.2% of cells infected
  • B ACH-2 cells at 0.5 hours post induction (p.i.) with 7.4% of cells positive for p24
  • C ACH-2 cells at 6 hours p.i, with 61.6% cells infected
  • D ACH-2 cells at 12, 18, and 24 hours p.i., respectively, showing complete infection.
  • Flow cytometric analysis was performed on all batches of cells to ensure active replication of HIV following induction with PMA. Data from one induction experiment is shown. Data indicate that viral replication occurs in an ordered manner post induction, and complete infection of cells is achieved within 12 hours post induction of chronically infected ACH-2 cells.
  • Figure 2 shows the levels of expression of multiply spliced (MS HIV-I) and unspliced (US HIV-I) mRNA prior and post induction of chronically infected ACH-2 cells.
  • Real time RT-PCR reactions were carried out using Taqman probes specific for early (multiply spliced) and late (unspliced) transcripts of HIV-I, tagged with FAM and TAMRA fluorescent dyes at the 5' and 3' ends respectively. Reactions were performed in triplicate for each time point as described in the Methods section and average values are shown. Maximal fold change in mRNA levels for early transcripts (MS HIV-I) was observed 8 hours post induction. Fold change for late transcripts (US HIV-I) showed maximal increase 18 hours post induction.
  • Figure 3 shows the hierarchical clustering of differentially expressed cellular genes before and after induction of chronically infected ACH-2 cells.
  • the figure shows the hierarchical clustering of the cellular genes that showed significant differential expression (p ⁇ 0.001) across the time course (before induction up to 96 hours post induction), following reactivation of chronically infected ACH-2 cells as per the criteria described in the Methods.
  • Genes that are shown on the color scale >1 showed up regulation, those on the color scale ⁇ 1 were down regulated, while those that did not show any change with respect to normalized matched control are shown in black.
  • the gray areas indicate missing data for the given gene and time point.
  • the magnified panels indicate selected kinetic profiles that are seen before and following induction into active viral replication.
  • A Up regulation of selected genes observed before induction;
  • B Up regulation of genes immediately following induction;
  • C Genes that are up regulated prior to induction and down regulated 12-24 hours post induction;
  • D Genes that are up regulated in the early stage following reactivation, but are down regulated in the intermediate stage;
  • E Genes that are down regulated before induction but are up regulated in the intermediate stage followed by down regulation in the late stage (48-96 hours p.i.).
  • Figure 4 shows the trends seen in pathways that show differential expression before and after induction of chronically infected ACH-2 cells. Pathway profiles observed prior to induction and following reactivation of ACH-2 cells with PMA over a period of 96 hours. The figure shows the number of genes in each pathway that were differentially expressed in a particular pathway, (A) indicates the pathways that were maximally altered prior to induction. (B) includes the pathways that showed maximum change during the early phase of the lytic cycle, (0.5-8 hours p.i.). (C) represents the pathways that showed maximal change during the period of 12-24 hours post induction. Most pathways did not show any change during the period of 48-96 hours post induction. The groups above are a selected representation of the various pathways that changed differentially prior to induction and/or over the time course studied. Classification of the altered genes into various pathways was performed using the CGAP pathway databases.
  • Figure 5 shows the hierarchical clustering of genes that show differential expression across three chronically infected cell lines prior to induction.
  • Hierarchical clustering of differentially expressed genes that show a significant change in expression p ⁇ 0.001
  • Genes shown on the color scale >1 are up regulated, those on the color scale ⁇ 1 exhibit down regulation, while black indicates normal expression.
  • dark gray areas indicate missing values.
  • Many genes are altered similarly across the cell lines.
  • Each cell line also shows some unique patterns of cellular expression. Data are the average of values from eight independent samples per cell line. The magnified portions of the cluster highlight some of the patterns of gene expression across the cell lines.
  • FIG. 6 shows the effects of specific agents on HIV p24 production in chronically infected ACH-2 cells.
  • Different concentrations of a proteasome inhibitor (A) clastolactacystin-beta-lactone or (B) Resveratrol, an Egrl activator or (C) Trichostatin, a HDAC inhibitor were tested in chronically infected ACH-2 cells treated with 250 nM AZT.
  • Figure 7 shows the effect of resveratrol on latently infected Jurkat clones.
  • Figure 8 shows the effect of resveratrol on reactivation of aviremic patient sample #1.
  • Figure 9 shows the effect of resveratrol (lOO ⁇ M) on reactivation of aviremic patient samples.
  • Egrl activators e.g., compounds of the formulae herein
  • an immunodeficiency virus preferably human cells infected with HIV and thus can be used for treatment in HIV infected individuals.
  • an "Egrl activator” means a compound or composition capable of increasing the amount or activity of Egrl in a cell or in a subject.
  • the compound or composition may increase transcription or translation of the Egrl protein, reduce or prevent degradation of the Egrl protein or reduce or prevent degradation of the Egrl protein transcript.
  • an Egrl activator may desirably increase translational or even post-translational processing of Egrl protein.
  • the compounds of the invention also include compounds which affect other factors downstream of Egrl .
  • Egrl controls p21 c ⁇ ; expression; thus, Egrl activators can include compounds that upregulate p21 Q - p; expression.
  • resveratrol acts upstream of the ERK pathway, and thus Egrl activators include compounds that act upstream of ERKl and/or ERK2.
  • Resveratrol is an Egrl activator (see, e.g., F.D. Ragione et al, J. Biol. Chem., Jun 2003; 278: 23360 - 23368).
  • Other Egrl activators include the anti-oxidants hydroxytyrosol and pyrrolidine dithiocarbamate (see, e.g., F.D. Ragione et al, FEBS Lett. 2002 Dec 18;532(3):289-94).
  • Egrl activators can be identified using screening assays, e.g., as described in the previosuly-cited references.
  • prodrugs include esters and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing the parent compounds described herein (see Goodman and Gilman's, The Pharmacological basis of Therapeutics, 8 th ed., McGraw-Hill, Int. Ed. 1992, "Biotransformation of Drugs”).
  • Resveratrol (3,5,4'-trihydroxystilbene) has been identified as a constituent not only of grape skins (Soleas et al . (1995) Am. J. Enol. Vitic. 46(3):346-352) but has also been found to be present in ground nuts, eucalyptus, and other plant species. Goldberg et al. (1995), Am. J. Enol. Vitic. 46(2):159-165. A great deal of interest has been focused on the compound's antifungal activity and its correlation with resistance to fungal infection. Id at 159. Resveratrol also has been shown to effective for cardiovascular health due to its anti-oxidant properties (see, e.g., Sieman, E. H., and Creasy, L. L.
  • Resveratrol may be obtained commercially (typically as the trans isomer, e.g. from the Sigma Chemical Company, St. Louis, Mo.), or it may be isolated from wine or grape skins, or it may be chemically synthesized.
  • Synthesis is typically carried out by a Wittig reaction linking two substituted phenols through a styrene double bond, as described by Moreno-Manas et al. (1985) Anal. Quim.
  • resveratrol is intended to mean either the cis-isomer of resveratrol, the trans-isomer of resveratrol, or a mixture of the two isomers.
  • the term is also intended to include both the naturally occurring active agent and the compound as it may be chemically synthesized in the laboratory.
  • Resveratrol may be administered in natural form, i.e., as isolated from grape skins, wine or other plant-derived compositions, or it may be administered as chemically synthesized in the laboratory (e.g., using the methods of Moreno-Manas et al., Jeandet et al., or Goldberg et al. (1994), cited earlier herein), or as obtained commercially, e.g., from the Sigma Chemical Company (St. Louis, Mo.).
  • Trans-resveratrol may be isolated from wine using the procedure of Goldberg et al. (1995) Am. J. Enol. Vitic. 46(2): 159-165, as follows: selected wine is maintained at 4. degree. Cs and tightly stoppered in the dark prior to the isolation procedure. Preferably, isolation of resveratrol from the wine is conducted within two weeks of obtaining the wine. Briefly, a predetermined quantity of wine is passed through a suitable cartridge, e.g., a preconditioned C-18 SP cartridge (Supelco), and the retained trans-resveratrol is eluted with ethyl acetate. The product may be purified using conventional crystallization or chromatographic techniques.
  • a suitable cartridge e.g., a preconditioned C-18 SP cartridge (Supelco)
  • the product may be purified using conventional crystallization or chromatographic techniques.
  • trans-resveratrol is synthesized from appropriately substituted phenols by means of a Wittig reaction modified by Waterhouse from the method of Moreno-Manas and Pleixats, cited supra. The final product is greater than approximately 95% pure as may be confirmed using NMR and UV spectroscopy.
  • trans-resveratrol as isolated from wine, or may be chemically synthesized, and may also be converted to the cis isomer by preparing a solution of the trans isomer in a suitable solvent, e.g., 0.2 M phosphoric acid-acetonitrile (4:1 v/v). The solution is then irradiated for 5-10 min at a wavelength of 254 nm and an intensity of 990 LuW/cm 2 . Ultraviolet spectroscopy will confirm that the trans-resveratrol peak, at 306 nm, to be substantially reduced following this procedure. (See Goldberg et al. (1995) J. Chromatog. 708:89-98.).
  • the compounds of the present invention can provide effective therapy of latently infected cells (i.e. cells infected by a virus which is an immunodeficiency virus such as FIV, SIV, HIV, etc.) as evidenced by the induction of lytic replication in latently infected cells.
  • latently infected cells i.e. cells infected by a virus which is an immunodeficiency virus such as FIV, SIV, HIV, etc.
  • the present invention can be used in treating those diagnosed as having AIDS as well as those having ARC, PGL and those seropositive but asymptomatic patients.
  • an effective amount of a compound can also be used prophylactically as a preventative for high risk individuals.
  • Compounds of the present invention can be used to treat cells, especially mammalian cells and in particular human cells, infected by an immunodeficiency virus such as HIV.
  • an immunodeficiency virus such as HIV.
  • P24 a major structural protein (product of gag), has been widely used for monitoring HTV-I replication in cells and vireamia in individuals.
  • Egrl activators e.g., resveratrol
  • concentrations that do not adversely affect cells can dramatically reduce the number of cells latently infected with HIV, e.g.
  • a reduction of HIV-I latently infected cells as shown by an increase (e.g., X-fold increase, where x is 2, 3, 4, 5, 10, 15, 20, 30, 40, or any integer between about 2 and about 50) in p24 levels in cells treated with test compound (e.g., Egr 1 activators) relative to levels in untreated latent HTV-infected cells, hi another aspect the increase can be determined relative to untreated uninfected cells.
  • an increase e.g., X-fold increase, where x is 2, 3, 4, 5, 10, 15, 20, 30, 40, or any integer between about 2 and about 50
  • test compound e.g., Egr 1 activators
  • the effective amount of a compound of the present invention used to obtain such a result can be at micromolar concentrations. Furthermore, the administration of the compounds of the present invention at effective concentrations, which enhance HIV expression, has not been found to adversely affect treated cells.
  • Resveratrol is an Egrl activator, and causes an increase in p24 expression, which may be the cause of the latency reactivation from latently HIV infected cells.
  • the compounds of the present invention can be administered to HTV infected individuals or to individuals at high risk for HIV infection, for example, those having sexual relations with an HTV infected partner, intravenous drug users, etc. Because of its effect of inducing lytic replication, the compounds of the present invention and a pharmaceutical compositions comprising one or more compounds of the formulae herein can be used prophylactically as a method of prevention for such individuals to minimize their risk of cells becoming latently infected. The compound is administered at an effective amount as set forth below by methodology such as described herein.
  • compounds of the present invention induce lytic replication of HIV-I LTR and HIV-I in latently infected cells.
  • compounds of the present invention in a dose- dependent fashion cause latently infected cells to lyrically replicate.
  • such induction is provided with essentially no adverse effects on cell survival or cellular mRNA or total cellular RNA synthesis.
  • compounds of the present invention will have utility in clearing latent infections of an HIV infection and other retroviral infections in cells and in a human, and (in preferred embodiments) to ultimately entirely clear virus from an infected subject.
  • one or more compounds of the invention is administered in an amount sufficient to activate lytic replication in at least about 25 percent of infected cells, more preferably an amount sufficient to induce lytic replication in at least about 50 percent of the infected cells and still more preferably induce lytic replication in at least about 75 percent of latently infected cells.
  • a preferred effective dose of one or more compounds of the present invention will be in the range 0.1 mg to 5g per kilogram body weight of recipient per day, more preferably in the range of 0.1 mg to 1,000 mg per kilogram body weight per day, and still more preferably in the range of 1 to 600 mg per kilogram of body weight per day.
  • a preferred effective dose of one or more therapeutic compounds can be readily determined based on known factors such as efficacy of the particular therapeutic agent used, age, weight and gender of the patient, and the like. See dosage guidelines as set forth e.g. in
  • the desired dose is suitably administered once or several more sub-doses administered at appropriate intervals throughout the day, or other appropriate schedule.
  • These sub-doses may be administered as unit dosage forms, for example, containing 100 to 4,000 mg, preferably 100 to 2,000 mg.
  • a compound e.g., of the formulae herein
  • a compound is used in accordance with the present invention in an isolated form distinct as it may be naturally found and in a comparatively pure form, e.g., at least 85% by weight pure, more preferably at least 95% pure.
  • the administered compound of the formulae herein be at least 98% or even greater than 99% pure.
  • Such a material would be considered sterile for pharmaceutical purposes.
  • Potential contaminants include side products that may result upon synthesis of a compound of the invention or materials that may be otherwise associated with the compound prior to its isolation and purification.
  • the present compounds should preferably be sterile and pyrogen free. Purification techniques known in the art may be employed, for example chromatography.
  • Administration of the compounds of the invention may be by any suitable route including oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal) with oral or parenteral being preferred. It will be appreciated that the preferred route may vary with, for example, the condition and age of the recipient.
  • the administered ingredients may be used in therapy in conjunction with other medicaments such as reverse transcriptase inhibitors such as dideoxynucleosides, e.g. zidovudine (AZT), 2',3 t -dideoxyinosine (ddl) and 2',3'-dideoxycytidine (ddC), lamivudine (3TC), stavudine (d4T), and TRIZrVTR (abacavir + zidovudine + lamivudine), nonnucleosides, e.g., efavirenz (DMP-266, DuPont Pharmaceuticals/Bristol Myers Squibb), nevirapine (Boehringer Ingleheim), and delaviridine (Pharmacia-Upjohn), TAT antagonists such as Ro 3-3335 and Ro 24- 7429, protease inhibitors, e.g., indinavir (Merck), ritonavir (Abbott), s
  • Egrl activators can be combined with the use of other lytic replication activators, for example, proteasome inhibitors or farnesyl transferase inhibitors, as disclosed in U.S. provisional patent application Nos. 60/587,810 and 60/587,771, respectively, both filed on July 13, 2004 and incorporated herein by reference.
  • one or more compounds of the invention are used in conjunction with one or more therapeutic agents useful for treatment or prevention of HIV, a symptom associated with HIV infection, or other disease or disease symptom such as a secondary infection or unusual tumor such as herpes, cytomegalovirus, Kaposi's sarcoma and Epstein-Barr virus-related lymphomas among others, that can result in HIV immuno-compromised subjects.
  • a symptom associated with HIV infection or other disease or disease symptom
  • a secondary infection or unusual tumor such as herpes, cytomegalovirus, Kaposi's sarcoma and Epstein-Barr virus-related lymphomas among others, that can result in HIV immuno-compromised subjects.
  • one or more compounds of the formulae herein are used in conjunction with a standard HIV antiviral treatment regimens (e.g., HAART).
  • a standard HIV antiviral treatment regimens e.g., HAART.
  • This combination is advantageous in that the compound(s) of the formulae herein can activate latent HIV infected cells to replicate by stimulating lytic viral replication, thus making them susceptible to the co ⁇ administered standard HIV antiviral treatment regimens.
  • the latent or secondary reservoirs of HTV-infected cells are depleted through "controlled” activation (rather then serendipitous or uncontrolled activation), resulting in more complete elimination of virus, while controlling the spread of viral infection..
  • the treatment methods herein include administration of a so-called HIV-drug "cocktail" or combination therapy, wherein a combination of reverse transcriptase inhibitor(s) and HIV protease inhibitor(s) is co-administered.
  • an Egrl activator is not combined with a nucleoside analog (such as ddl, zalcitabine (ddC), and zidovudine (AZT)).
  • a combination therapy according to the invention includes administration of an Egrl activator together with an abl kinase inhibitor such as imatinib (use of imatinib for HIV treatment is described more fully in co-pending U.S. Provisional Patent Application No. 60/588,015, filed June 13, 2004).
  • the methods involve modulation of any gene that exhibits altered expression in chronically HPV-infected cells compared to uninfected parental cells, prior to induction into lytic replication.
  • the methods herein can involve, or target, any of the genes listed in the tables herein. This modulation can be direct or indirect, that is, it can be by direct control of expression or binding activity of the target, or by indirect control of the expression or binding activity of the target.
  • another aspect is modulation of replication activity of latent HIV-infected cells.
  • the methods involve modulation of lyn, cdc42, MNDA, CEBP alpha or Meisl by administration of the compounds of the formulae herein.
  • Egrl (early growth response 1) is a cell cycle modulator. It has recently been shown that resveratrol, an anti-oxidant stilbene (3, 5, 4'-trihydroxystilbene), can activate Egrl, thereby modulating p21cip expression and thus exert an effect on cell cycle regulation. Egrl expression also causes growth arrest (Gl-S, G2-M phases)
  • Egi 1 was down regulated during viral latency in ACH-2 cells.
  • Resveratrol an Egrl activator, caused viral reactivation.
  • Increased levels of Egrl leads to growth arrest (Gl/S, G2M) (52), similar to that observed during viral replication.
  • Down regulation of Egrl may lead to decreased Egrl levels thus preventing growth arrest and creating conditions unfavorable for viral replication.
  • the effect observed with resveratrol may be a combination of manipulating the latent cellular environment and mimicking the effects of active HIV replication on the cell cycle, thus providing favorable conditions for viral replication.
  • Resveratrol has been shown to not affect the levels of NFkappa-B in other cell lines studied (52), suggesting that mechanisms other than NFkappa-B modulation may contribute to viral latency and reactivation.
  • the present invention includes use of both racemic mixtures and optically active stereoisomers of compounds of the formulae herein.
  • compositions of compounds of the invention used in combination with other compounds (e.g., reverse transcriptase inhibitors, protease inhibitors, and the like) may be employed alone or in combination with acceptable carriers such as those described below.
  • a suitable effective dose of a compound in such a composition will be in the range of 1 to 5,000 mg per kilogram body weight of recipient per day, preferably in the range of 10 to 4,000 mg per kilogram body weight of recipient per day. When multiple compounds having complementary activity are administered it is expected one can use the lower portion of these ranges (or even less).
  • the pharmaceutically acceptable salts of compounds for use in the methods of the invention can be synthesized from the corresponding compound of this invention which contain a basic moiety by conventional chemical methods.
  • the salts are prepared by reacting the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents.
  • One or more compounds of the formulae herein may be administered alone, or as part of a pharmaceutical composition, comprising at least one Egrl activator (e.g., a compound of the formulae herein) together with one or more acceptable carriers thereof and optionally other therapeutic ingredients, including those therapeutic agents discussed supra.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • stable refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., formulation into therapeutic products, intermediates for use in production of therapeutic compounds, isolatable or storable intermediate compounds).
  • the compounds delineated herein are commercially available or readily synthesized by one of ordinary skill in the art using methodology know in the art.
  • Some of the compounds of this invention have one or more double bonds, or one or more asymmetric centers. Such compounds can occur as racemates, racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans- or E- or Z- double isomeric forms. All such isomeric forms of these compounds are expressly included in the present invention.
  • the compounds of this invention may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein (e.g., alkylation of a ring system may result in alkylation at multiple sites, the invention expressly includes all such reaction products). All such isomeric forms of such compounds are expressly included in the present invention. All crystal forms of the compounds described herein are expressly included in the present invention.
  • compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and in liposomes, and may be prepared by any methods well known in the art of pharmacy.
  • Such methods include the step of bringing into association the active ingredients to be administered and the carrier which constitutes one or more accessory ingredients.
  • the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers or both, and then if necessary shaping the product.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, or packed in liposomes and as a bolus, etc.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.
  • compositions suitable for topical administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the ingredient to be administered in a suitable liquid carrier.
  • compositions suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising one or more compounds of the present invention and a pharmaceutically acceptable carrier.
  • a suitable topical delivery system is a transdermal patch containing the ingredient to be administered.
  • compositions suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
  • compositions suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size, for example, in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Suitable formulations wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.
  • compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • Compositions suitable for parenteral administration include aqueous and non ⁇ aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • sterile liquid carrier for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include flavoring agents.
  • ACH-2, A3.01, Jl.1, and Ul cells were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.
  • U-937 cells were obtained from American Type Culture Collection (Manassas, VA).
  • ACH-2, Jl.1 and Ul are chronically infected cell lines harboring HIV-I LAV strain, while A3.01, Jurkat, and U-937 are the corresponding parental uninfected cell lines.
  • Cells were grown in RPMI-1640 (Invitrogen, San Diego, CA) with 10% fetal bovine serum (FBS, Invitrogen), 5% penicillin-streptomycin (Invitrogen), and 2mM glutamine (Invitrogen). Cells were maintained at a concentration of 1x10 6 cells/ml in T-175 flasks. Cell concentrations and cell viability were monitored throughout the experiment at all time points studied. Cells were induced by addition of 20 ng/mL of phorbol myristyl acetate (PMA or TPA, Sigma, St Louis, MO) for one hour, after which the cells were washed with phosphate buffered saline (PBS).
  • PMA or TPA phorbol myristyl acetate
  • ACH-2 and A3.01 were washed twice with ice-cold PBS and suspended in 50 ⁇ L ice-cold permeabilization buffer (BD Biosciences, San Jose, CA), and incubated at 4 0 C in the dark for 30 minutes. The cells were fixed using the CytoFix/CytoPerm kit (BD Biosciences) and 5 ⁇ L KC57-FITC-labeled p24 antibody (Beckman Coulter), was added to detect intracellular p24.
  • RNA from the samples was subjected to DNase treatment to remove contaminating DNA, and the DNAse was inactivated using the DNase Free kit (Amersham Biosciences, Piscataway, NJ) according to manufacturer's protocols. 2 ⁇ g of RNA was reverse transcribed using the Taqman RT kit from ABI per manufacturer's specifications.
  • reaction mixture 50 ⁇ l was incubated at 65 0 C for 5 minutes followed by 37 0 C for 45 minutes, 94 0 C for 5 minutes, and then cooled on ice. 1/40 th aliquots of the corresponding samples were then used in a real- time PCR reaction using Taqman probes labeled with FAM and TAMRA at the 5' and 3' ends respectively. Primer probe pairs were designed using PrimerExpress (ABI). The reactions were carried out in triplicate for each time point and the fold changes observed were normalized to GAPDH, for each time point.
  • sequence detection primers for unspliced or late RNA were SK38 and SK39 from the HIV-I Gene Amplimer kit (ABI).
  • a TAMRA labeled probe with sequence identical to SK 19 (ABI) was used for Real time PCR quantitation of the late viral RNA species.
  • Standards from the kit were diluted to calculate copy number of virus based on gag mRNA concentrations.
  • Real time RT-PCR analysis was also carried out for selected cellular genes using gene specific primer probe pairs and Taqman detection primers.
  • RNA obtained from induced chronically infected and corresponding uninfected parental cells were used for microarray experiments. For each time point, RNA from the induced chronically infected ACH-2 cells and RNA from the corresponding induced, uninfected A3.01 cells were compared to minimize effects due to PMA induction.
  • Microarray s were obtained from the National Cancer Institute Microarray Facility, Advanced Technology Center (Gaithersburg, MD). The microarrays (Hs. UniGem2) contained 10,395 cDNA spots on each glass slide.
  • cDNAs were selected for spotting on the slides based on their known or probable involvement in oncogenesis, signal transduction, apoptosis, immune function, inflammatory pathways, cellular transport, transcription, protein translation and other important cellular functions.
  • a number of expressed sequence tags (ESTs) from unknown genes homologous to known genes and cDNAs encoding housekeeping genes were also included in these gene sets.
  • ESTs expressed sequence tags
  • 50 ⁇ g of total RNA from PMA induced ACH-2 cells and 70 ⁇ g of total RNA from PMA induced A3.01 cells was labeled with Cy-3-dUTP and Cy-5-dUTP respectively as previously described (34, 60). Higher amounts of RNA were used for Cy-5 labeling to minimize the disparities in dye incorporation.
  • RNA from PMA- induced, infected cells from a particular time point was compared with RNA from the corresponding PMA-induced, uninfected cells from the same time point for subsequent hybridization to the same array to ensure accurate comparisons and to eliminate inter-array variability.
  • the labeled cDNAs were then combined and purified using MicroCon YM-30 (Millipore, Bedford, MA) spin column Filters, to remove any unincorporated nucleotides. 8-10 ⁇ g each of Cot-1 DNA, (Boehringer Mannheim, Indianapolis, IN), yeast tRNA (Sigma) and polyA (Amersham Biosciences) were added to the reaction mixture and heated at 100 0 C for 1 minute.
  • Hybridization of the labeled cDNA to the microarray was carried out at 65 0 C overnight, followed by washes with IX SSC, 0.2X SSC and 0.05X SSC respectively. The slides were dried by centrifugation at 1000 rpm for 3 minutes and then scanned as described below. RNA samples from three identical but independently conducted time course experiments were tested. Microarray experiments were performed at least twice for each time point (technical replicates) of each experiment. We also compared AZT-treated ACH-2 cells to untreated ACH-2 cells to determine whether , any differences in gene expression might be solely due to AZT.
  • RNA from the same samples labeled with Cy5 (70 ⁇ g RNA) and Cy3 (50 ⁇ g RNA) were co-hybridized to the same array, scanned, and data were analyzed for all the cell lines studied, using identical filtering and statistical tests, and genes showing dye incorporation bias were eliminated from further analysis as described below.
  • Microarray Scanning and Data Analysis The slides were scanned using an Axon GenePix 4000 scanner (Axon Instruments, Union City, CA). The photomultiplier tube values (PMT) were adjusted to obtain equivalent intensities at both wavelengths used, 635 nm and 532 nm for the Cy5 and Cy3 channels respectively. Image analysis was performed using GenePix analysis software (Axon Instruments) and data analysis was performed using the microArray Database (mAdb) system hosted by the Center for Information Technology and Center for Cancer Research at NIH (http://nciarray.nci.nih.gov). Each array was normalized using Lowess normalization (71).
  • Filtering criteria were as follows: a) For each spot, signal intensity must be at least twice that of the background intensity; b) Each gene must have values in at least 70% of the arrays; c). Each array must have values for at least 70% of the gene spots. Genes that showed dye labeling bias in a particular cell line after normalization were excluded from that gene set prior to further analysis. This was determined using a one sample t-test on mean log ratios for replicate arrays with the same sample labeled with both Cy3 and Cy5.
  • Hierarchical clustering analyses on the resulting data sets were done using the mAdb system as well as Cluster and TreeView software programs (Stanford University, CA). Since statistically significant gene classes need not necessarily indicate the biological relevance of genes in a particular class, pathway analysis of the various genes that showed significant differential expression was performed by utilizing analysis tools provided by the NIH mAdb database (http://nciarray.nci.nih.gov) and querying the database of the Cancer Genome Anatomy Project (CGAP), (http://cgap.nci.nih.gov ⁇ with pathway information provided by KEGG (www, genome.ad.i p/kegg ⁇ and Biocarta (www.biocarta.com) pathway databases.
  • CGAP Cancer Genome Anatomy Project
  • AZT (250 nM) was added to the chronically infected cells in order to inhibit p24 production that may be caused due to low levels of actively replicating virus present along with the chronically infected cells and to ensure that any increases in p24 expression would be attributable to activation of latent provirus and not due to subsequent amplification via additional rounds of viral replication.
  • Cells were incubated with different concentrations of either CLBL, resveratrol, or trichostatin at 37 0 C. 200 ⁇ L samples of cell supernatant were collected at 24 hours after treatment.
  • TNF-8& 0.5 ⁇ g/mL tumor necrosis factor alpha
  • the plates were washed, and rabbit anti-HIV p24 antibody was added at 1:400 dilution. Following incubation for one hour, the plates were washed and goat anti-rabbit IgG peroxidase labeled antibody at 1:300 dilution was added. The plates were incubated for one hour at 37 0 C, followed by washing and addition of a two-component substrate.
  • Substrate solution consisted of equal volumes of TMB peroxidase substrate and peroxidase solution B (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Samples were incubated for 30 minutes at room temperature and reactions were stopped by addition of IN hydrochloric acid solution. The absorbance was measured at 450 nm using a SpectraMax250 spectrophotometer (Molecular Devices Corporation, Sunnyvale, CA). The samples were assayed in duplicate and experiments were performed at least thrice using independent cell samples.
  • the late time period (48-96 hours p.i) showed the least change with 566 genes exhibiting significant altered expression (p ⁇ 0.001).
  • Many of the genes that were differentially expressed in the early time period also showed either similar or the opposite trend in their expression patterns during the other time periods, hence some genes were included in the analysis of both the time periods.
  • a number of discrete patterns of gene regulation were observed.
  • Several cellular genes showed distinct temporal expression patterns during the lytic replication cycle, an expected finding, but more interestingly, a smaller number of genes appeared to be differentially expressed in the latently infected ACH-2 cells compared to their parental, uninfected cells, even before induction of the lytic cycle.
  • genes encoding transcription factors, components of proteasomes, factors that control immune function, apoptosis and other functional classes were included.
  • gene classes that were annotated in the gene ontology database GO database, www.geneontology.org
  • observed/expected ratio for the number of genes within a functional class that were differentially expressed was set at greater than one (O/E > 1), so as to eliminate functional classes where the number of genes differentially expressed was not greater than that randomly expected.
  • O/E > 1 the gene ontology database
  • Table 1 An abbreviated listing of the genes grouped according to known functions that were differentially expressed before induction is given in Table 1.
  • Table 1 Functionally related genes that were differentially expressed prior to induction in chronically infected ACH-2 cells.
  • Table 1 Functionally related genes that were differentially expressed prior to induction in chronically infected ACH-2 cells. List of selected classes of genes based on known function that are differentially expressed in latently infected ACH-2 cells, relative to uninfected parental cell line, A3.01. A number of genes involved in similar cellular functions previously not associated with presence of proviral HIV were altered coordinately even during the latent non-replicative stage. EXAMPLE 6
  • PSMB4 has peptidase activity, which is inhibited by Tat during viral replication.
  • Tat competes with the 11 S regulatory subunit, for binding to the 2OS core complex due to presence of a common binding site in Tat and the 1 IS regulator alpha subunit (32, 59).
  • Proteasomes are also involved in processing certain regions of HIV-I Nef preferentially, which leads to production of Nef-specific CTLs (cytotoxic T-lymphocytes) (44).
  • Many other classes of genes encoding immune response modulators, integrins, cell cycle modulators (such as Egrl), nuclear import factors, and G-protein signaling molecules were also differentially expressed.
  • a listing of genes that were differentially expressed prior to induction, based on their functional classification is given (Table 1).
  • a list of pathways that were affected in the uninduced, chronically infected cells is given (supplemental data, Table S2).
  • the proteins encoded by these genes are known to be critical in the progress of certain leukemias (18, 54, 65), but have not been hitherto related to HIV latency. Certain genes show similar differential expression in ACH-2 and J 1.1 but not in Ul cells. Also, some genes show opposite trends in the cell lines tested. For example, proteasome subunits are up regulated in ACH-2, while they are down regulated in Ul. A list of common pathways affected and some pathways that change selectively is given (supplemental data, Table S4). The list of differentially expressed genes common to all three cell lines is given along with their expression ratios (supplemental data, Table S5).
  • HDACl histone deacetylases
  • HDAC2 histone deacetylases
  • proteasome class of genes showed increased expression in ACH-2 cells even prior to induction, but not so in J 1.1 and Ul cell lines. Hence, we sought to determine whether inhibition of proteasomes would induce latent provirus into lytic replication.
  • a proteasome inhibitor clastolactacystin-beta-lactone (19)
  • Egr 1 activators are capable of increasing the efficiency of an acute HIV infection (58).
  • PS-341 a highly specific Egr 1 activator that binds to the Beta-5 subunit of the proteasome and is approved for clinical treatment of multiple myeloma (1, 3).
  • PS-341 due to the extreme cytotoxicity of PS-341 even at low concentration (100 nM) we were unable to observe any changes in p24 expression under our experimental conditions (1-1000 nM) in latently infected cells (data not shown).
  • Egrl (early growth response 1) was down regulated in ACH-2 cells prior to induction and more importantly, up regulated during lytic replication. Egrl is involved in cell cycle regulation and cell differentiation, in response to a number of different growth factors (56, 64). Egrl activity is suppressed in many cancers including breast cancer and brain tumors, indicating that its activity is essential in cell cycle regulation (42),(38). It has recently been shown that resveratrol, an anti-oxidant stilbene (3, 5, 4'-trihydroxystilbene), can activate Egrl, thereby modulating p21cip expression and thus exert an effect on cell cycle regulation. Egrl expression also causes growth arrest (Gl-S, G2-M phases) (52).
  • Table Sl List of sequence detection primers and probe pairs for real time RT- PCR quantitation of selected genes.
  • Table Sl List of sequence detection primers and probe pairs for real time RT- PCR quantitation of selected genes.
  • PrimerExpress software from Applied Biosystems (ABI).
  • Table S2 Pathways that changed significantly prior and post induction of ACH-2 cells.
  • Table S3 Verification of differentially expressed gene expression levels by real time RT-PCR quantitation.
  • Table S3 Verification of differentially expressed gene expression levels by real time RT-PCR quantitation.
  • RT-PCR data for the selected genes was normalized to data for GAPDH for each cell line, before assessing fold change in ACH-2 cells with respect to A3.01 cells. Differential expression of the selected genes was confirmed by RT-PCR quantitation.
  • Table S4 Pathways that change significantly in three chronically infected cell lines.
  • Table S4 Pathways that changed significantly in three chronically infected cell lines.
  • Table S5 Genes that showed similar differential expression in all three chronically infected cell lines.
  • Table S5 Genes that showed similar differential expression in all three chronically infected cell lines.
  • Enriched CD4+ T-cells were obtained from patients (see, e.g., Chun TW, Finzi D. Margolick J, Chadwick K. Schwartz D. Siliciano RF. In vivo fate of HIV-I - infected T cells: quantitative analysis of the transition to stable latency .Nat Med. 1995 Dec;l (12): 1284-90) who had been treated with highly active antiretroviral threrapy (HAART) for some time and had viral loads below the limits of detection, but presumably had reservoirs of cells latently infected with HIV.
  • HAART highly active antiretroviral threrapy
  • resveratrol caused reactivation of latent provirus from latently infected cell lines as well as from aviremic patient samples.
  • MNDA human myeloid cell nuclear differentiation antigen
  • Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone. Proc Natl Acad Sci U S A. 86:2365-8.
  • HIV-I human immunodeficiency virus type 1
  • T lymphocyte epitopes of HIV-I Nef Generation of multiple definitive major histocompatibility complex class I ligands by proteasomes. J Exp Med 191:239-52.
  • ERK MAP kinase links cytokine signals to activation of latent HIV-I infection by stimulating a cooperative interaction of AP-I and NF-kappaB. J Biol Chem 274:27981-8.

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Abstract

L'invention concerne un traitement de cellules ou d'humains porteurs d'un virus ou infectés par un virus pouvant provoquer une maladie immunodéficiente. Le traitement de l'invention fait appel à des composés particuliers, notamment des activateurs Egr 1.
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WO2023069981A1 (fr) * 2021-10-19 2023-04-27 Phyto Tech Corp. Utilisations de la taxifoline pour la santé respiratoire
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WO2023235826A3 (fr) * 2022-06-02 2024-02-01 The Regents Of The University Ofcolorado A Body Corporate Procédés de modulation d'endonucléase g à l'aide de resvératrol et de ses dérivés

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