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WO2006017353A2 - Traitement d'infections virales - Google Patents

Traitement d'infections virales Download PDF

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Publication number
WO2006017353A2
WO2006017353A2 PCT/US2005/024922 US2005024922W WO2006017353A2 WO 2006017353 A2 WO2006017353 A2 WO 2006017353A2 US 2005024922 W US2005024922 W US 2005024922W WO 2006017353 A2 WO2006017353 A2 WO 2006017353A2
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WIPO (PCT)
Prior art keywords
substituted
lower alkyl
amino
radical
hydrogen
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PCT/US2005/024922
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English (en)
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WO2006017353A3 (fr
Inventor
Steven Zeichner
Vyjayanthi Krishnan
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GOVERNMENT OF THE UNITED STATES, as represented byTHE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES
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Publication of WO2006017353A2 publication Critical patent/WO2006017353A2/fr
Publication of WO2006017353A3 publication Critical patent/WO2006017353A3/fr

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Classifications

    • 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
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • 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
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K31/655—Azo (—N=N—), diazo (=N2), azoxy (>N—O—N< or N(=O)—N<), azido (—N3) or diazoamino (—N=N—N<) compounds
    • 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

  • HTV-I human immunodeficiency virus type 1
  • HAV-JJJ/LAV human immunodeficiency virus type 2
  • ADDS acquired immune deficiency syndrome
  • This disease is characterized by a long asymptomatic period followed by the progressive degeneration of the immune system and the central nervous system.
  • Studies of the virus indicate that replication is highly regulated, and both latent and lytic infection of the CD4 positive helper subset of T-lymphocytes occur in tissue culture. Zagury, et al., Science, 211:850-853 (1986).
  • the expression of the virus in infected patients also appears to be regulated as the titer of infectious virus remains low throughout the course of the disease.
  • Both HIV-I and 2 share a similar structural and function genomic organization, having regulatory genes such as ial, rey, nef, in addition to structural genes such as env, gag and p ⁇ l.
  • 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 HIV are believed to be persistently infective to others.
  • PDL persistent generalized lymphadenopathy syndrome
  • ARC AIDS related complex
  • Ri is 4-pyrazinyl, 1 -methyl- lH-pyrrolyl, amino- or amino-lower alkyl-substituted phenyl wherein the amino group in each case is free, alkylated or acylated, lH-indolyl or lH-imidazolyl bonded at a f ⁇ ve-membered ring carbon atom, or unsubstituted or lower alkyl-substituted pyridyl bonded at a ring carbon atom and unsubstituted or substituted at the nitrogen atom by oxygen,
  • R 2 and R 3 are each independently of the other hydrogen or lower alkyl ⁇ one or two of the radicals R 4 , R 5 , R 6 , R7 and Rs are each nitro, fluoro-substituted lower alkoxy or a radical of formula II
  • R 9 is hydrogen or lower alkyl
  • X is oxo, thio, imino, N-lower alkyl-imino, hydroximino or O-lower alkyl- hydroximino,
  • Y is oxygen or the group NH, n is 0 or 1 and
  • Ri 0 is (a) an aliphatic radical having at least 5 carbon atoms, or an aromatic, aromatic-aliphatic, cycloaliphatic, cycloaliphatic-aliphatic, heterocyclic or hetero-cyclicaliphatic radical,
  • phenyl which is substituted by an unsubstituted or substituted radical selected from the group consisting of benzylamino; benzoylamino; pyrrolidinyl; piperidyl; piperazinyl; piperazinyl-carbonyl; morpholinyl; and lower alkyl substituted by benzylamino, benzoylamino, pyrrolidinyl, piperidyl, piperazinyl or morpholinyl, the substituents of said substituted radical being selected from the group consisting of cyano; lower alkyl; hydroxy- or amino- substituted lower alkyl; trifluoromethyl; hydroxy; lower alkoxy; lower alkanoyloxy; amino; mono- or di-lower alkylamino; lower alkanoylamino; benzoylamino; carboxy; lower alkoxycarbonyl and halogen, and
  • phenyl which is optionally further substituted by one or more radicals selected from the group consisting of cyano; lower alkyl; hydroxy- or ammo- substituted lower alkyl; trifluoromethyl; hydroxy; lower alkoxy; lower alkanoyloxy; amino; mono- or di-lower alkylamino; lower alkanoylamino; benzoylamino; carboxy; lower alkoxycarbonyl and halogen,
  • R 4 , R 5 , Re, R7 and R 8 are each independently of the others hydrogen, lower alkyl that is unsubstituted or substituted by free or alkylated amino, piperazinyl, piperidinyl, pyrrolidinyl or by morpholinyl, or lower alkanoyl, trifluoromethyl, free, etherified or esterifed hydroxy, free, alkylated or acylated amino or free or esterif ⁇ ed carboxy,
  • R 7 and R 8 are each nitro or a radical of formula ⁇ , wherein
  • R9 is hydrogen or lower alkyl
  • X is oxo, thio, imino, N-lower alkyl-imino, hydroximino or O-lower alkyl- hydroximino,
  • Y is oxygen or the group NH
  • n O or l
  • Ri is 4-pyrazinyl, 1 -methyl- lH-pyrrolyl, amino- or amino-lower alkyl-substituted phenyl wherein the amino group in each case is free, alkylated by one or two lower alkyl radicals or acylated by lower alkanoyl or by benzoyl, lH-indolyl or IH- imidazolyl bonded at a five-membered ring carbon atom, or unsubstituted or lower alkyl-substituted pyridyl bonded at a ring carbon atom and unsubstituted or substituted at the nitrogen atom by oxygen,
  • R 2 and R 3 are each independently of the other hydrogen or lower alkyl, one or two of the radicals R 4 , Rs, R 6 , R7 and R 8 are each nitro, fluoro-substituted lower alkoxy or a radical of formula II wherein
  • Rg is hydrogen or lower alkyl
  • X is oxo, thio, imino, N-lower alkyl-imino, hydroximino or O-lower alkyl- hydroximino,
  • Y is oxygen or the group NH
  • n 0 or 1
  • Rio is an aliphatic hydrocarbon radical having 5-22 carbon atoms, a phenyl or naphthyl radical each of which is unsubstituted or substituted by cyano, lower alkyl, hydroxy-lower alkyl, amino-lower alkyl, (4-methyl-piperazinyl)-lower alkyl, trifluoromethyl, hydroxy, lower alkoxy, lower alkanoyloxy, halogen, amino, lower alkylarhino, di-lower alkylamino, lower alkanoylamino, benzoylamino, carboxy or by lower alkoxycarbonyl, or phenyl-lower alkyl wherein the phenyl radical is unsubstituted or substituted as indicated above, a cycloalkyl or cycloalkenyl radical having up to 30 carbon atoms, cycloalkyl-lower alkyl or cycloalkenyl-lower alkyl each having up to 30 carbon
  • R 4 , Rs, R ⁇ , R? and Rs are each independently of the others hydrogen, lower alkyl that is unsubstituted or substituted by amino, lower alkylamino, di-lower alkylamino, piperazinyl, piperidinyl, pyrrolidinyl or by morpholinyl, or lower alkanoyl, trifluoromethyl, hydroxy, lower alkoxy, lower alkanoyloxy, halogen, amino, lower alkylamino, di-lower alkylamino, lower alkanoylamino, benzoylamino, carboxy or lower alkoxycarbonyl,
  • Ri is pyridyl bonded at a ring carbon atom and unsubstituted or substituted at the nitrogen atom by oxygen,
  • R 2 and R 3 are each hydrogen
  • R 4 is hydrogen or lower alkyl
  • R5 is hydrogen, lower alkyl or fluoro-substituted lower alkoxy
  • R 6 is hydrogen
  • R 7 is nitro, fluoro-substituted lower alkoxy or a radical of formula ⁇ wherein
  • R9 is hydrogen
  • n O and
  • Rio is an aliphatic hydrocarbon radical having 5-22 carbon atoms, a phenyl radical that is unsubstituted or substituted by cyano, lower alkyl, (4-methyl-piperazinyl)-lower alkyl, lower alkoxy, halogen or by carboxy; a cycloalkyl radical having up to 30 carbon atoms or a monocyclic radical having 5 or 6 ring members and 1-3 sulfur ring atoms, and
  • R 8 is hydrogen
  • Ri is pyridyl or N-oxido-pyridyl each of which is bonded at a carbon atom
  • R 2 and R 3 are each hydrogen
  • R 4 is hydrogen or lower alkyl
  • Rs is hydrogen, lower alkyl or trifluoromethyl
  • R 7 is nitro, fluoro-substituted lower alkoxy or a radical of formula II wherein
  • R9 is. hydrogen
  • n is the number 0
  • Rio is pyridyl bonded at a carbon atom, phenyl that is unsubstituted or substituted by halogen, cyano, lower alkoxy, carboxy, lower alkyl or by 4-methyl-piperazinylmethyl, or C5 -C 7 alkyl, thienyl, 2-naphthyl or cyclohexyl, and
  • R 8 is hydrogen
  • Ri is unsubstituted or lower alkyl-substituted pyridyl bonded at a ring carbon atom and unsubstituted or substituted at the nitrogen atom by oxygen
  • R 2 and R 3 are each independently of the other hydrogen or lower alkyl
  • one of the radicals R 4 , R5, R 6 , R 7 and R 8 is a radical of formula II, wherein R9 is hydrogen or lower alkyl
  • X is oxo, thio, imino, N-lower alkyl-imino, hydroximino or O-lower alkyl- hydroximino,
  • n 0
  • R 4 , Rs, R 6 , R7 and R 8 are each independently of the others hydrogen; lower alkyl that is unsubstituted or substituted by free or alkylated amino, piperazinyl, piperidyl, pyrrolidinyl or morpholinyl; lower alkanoyl; trifluoromethyl; free, etherified or esterifed hydroxy; free, alkylated or acylated amino; or free or esterified carboxy,
  • Ri is unsubstituted or lower alkyl-substituted pyridyl bonded at a ring carbon atom and unsubstituted or substituted at the nitrogen atom by oxygen,
  • R 2 and R 3 are each independently of the other hydrogen or lower alkyl, R5 or R 7 ia a radical of formula II, wherein
  • R9 is hydrogen
  • n 0
  • piperazinyl-lower alkyl which is optionally substituted by one or more radicals selected from the group consisting of cyano; C 3 -C 7 -lower alkyl; hydroxy- or amino-substituted lower alkyl; trifluoromethyl; hydroxy; lower alkoxy; lower alkanoyloxy; amino; mono- or di-lower alkylamino; lower alkanoylamino; benzoylamino; carboxy; lower alkoxycarbonyl and halogen, and
  • R 4 , R 5 , Re, R7 and- R 8 are each independently of the others hydrogen; lower alkyl that is unsubstituted or substituted by free or alkylated amino, piperazinyl, piperidyl, pyrrolidinyl or morpholinyl; lower alkanoyl; trifluoromethyl; free, etherif ⁇ ed or esterifed hydroxy; free, alkylated or acylated amino; or free or esterified carboxy,
  • Ri is unsubstituted or lower alkyl-substituted pyridyl bonded at a ring carbon atom and unsubstituted or substituted at the nitrogen atom by oxygen,
  • R 2 and R 3 are both hydrogen, R 4 is lower alkyl,
  • R 5 and R 6 are both hydrogen
  • R 7 is a radical of formula ⁇ , wherein
  • R9 is hydrogen, X is oxo, n is 0 and
  • R 8 is hydrogen, or a salt of such a compound having at least one salt-forming group.
  • Ri 0 is phenyl which is substituted by piperazinyl-lower alkyl which is optionally substituted in the piperazine ring by one or more radicals selected from the group consisting of cyano; C 3 -C 7 -lower alkyl; hydroxy- or amino-substituted lower alkyl; trifluoromethyl; hydroxy; lower alkoxy; lower alkanoyloxy; amino; mono- or di-lower alkylamino; lower alkanoylamino; benzoylamino; carboxy; lower alkoxycarbonyl and halogen, and
  • compounds of the formulae herein are used for purposes of inhibiting viral replication of immunodeficiency viruses, preferably human immunodeficiency viruses such as HIV:
  • the invention provides a method of modulating lytic replication in an HlV-infected cell in a subject comprising administration to the subject of an effective amount of a direct or indirect NF-kB modulator.
  • a method of reducing HIV viral load in an HIV-infected subject comprises administration to the subject of an effective amount of a direct or indirect modulator ofNF-kB.
  • the invention provides a method of treating or preventing
  • the abl kinase inhibitor is a compound according to formula I or m as defined herein.
  • the invention provides a method of inhibiting HIV replication in a cell comprising administration to the cell of an effective amount of an abl kinase inhibitor.
  • the invention provides a method of modulating lytic replication in an HlV-infected cell in a subject comprising administration to the subject of an effective amount of an abl kinase inhibitor.
  • the invention provides a method of modulating lytic replication in an HlV-infected cell in a subject comprising administration to the subject of an effective amount of a direct or indirect NF-kB modulator.
  • the cell is a lymphocytic cell. In other preferred embodiments, the cell is a monocytic cell.
  • the invention provides a method of reducing HIV viral load in an HlV-infected subject comprising administration to the subject of an effective amount of an abl kinase inhibitor.
  • the invention provides a method of reducing HIV viral load in an HIV-infected subject comprising administration to the subject of an effective amount of a direct or indirect modulator of NF-kB .
  • the method includes the step of administration of one or more additional anti-HIV therapeutic agents (i.e., to the subject or the cell), hi certain preferred embodiments, the additional agent(s) are a reverse transcriptase inhibitor, a protease inhibitor, or combination thereof.
  • the method includes the step of identifying the subject or cell as being in need of such treatment.
  • the compounds of the present invention can inhibit viral replication in a subject infected with an immunodeficiency virus such as HIV by targeting the abl-kinase, to decrease the amount of NF-kB.
  • NF-kB is a known positive regulator of HTV replication.
  • the compounds of the present invention can treat cells infected acutely and chronically by immunodeficiency viruses, for example, HFV, preferably HIV- 1 , and thus can be used to treat humans infected by HIV.
  • immunodeficiency viruses for example, HFV, preferably HIV- 1
  • HFV immunodeficiency virus
  • HIV- 1 HIV- 1
  • the invention also provides pharmaceutical compositions comprising a compound of formulas I and DI and a suitable carrier therefore for use in the conditions referred to above.
  • aspects of the invention are methods of treating or preventing HTV infection in a subject comprising administration of an effective amount of viral replication inhibitor, including those having the structure of the formulae herein.
  • the compounds of the present invention can treat cells infected acutely and chronically by immunodeficiency viruses, for example, HTV, preferably HTV-I, and thus can be used to treat humans infected by HTV.
  • immunodeficiency viruses for example, HTV, preferably HTV-I
  • HTV immunodeficiency virus
  • HTV-I immunodeficiency virus
  • the compounds of the invention are used to treat lyrically replicating cells or cells that have been activated into lytic replication from latent infection.
  • the methods of the invention include the use of the compounds of the invention either alone or preferably in combination with other anti-retroviral compounds, hi certain embodiments, the immunodeficiency virus (e.g., HTV-I) is a drug-resistant strain, e.g., a saquinavir-resistant virus type).
  • the methods delineated herein can further include administration of one or more additional anti-HF/ therapeutic agents.
  • the additional agent(s) can be a reverse transcriptase inhibitor, a protease inhibitor, lytic replication activators or combinations thereof.
  • the methods delineated herein include administering to a cell or a subject (e.g., a human or an animal) in need thereof an effective amount of one or more compounds as delineated herein.
  • the subject is not suffering from (and/or has not been diagnosed as suffering from) leukemia, e.g., chronic myeloid leukemia.
  • the subject is not suffering from (and/or has not been diagnosed as suffering from) Kaposi's sarcoma.
  • the methods can also include, in certain preferred embodiments, the step of identifying that the subject is in need of treatment of diseases or disorders described herein.
  • 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).
  • 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 HTV infection marker.
  • a marker including a marker or diagnostic measure of HIV infection, HIV replication, viral load, or expression of an HTV infection marker.
  • 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 professional.
  • 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).
  • a farm animal e.g., a horse, a cow, a pig, etc.
  • 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 illustrates the effect of a compound of formula IJJ (i.e., imatinib) on HIV replication induced in three HIV latently infected cell lines, ACH-2 and J 1.1, and a monocytic cell line, Ul.
  • HIV-Latently infected cells ACH-2, Jl .1, or Ul
  • TNF-a Tumor necrosis factor -alpha
  • Imatinib mesylate also called Gleevec, STI-571
  • Cells without TNF- ⁇ treatment were used to determine the basal p24 expression in the latently infected cells.
  • Cells treated with TNF- a alone were assayed to determine the maximal level of HIV p24 expression in the absence of Imatinib mesylate.
  • Figure 2 shows the effect of Imatinib mesylate on viral replication 24 hours following an acute HIV infection of A3.01 and Jurkat cells.
  • Cells infected with HIV-I were used as positive control to determine maximal HTV-p24 expression in the absence of Imatinib mesylate.
  • a dose dependent decrease in p24 expression was observed in cells treated with Imatinib mesylate, indicating inhibition of HIV replication in the drug treated cells.
  • the effect of imatinib mesylate in inhibiting HIV replication was determined by calculating the amount of p24 expression in imatinib treated HIV-infected cells and expressing it as a percentage of p24 expression caused by HIV infection alone.
  • Imatinib For example, a 5 ⁇ M Imatinib treatment of infected Jurkat cells produced 32% p24 levels as compared to infected Jurkat cells alone (100%), indicating a 68% inhibition.
  • the data showed that Imatinib mesylate caused a dose-dependent decrease in p24 expression in HIV infected cells, indicating that Imatinib was effective in inhibiting HIV replication following an acute infection.
  • Figure 3 shown 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 4 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 HTV-I) was observed 8 hours post induction. Fold change for late transcripts (US HIV-I) showed maximal increase 18 hours post induction.
  • Figure 5 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 in on the color scale >1 showed up regulation, those in 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 6 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 7 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.
  • (A) shows genes that are up regulated in all three cell lines; (B) shows genes that are down regulated in all three cell lines; (C) indicates the genes that are up regulated in ACH-2 and Jl .1 and down regulated in Ul ; (D) indicates genes which show no significant similarity in their expression in the three cell lines.
  • Figure 8 shows the effect of imatinib and saquinavir on HIV-infected Jurkat cells.
  • Figure 9 shows the effect of imatinib and saquinavir on HTV-infected MT-2 cells.
  • Figure 10 illustrates the effect of imatinib and a combination of imatinib and saquinavir on saquinavir-resistant HIV infection in MT-2 cells.
  • Figure 11 shows the effect of imatinib on four PBMC samples acutely infected with HF/-NL4-3.
  • Figure 12 shows the effect of a combination of imatinib and AZT on HTV infection in Jurkat cells.
  • DF.TATT .FD DF.SCK TPTTON OF THF. TNVFNTTON We have discovered that compounds of the following formula I can be used to treat cells infected by an immunodeficiency virus, preferably human cells infected with HTV and thus can be used for treatment in HTV infected individuals.
  • compounds of formula I or other compounds which target, decrease or inhibit the activity of c-Abl family members and gene products and their gene fusion products are, e.g. a N-phenyl-2-pyrimidine-amine derivative, e.g.
  • PD180970 a pyrido[2,3-d]pyrimidme inhibitor of Bcr- AbI kinase
  • AG957 (4-(N-2,5-dihydroxybenzyl)amino methyl benzoate); or ⁇ SC 680410 (adaphostin).
  • substituted refers to one or more substituents (which may be the same or different), each replacing a hydrogen atom.
  • substituents include, but are not limited to, halogen (F, Cl, Br, or T), hydroxyl, amino, alkylamino, arylamino, dialkylamino, diarylamino, cyano, nitro, mercapto, oxo (i.e., carbonyl), thio, imino, formyl, carbamido, carbamyl, carboxyl, ester, N-alkyl-substituted amido, alkoxycarbonyl, alkylcarbonyl, alkyl, alkenyl, or alkyloxy.
  • 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”).
  • a direct or indirect modulator of NF-kB refers to any compound that is capable of affecting the amount or activity of NF-kB, for example, an abl-kinase inhibitor.
  • the compound or composition may be effective through direct interaction with NF-kB or may, in combination or synergy with another compound, directly or indirectly interact with NF-kB.
  • the composition may also interact with another composition that in turn modulates NF-kB activity.
  • the use of a compound such as imatinib (or related compounds) according to the invention poses little health risk and has at most minor side effects.
  • 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'-dideoxyinosine (ddl) and 2',3'-dideoxycytidine (ddC), lamivudine (3TC), stavudine (d4T), and TRIZIVTR (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), saqui
  • acyclovir 9-(2- hydroxyethoxymethyl)guanme
  • interferon e.g., alpha-interferon, interleukin ⁇ , and phosphonoformate (Foscarnet)
  • inducers of lytic viral replication in latently infected cells including Egrl activators such as resveratrol, proteasome inhibitors such as bortezomib or clastolactacystin-beta-lactone, farnesyl transferase inhibitors such as L-744832, or in conjunction with other immune modulation agents or treatments including bone marrow or lymphocyte transplants or other medications such as levamisol or thymosin which would increase lymphocyte numbers and/or function as is appropriate.
  • Egrl activators such as resveratrol, proteasome inhibitors such as bortezomib or clastolactacystin-beta-lactone, farnesyl transferase inhibitor
  • 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, 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 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.
  • the subject is not suffering from (and/or has not been diagnosed as suffering from) Kaposi's sarcoma.
  • the subject is not infected with (and/or has not been diagnosed as being infected with) HHV-8 (human herpes virus 8, also known as Kaposi sarcoma herpes virus (KSHV)), a virus associated with development of Kaposi's sarcoma.
  • HHV-8 human herpes virus 8, also known as Kaposi sarcoma herpes virus (KSHV)
  • KSHV Kaposi sarcoma herpes virus
  • the HIV is a drug-resistant strain of HIV.
  • one or more compounds of the formulae herein are used in conjunction with a standard HTV antiviral treatment regimen.
  • This combination is advantageous in that the abl kinase inhibitors of the invention (e.g., compound(s) of the formulae herein, e.g., imatinib) are believed to act by targeting, a cellular co-factor rather than a viral protein and therefore may have activity against viral strains that are resistant to current anti-retrovirals.
  • the treatment methods herein include administration of a so-called HIV-drug "cocktail” or combination therapy (including HAART), wherein a compound according to the invention is administered in conjunction with (or co-administered with) a combination of reverse transcriptase inhibitor(s) and HIV protease inhibitor(s).
  • Preferred drug combinations include HAART therapies such as: 1) efavirenz + (lavimudine or emtricitabine) + (zidovudine or tenofovir DF); and 2) lopinavir/ritonavir + (lavimudine or emtricitabine) + zidovudine.
  • the present invention includes use of both racemic mixtures and optically active stereoisomers of compounds of the formulae herein.
  • compositions of compounds of the formulae herein used in combination with other compounds 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.
  • One or more compounds of the formulae herein may be administered alone, or as part of a pharmaceutical composition, comprising at least one 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.
  • 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.
  • hnatinib targets abl-kinase and may serve as an upstream regulator of Ras proteins. Ras and other Ras related genes are up-regulated in the HIV lyrically replicating cells, as shown in the examples and figures, which follow.
  • abl-kinase and Ras synergistically activate NF-kB which is a regulator of HTV replication. It has now been found that treatment of infected cells with imatinib causes a dose dependent decrease in HIV viral replication. The finding was observed in three different latently infected cell lines that were induced into lytic replication by TNF-alpha.
  • abl-kinase and Ras synergistically activate NF- kB which is a well-known potent positive regulator of HIV gene expression.
  • abl- kinase, c-kit and/or PDGF or possibly other factor(s)
  • HIV proteins such as the HIV reverse transcriptase, integrase, or proteases.
  • HIV latently infected cell lines were induced into lytic replication with TNF- ⁇ in the presence of differing amounts of imatinib. It was found that imatinib caused a dose-dependent decrease in HIV viral replication, as judged by p24 production with a p24 ELISA assay ( Figure 1). These cell lines include two lymphocytic cell lines, ACH-2 and J 1,1, and a monocytic cell line, Ul. Therefore, targeting cellular genes that play a role in HIV lytic replication may inhibit HIV replication, offering the potential for new anti-retroviral therapies.
  • Micorarray analysis shown in Figures 5 and 6, of latently infected cells and of lyrically replicating cells shows that there is one set of cellular conditions for normal cell growth and homeostasis, another set of cellular conditions for supporting viral replication, and one set of conditions for latent retroviral infection.
  • HIV has evolved ways of altering the host cell environment to better support viral replication, and targeting those alterations in the host cell may inhibit viral replication.
  • Therapies targeting host cell functions may potentially be less troubled by the development of resistance than currently available antiretroviral drugs that target viral functions because they may be required for selection of a mutant virus that no longer required a host cell.
  • Table 2 shows the results of data graphically displayed in Figure 2.
  • the effect of imatinib mesylate in inhibiting HTV replication was determined by calculating the amount of p24 expression in imatinib treated HIV-infected cells and expressing it as a percentage of p24 expression caused by HIV infection alone. For example, a 5 ⁇ M Imatinib treatment of infected Jurkat cells produced 32% p24 levels as compared to infected Jurkat cells alone (100%), indicating a 68% inhibition. The data showed that Imatinib mesylate caused a dose-dependent decrease in p24 expression in HTV infected cells, indicating that Imatinib was effective in inhibiting
  • HTV replication following an acute infection See Table 2 and Figure 2; see also Examples 11-15 and Figures 8-12.
  • the methods and compounds of the present invention can provide effective therapy for inhibiting HIV replication and thereby reducing the viral load of a subject.
  • 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.
  • a preventative it can also be used prophylactically as a preventative for high risk individuals.
  • the methods and 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.
  • the methods and compositions of the invention can inhibit viral replication.
  • the viral load of a subject can be significantly reduced.
  • P24 a major structural protein (product of gag)
  • gag a major structural protein
  • Use of present compounds such as the compounds of the invention, at concentrations that do not adversely affect cells, can dramatically reduce HIV-I replication, e.g. preferably a reduction of HTV-I replication of more than 25% as determined by P24 levels, more preferably a reduction of more than 50%, and still more preferably a reduction of HIV-I replication of more than 80% as determined by P24 levels.
  • the effective amount of a compound of the present invention used to obtain such a result can be at micromolar concentrations. Furthermore, the use of the compounds of the present invention at concentrations which inhibit HIV expression has not been found to adversely affect treated cells in vitro.
  • the compounds of the present invention can be administered to HTV infected individuals or to individuals at high risk for HTV infection. For example, those having, sexual relations with an HTV infected partner, intravenous drug users, etc. Because of its inhibitory effect, the compounds of the present invention and pharmaceutical compositions according to invention (e.g., comprising one or more compounds of formula I) can be used prophylactically as a method of prevention for such individuals to minimize their risk. An effective amount of the compound is administered as set forth below by methodology such as described herein.
  • compounds, of the present invention can inhibit replication or suppress activity of HIV-I in infected cells.
  • compounds of the present invention in a dose dependent fashion inhibit HTV replication.
  • such inhibition is provided with essentially no adverse effects on cell survival or cellular mRNA or total cellular RNA synthesis in vitro.
  • compounds of the present invention have utility in inhibiting the progression of an HTV infection and other retroviral infections in cells and in a human, including utility in extending the latency of an HTV infection in subjects (including humans) and delaying the onset of symptoms.
  • one or more compounds of the invention is administered in an amount sufficient to reduce the amount of protein expressed by the gene by at least about 25 percent relative to an untreated cell, more preferably an amount sufficient to reduce the amount of protein by at least about 50 percent and still more preferably a reduction of the amount of protein expressed by at least about 75 percent relative to an untreated cell.
  • a preferred effective dose of one or more compounds of the present invention, in particular compounds of formula I 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.
  • 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 of formula I 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.
  • a comparatively pure form e.g., at least 85% by weight pure, more preferably at least 95% pure.
  • administered compound of formula I 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.
  • compositions of compounds of formula I or formula EI used in combination with other compounds may be employed alone or in combination with acceptable carriers such as those described below.
  • acceptable carriers such as those described below.
  • a suitable effective dose of other compounds 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.
  • the compounds are administered together it is expected one can use the lower portion of these ranges (or even less) of one or more of the compounds with excellent results.
  • One or more compounds of formula I or formula IH may be administered alone, or as part of a pharmaceutical composition, comprising at least one compound of formula I 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.
  • 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.
  • 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 maybe 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 maybe 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.
  • 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.
  • HIV-Latently infected cells (ACH-2, J 1.1, or Ul) were induced into lytic replication using Tumor necrosis factor -alpha (TNF-a, 0.5 ⁇ g/ml) and treated with different concentrations of imatinib mesylate.
  • TNF-a Tumor necrosis factor -alpha
  • Cells without TNF- ⁇ treatment were used to determine the basal p24 expression in the latently infected cells.
  • Cells treated with TNF- a alone were assayed to determine the maximal level of HIV p24 expression in the absence of Imatinib mesylate.
  • HIV-p24 ELISA antigen capture assay After 24 hours incubation at 37 0 C, cell supernatants were assayed for HIV-p24 protein levels by HIV-p24 ELISA antigen capture assay per manufacturer's protocol. Samples were mixed with lysing buffer (10% Triton-X-100, Sigma) to inactivate virus and diluted 5-fold with sample diluent (1% bovine serum albumin, 0.2% Tween- 20 in RPMI- 1640). p24 expression was assayed by ELISA using HIV-I p24 antigen capture kits (AIDS Vaccine Program, MD) per manufacturer's specifications. Briefly, plates were washed with plate wash buffer and samples were added in duplicate wells.
  • the diluted samples (100 ⁇ L) were incubated for 2 hours at 37 0 C. 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 effect of imatinib mesylate in inhibiting HTV replication was determined by calculating the amount of p24 expression in imatinib treated cells and expressing it as a percentage of p24 expression caused by TNF-a alone. For example, a 5 ⁇ M Imatinib treatment of TNF- ⁇ treated Jl.1 cells produced 80% p24 levels as compared to TNF- ⁇ alone, indicating a 20% inhibition.
  • Uninfected cells (A3.01, Jurkat) were infected with HIV-I (NL4-3 strain) and treated with different concentrations of Imatinib mesylate. Uninfected cells of each cell type were used as negative control. HTV-I infected cells with no drug treatment were assayed to determine the maximal level of HIV p24 expression in the absence of
  • sample diluent 1% bovine serum albumin, 0.2% Tween-20 in RPMI-1640.
  • p24 expression was assayed by ELISA using HIV-I ⁇ 24 antigen capture kits (AIDS Vaccine Program, Frederick, MD) per manufacturer's specifications. Briefly, plates were washed with plate wash buffer and samples were added in duplicate wells. The diluted samples (100 ⁇ L) were incubated for 2 hours at 37 0 C. The plates were washed, and rabbit anti-HIV p24 antibody was added at 1 :400 dilution.
  • the effect of imatinib mesylate in inhibiting HIV replication was determined by calculating the amount of p24 expression in imatinib treated HIV-infected cells and expressing it as a percentage of p24 expression caused by HIV infection alone. For example, a 5 ⁇ M Imatinib treatment of infected Jurkat cells produced 32% p24 levels as compared to infected Jurkat cells alone (100%), indicating a 68% inhibition. The data showed that Imatinib mesylate caused a dose-dependent decrease in p24 expression in HIV infected cells, indicating that Imatinib was effective in inhibiting HIV replication following an acute infection.
  • ACH-2, A3.01, Jl.1, and Ul cells were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AJDS, 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 HJV-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 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
  • fflV-infected and uninfected cells maintained and harvested in parallel with the PMA- treated cells but not induced with PMA were also harvested.
  • 3'-azido- 3 '-deoxythymidine (AZT, Sigma) was not added to the ACH-2 or A3.01 cells, in order to keep conditions as close to an acute infection as allowed by the experimental model.
  • Flow Cytometry To confirm viral replication following PMA induction, we measured the accumulation of intracellular p24 over a period of 48 hours by measuring cell populations labeled with anti-p24 FITC-labeled antibody by flow cytometry.
  • Cells (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.
  • Total RNA Extraction Total RNA was extracted using RNEasy Midiprep
  • RNA concentrations and purity were measured by spectrophotometry, RNA quality (absence of RNA degradation) was assessed by gel electrophoresis. RNA concentration was adjusted to the levels required for subsequent microarray experiment protocols by concentration in a SpeedVac (Savant Instruments, Holbrook, CA). RNA samples (6-7 ⁇ g/ ⁇ L) were stored in 100 ⁇ L TE buffer at -8O 0 C.
  • 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.
  • the sequence for the labeled probe was 5'CTTCTCTATCAAAGCAGACCCACCTCCS' which overlapped with the splice site of HIV-I Rev sequence.
  • the 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 SKl 9 (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. Fold differences in mRNA expression in uninduced ACH-2 samples and the corresponding A3.01 samples, was determined using the protocol described for quantitation of viral mRNA. Real time RT-PCR quantitation was performed for genes PSMC5, p44sl ⁇ (proteasome subunits) Egrl (early growth response 1), HDACl (histone deacetylase 1), NK4, EIF4, SFRS3, to confirm that these genes were differentially expressed in the latently infected ACH-2 cells compared to the uninfected A3.01 parental cells. Primer-probe pairs specific for each gene were designed using PrimerExpress (ABI). The sequences for the detection primers and probes for each gene are available as supplemental data, Table Sl.
  • 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. Microarrays were obtained from the National
  • the microarrays contained 10,395 cDNA spots on each glass slide.
  • the 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.
  • RNA from PMA induced ACH-2 cells 50 ⁇ g 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.
  • Each sample of 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°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.
  • ArrayTools (62). Since RNA for infected and uninfected cell lines corresponding to the same time point were paired and co-hybridized on the same array, inter-array sources of variation were minimized and differential expression could be detected by a statistically significant non-zero mean log-ratio in biologically independent replicates. All biological replicates that passed the filtering criteria described above were used in the analyses. Technical replicates were averaged. The random variance model enabled variance information to be shared across genes without assuming that all genes have the same variance (69). For comparison of expression for latently infected versus uninfected cell lines, significance was based on p ⁇ 0.001 for a parametric one-sample random variance t-test.
  • 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.jp/kegg/) and Biocarta (www.biocarta.com) pathway databases.
  • CGAP Cancer Genome Anatomy Project
  • DMSO DMSO
  • 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- ⁇ tumor necrosis factor alpha
  • AZT tumor necrosis factor alpha
  • Samples were mixed with lysing buffer (10% Triton-X-100, Sigma) to inactivate virus and diluted 5-fold with sample diluent (1% bovine serum albumin, 0.2% Tween-20 in RPMI- 1640).
  • sample diluent 1% bovine serum albumin, 0.2% Tween-20 in RPMI- 1640.
  • p24 expression was assayed by ELISA using HIV-I p24 antigen capture kits (AIDS Vaccine Program, Frederick, MD) per manufacturer's specifications.
  • 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.
  • PSMB4 has peptidase activity, which is inhibited by Tat during viral replication. Tat competes with the 1 IS 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).
  • Nef-specif ⁇ c CTLs cytotoxic T-lymphocytes
  • 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) .
  • Table 1 Functionally related genes that were differentially expressed prior to induction in chronically infected ACH-2 cells.
  • HTV cell lines including J 1.1, a chronically infected T-lymphocytic cell line derived from Jurkat cells and Ul, a promonocytic chronically infected cell line derived from U937 cells, were studied using microarrays to determine the similarities and differences in their expression profiles.
  • the p24 expression in all the latently infected cell lines was below lng/mL (0.2-0.8 ng/mL) indicating that the cells were not lyrically active at the time of harvesting the cells.
  • Experiments were performed on eight independent cell samples for each cell line and similar parameters were applied for filter criteria, gene selection and statistical analysis as with the ACH-2 cell line, as described in the Methods section.
  • 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 JLl 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).
  • 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.
  • FX A MPT .F, 1 1 Effect of imatinib and saquinavir on HIV-infected Jurkat cells.
  • imatinib caused a dose dependent decrease in p24 expression in HTV infected Jurkat cells with an IC50 of about 2.5 ⁇ M.
  • the combination of imatinib with varying concentrations of saquinavir (an HIV protease inhibitor) caused a marked decrease in HJV p24 expression, greater than that caused by either drug alone, indicating an additive or synergistic effect.
  • imatinib alone or in combination with saquinavir on HlV-infected MT-2 cells was studied using methodology similar to that described in Example 1 , supra.
  • imatinib caused a dose dependent decrease in p24 expression in HIV infected MT-2 cells with an IC50 of about 1 ⁇ M.
  • the IC50 of saquinavir (a HIV protease inhibitor), alone was 0.5 ⁇ M.
  • the combination of imatinib with varying concentrations of saquinavir caused a marked decrease in HIV p24 expression, greater than that caused by either drug alone, indicating an additive or synergistic effect.
  • imatinib alone or in combination with saquinavir on drug- resistant (saquinavir-resistant) HIV-infected MT-2 cells was studied using methodology similar to that described in Example 1 , supra.
  • imatinib caused a dose dependent decrease in p24 expression in saquinavir-resistant HIV-infected MT-2 cells with an IC50 of about 1 ⁇ M.
  • Combination of imatinib with varying concentrations of saquinavir (a HTV protease inhibitor), caused a marked decrease in HIV p24 expression, greater than that caused by either drug alone, indicating an additive or synergistic effect.
  • Saquinavir alone was unable to inhibit replication of saquinavir-resistant virus, as expected.
  • the IC 50 of saquinavir alone (> 5 ⁇ M) was shifted greater than 10-fold than that observed with wild type HIV (0.5 ⁇ M, see Figure 9).
  • the effect of the combination of saquinavir and imatinib is greater than either of the drugs tested alone, indicating an additive or a synergistic effect in the range of 1-2.5 ⁇ M imatinib and 0.1-0.5 ⁇ M of saquinavir.
  • PBMC samples was studied using methodology similar to that described in Example 1, supra.
  • PBMC peripheral blood mononuclear cells
  • Imatinib caused a dose dependent decrease in p24 expression in all the four infected samples, though the extent of inhibition varied between the donor samples.
  • the dose at which imatinib inhibits HIV replication is much lower than in cell lines, however there is considerable variability in efficacy of imatinib between donor samples. In all cases, cellular viability was not an issue at the concentrations of imatinib tested.
  • Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone.
  • Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus interacts with human myeloid cell nuclear differentiation antigen induced by interferon alpha. Virus Genes 27:237-47.
  • HTV- 1 Tat protein and the HS regulator subunit alpha is crucial for their effects on proteasome function including antigen processing. J MoI Biol 323:771-82.

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Traitement de cellules ou d'humains porteurs d'un virus, ou infectés par ce dernier, capable de provoquer une maladie immunodéficitaire, au moyen de composés définis, y compris des dérivés de N-phényl-2-pyrimidine-amine représentés par la formule (I), dont les substituants sont indiqués dans le descriptif, et de médicaments contenant ces composés et leurs utilisations.
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WO2008124129A2 (fr) * 2007-04-09 2008-10-16 University Of Massachusetts Procédés de traitement du vih
US8242271B2 (en) 2007-06-04 2012-08-14 Avila Therapeutics, Inc. Heterocyclic compounds and uses thereof

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US20040102453A1 (en) * 2000-09-13 2004-05-27 Buerger Hans Michael N-phenyl-2-pyrimidine-amine derivatives
US20040126877A1 (en) * 2001-04-20 2004-07-01 Man-Wook Hur Repressors for hiv transcription and methods thereof
WO2005072826A2 (fr) * 2004-01-21 2005-08-11 Emory University Compositions et procedes pour utiliser des inhibiteurs de tyrosine kinase afin de traiter une infection pathogenique

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US20040102453A1 (en) * 2000-09-13 2004-05-27 Buerger Hans Michael N-phenyl-2-pyrimidine-amine derivatives
US20040126877A1 (en) * 2001-04-20 2004-07-01 Man-Wook Hur Repressors for hiv transcription and methods thereof
WO2005072826A2 (fr) * 2004-01-21 2005-08-11 Emory University Compositions et procedes pour utiliser des inhibiteurs de tyrosine kinase afin de traiter une infection pathogenique

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TSIMBERIDOU APOSTOLIA-MARIA ET AL: "Chronic myeloid leukemia in a patient with acquired immune deficiency syndrome: complete cytogenetic response with imatinib mesylate: report of a case and review of the literature" LEUKEMIA RESEARCH, vol. 28, no. 6, June 2004 (2004-06), pages 657-660, XP002361027 ISSN: 0145-2126 *

Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO2008124129A2 (fr) * 2007-04-09 2008-10-16 University Of Massachusetts Procédés de traitement du vih
WO2008124129A3 (fr) * 2007-04-09 2009-04-30 Univ Massachusetts Procédés de traitement du vih
US8242271B2 (en) 2007-06-04 2012-08-14 Avila Therapeutics, Inc. Heterocyclic compounds and uses thereof
US8586600B2 (en) 2007-06-04 2013-11-19 Celgene Avilomics Research, Inc. Heterocyclic compounds and uses thereof
US9067929B2 (en) 2007-06-04 2015-06-30 Celgene Avilomics Research, Inc. Heterocyclic compounds and uses thereof

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