EP0676958A1 - Method for inhibiting intracellular viral replication - Google Patents

Method for inhibiting intracellular viral replication

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Publication number
EP0676958A1
EP0676958A1 EP95901093A EP95901093A EP0676958A1 EP 0676958 A1 EP0676958 A1 EP 0676958A1 EP 95901093 A EP95901093 A EP 95901093A EP 95901093 A EP95901093 A EP 95901093A EP 0676958 A1 EP0676958 A1 EP 0676958A1
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EP
European Patent Office
Prior art keywords
cells
htv
viral
expression
virus
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP95901093A
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German (de)
French (fr)
Inventor
David W. Leung
Gail E. Underiner
Jack W. Singer
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Cell Therapeutics Inc
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Cell Therapeutics Inc
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Publication of EP0676958A1 publication Critical patent/EP0676958A1/en
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435Heterocyclic 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
    • A61K31/44Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445Non condensed piperidines, e.g. piperocaine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435Heterocyclic 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
    • A61K31/47Quinolines; Isoquinolines
    • A61K31/472Non-condensed isoquinolines, e.g. papaverine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495Heterocyclic 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
    • A61K31/505Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495Heterocyclic 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
    • A61K31/505Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495Heterocyclic 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
    • A61K31/505Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52Purines, e.g. adenine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • A61P37/04Immunostimulants
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention provides a method of preventing or delaying the occurrence of acquired immunodeficiency syndrome (AIDS) in human immunodeficiency virus (HTV) seropositive humans by administering an effective amount of a compound that inhibits cellular signaling through a specific phospholipid-based cellular signaling and signal amplification pathway.
  • the invention further provides a method for preventing or delaying clinical symptoms of a group of viral diseases wherein the viral disease is mediated by host cell viral replication.
  • the invention provides an advantage by attacking host cellular signaling mechanisms to prevent the development of drug resistance from rapidly mutating viruses.
  • Drugs currently approved for AIDS therapy belong to classes of compounds that directly attack viral proteins, such as reverse transcriptase (e.g., AZT or zidovudine) or HTV protease.
  • viruses such as reverse transcriptase (e.g., AZT or zidovudine) or HTV protease.
  • reverse transcriptase e.g., AZT or zidovudine
  • HTV protease e.g., AZT or zidovudine
  • Such compounds have the problem of rapidly developing drug resistance due to rapid transformation of the infecting viral genome.
  • such drugs have been used to treat HTV seropositive individuals to help prevent the spread and progression of the infection into the clinical symptoms of AIDS.
  • HIV infection leads to progressive depletion of CD4 + T lymphocytes and eventual development of clinical symptoms associated with AIDS.
  • After the primary HTV infection there is a prolonged incubation period of clinical latency that can last as long as twelve years (Weiss, Science 260:1273-1279, 1993).
  • Recent studies indicate viral replication occurs continuously (Pantaleo et al., Nature 362:355-358, 1993) and multiple components of the immune system are chronically activated (Fauci, Science 262:1011-1018, 1993; Bass et al., Clin Immunol Immunopathol. 64:63-70, 1992) during this asymptomatic phase of disease.
  • Persistent activation of the immune system may lead to overproduction of a number of cytokines (Fauci, Science 262:1011-1018, 1993), induction of HIV expression in latently infected cells (Rosenberg et al., AIDS Res. Hum. Retroviruses 5:1-4, 1989), and apoptosis of T cells (Gougeon, Science 260:1269-1270, 1993).
  • cytokines Fauci, Science 262:1011-1018, 1993
  • induction of HIV expression in latently infected cells Ros. Hum. Retroviruses 5:1-4, 1989
  • apoptosis of T cells Gougeon, Science 260:1269-1270, 1993.
  • LTR long terminal repeat
  • the LTR promoter contains cw-acting repressive sequences that inhibit HIV transcription initiation in resting T cells (Lu et al., J. Virol. 64:5226-5229, 1990). Mitogenic stimulation of T cells can activate the LTR promoter to synthesize large amounts of viral RNA.
  • Cytokines such as tumor necrosis factor (TNF- ⁇ ) and interleukin-6 (IL-6), are known to induce HTV synthesis in infected cells (Poli et al., Semin. Immunol. 5: 165-173, 1993).
  • HTV infection leads to progressive depletion of specific populations of T lymphocytes and progression of the infection to clinical symptoms associated with AIDS (acquired immunodeficiency syndrome), including HJN-related dementia.
  • AIDS immunodeficiency syndrome
  • HJN-related dementia Currently, there is no effective treatment for HIV-related dementia, although current therapy, AZT, may have some minor short-term effectiveness.
  • Microglial cells and T ⁇ F ⁇ production have been implicated in the neuropathogenesis of HIV-related dementia. Moreover, microglia and T ⁇ F ⁇ release have been suggested to play a role in other neurodegenerative disorders, such as Alzheimer's disease and multiple sclerosis.
  • immunosuppressive and immunomodulatory agents have been shown to suppress viral replication.
  • immunomodulating CD8 lymphocytes have been shown to suppress replication of HIV in peripheral blood mononuclear cells (Waler et al., Science 234:1563, 1986) and activated CD8+ T cells have been shown to inhibit replication of HTV in cultures of CD4+ cells from asymptomatic HIV seropositive individuals (Brinchmann et al., /. Immunol. 144:2691, 1990).
  • the immunosuppressive compound, cyclosporin A (CyA) has a protective effect in several animal models of viral infection.
  • the HIV genome encodes at least seven groups of viral proteins (Haseltine, FASEB 5:2349, 1991). These groups of proteins include the three classes of polypeptides present in the majority of animal retroviruses: (1) structural, nonenvelope polypeptides encoded by the gag gene; (2) enzymes required for viron replication (reverse transcriptase/rt) and for cleavage of viral precursor proteins (protease/pr), both encoded by the pol gene; and (3) envelope polypeptides encoded by the env genes. In addition, HIV's possess genes that encode four sets of polypeptides not found in the majority of typical retroviruses.
  • RNA translation a transactivating regulator of RNA translation (tat)
  • nef a cw-acting downregulator of RNA transcription
  • rev structural proteins
  • vpr, vpu two proteins whose functions are still unclear.
  • the genomic order of viral genes encoding the initial portion of each of these proteins (5' to 3') is gag-pr-pol-vif-tat-rev-env-nef.
  • HIN-1 HIV type 1
  • LTR long term repeat
  • the tat gene encodes a 14 kD protein that transactivates and increases transcription of D ⁇ A in HTN-infected cells.
  • the tat gene consists of two separate segments that have been mapped to a location in the HTN genome between the vif and nef regions. It encodes for a 14 kD protein that exists as a metal-linked dimer in infected cells. Products of the tat gene have been shown to greatly augment the rate of viral protein synthesis in HTN-infected cells, and thus to increase the production of HIN virons.
  • T ⁇ F tumor necrosis factor
  • the present invention provides a method for treating viral infection by preventing viral replication in infected cells by inhibiting host cell viral replication signaling, comprising administering an effective amount of a compound that inhibits formation of myristilated PA (Phosphatidic acid) in response to an inflammatory stimulus.
  • An inflammatory stimulus can be mediated by, for example, TNF or TL-1.
  • the compound that can inhibit myristilated PA (myrPA) in either the sn-1 or sn-2 positions or both can be determined by following an assay procedure described herein.
  • the compound is a small organic molecule that can mimic binding to a complex of enzymes that mediate signal amplification.
  • the compounds include resolved enantiomers and/or diastereomers, hydrates, salts, solvates and mixtures thereof, the compounds having a straight or branched aliphatic hydrocarbon structure of formula I:
  • n is an integer from one to four and m is an integer from four to twenty.
  • R ⁇ and R2 are hydrogen, a straight or branched chain alkyl, alkenyl or alkynyl of up to twenty carbon atoms in length or -(CH2) W 5- If Ri or R2 is -(CH2) W R5, w may be an integer from one to twenty and R5 may be an hydroxyl, halo, Ci _g alkoxyl group or a substituted or unsubstituted carbocycle or heterocycle.
  • Ri and R2 may jointly form a substituted or unsubstituted, saturated or unsaturated heterocycle having from four to eight carbon atoms, N being a hetero atom of the resulting heterocycle.
  • R3 may be hydrogen, a hydroxy group, a Ci .3 straight or branched alkyl, or a C 1.3 alkoxy.
  • Preferred compounds may have one of R ⁇ or R2 and R3 that form a substituted or unsubstituted linking carbon chain, having from one to four carbon atoms. This R1/R3 or R2 R3 linking chain will join the O and N in a cyclic structure, an integer sum equal to n + a number of carbon atoms in the linking carbon chain being less than six.
  • R4 is a terminal moiety comprising a substituted or unsubstituted, heterocyclic moiety, wherein the heterocyclic moiety consists essentially of one to three ring structures having 5-7 members each, a heteroatom, and a predominantly planar structure or essentially aromatic. However, if R4 is phthalimide, m of formula I is not less than five.
  • the compounds may include resolved enantiomers and/or diastereomers, hydrates, salts, solvates and mixtures thereof that have a straight or branched aliphatic hydrocarbon structure of formula II: R
  • n, m, R3, and R4 are defined as provided in formula I above.
  • Rg and R7 are hydrogen, a straight or branched chain alkane, alkene or alkyne of up to twenty carbon atoms in length, or -(CH2) X R8» at least one of Rg or R7 being -(CH2) ⁇ Rs-
  • x is an integer from zero to fourteen and Rg is a moiety having a general structure as provided in formula HI
  • m, R3, and R4 are defined as provided in formula I above.
  • Z is N or CH and p is an integer from zero to four.
  • R9 is H or a straight or branched chain alkane, alkene or alkyne of up to twenty carbon atoms in length.
  • the present invention provides a method for treating viral infections by attacking host cell mechanisms that contribute to intracellular viral replication without attacking directly any viral proteins involved in viral replication.
  • the illustrated compounds provide an unexpected advantage of avoiding drug resistance from rapid viral mutation that has plagued current AIDS therapies that attack viral reverse transcriptase and protease enzymes.
  • the compounds are active against a very wide range of retroviruses because there is no need for viral specificity when targeting common host cell mechanisms.
  • the method of treatment is designed to prevent further progression of the virally-mediated disease but not to eradicate the virus. Therefore, the present inventive method includes combination therapeutic regimens that combine an agent designed to prevent host cell viral replication by targeting host cell signaling together with an agent or group of agents that are directly antiviral.
  • the compounds described herein exert their antiviral activity by inhibiting one or a plurality of enzymes or an enzyme complex, resulting in a diminution of host (or infected) cell PA (phosphatidic acid) formation in response to an inflammatory stimulus (e.g., TNF or IL-1) mediated through a Type I TNF or IL-1 receptor on the host cell surface. Therefore, the inventive compounds describe a genus of compounds that share a common mechanism of action, on a cellular level, that results in decreased intracellular signaling within a virally- infected cell, that does not transmit an inflammatory signal that would otherwise (in the absence of drug) signal an infected cell to begin the process of viron replication, assembly and viron shedding.
  • an inflammatory stimulus e.g., TNF or IL-1
  • This cellular and biochemical mechanism of action as illustrated by cells infected by the HTN virus as described herein, provides evidence for a general antiviral activity of the genus of compounds described herein, or other compounds that exert a similar mechanism of action (that is, prevention of infected host cell PA accumulation in response to an inflammatory stimulus) by virtue of the ability of the drug to prevent normal viral replication within an infected host cell.
  • viruses that also replicated in an infected host cell in response to inflammatory stimuli (usually mediated by the cytokines T ⁇ F or IL-1) include, for example, cytomegalovirus (CMN), herpes family of viruses, including herpes simplex virus (HSN) 1, 2 and 6, hepatitis A, B, C and D, HTV 1 and 2, Epstein Barr virus (EBV), human T cell leukemia virus (HTLV), human papiloma virus, influenza, parainfluenza, respiratory syncytial virus, all adenoviruses, and rhinoviruses.
  • CPN cytomegalovirus
  • HSN herpes simplex virus
  • HTV 1 and 2 hepatitis A, B, C and D
  • Epstein Barr virus EBV
  • human T cell leukemia virus HTLV
  • human papiloma virus influenza, parainfluenza, respiratory syncytial virus, all adenoviruses, and rhinoviruses.
  • the inventive method results in prevention of progression and treatment of a viral disease, comprising administering an effective amount of a compound that can reduce intracellular myrPA concentrations in response to an inflammatory stimulus.
  • viral diseases include, for example, CMV retinitis, AIDS, ARDS, systemic viral diseases affecting immunocompromised individuals (e.g., AIDS or transplant recipients), cold sores (HSV-1), genital herpes (HSV- 2), hepatitis (A, B or C, or HSV-6), genital warts (human papiloma virus), infectious mononucleosis and some lymphomas (EBV), shingles (Varicella zoster), pericarditis (coxsackie virus), influenza, and cold and flu (rhinoviruses and adenoviruses).
  • the inventive method further provides a method for treating a consequence of viral infection of the retroviral variety, wherein consequences of retroviral infections include, but are not limited to, cachexia associated with HTV infection, cachexia associated with EBV infection, HTV, EBV and HTLV related malignancies such as Kaposi's sarcoma and lymphoma, ATDS-related opportunistic infections (PCP, MAI, etc.) and for prophylaxis of opportunistic infection in AIDS patients.
  • consequences of retroviral infections include, but are not limited to, cachexia associated with HTV infection, cachexia associated with EBV infection, HTV, EBV and HTLV related malignancies such as Kaposi's sarcoma and lymphoma, ATDS-related opportunistic infections (PCP, MAI, etc.) and for prophylaxis of opportunistic infection in AIDS patients.
  • Figure 1 shows AP reporter gene activity in 293-EB ⁇ A cells transiently transfected with pHIV. AP alone or in combination with pSVt t72. The expression level of AP increased by more than 50 fold in the presence of the tat expression vector, indicating transactivation of the HTV-LTR promoter by the 72 amino acid tat protein.
  • Figure 2 shows an example of assay results of three compounds (CT1501R, CT1827, and CT1829, see Table 2 for chemical structures). Both CT1827 and CT1829 inhibited AP reporter gene expression in 239-EBNA cells with ID50 values in the range of 4 to 6 ⁇ M using the HTV-LTR promoter construct transfected with a tat expression vector. No appreciable inhibition of AP expression was observed with CT1501R.
  • Figure 3 shows the results of the above-noted compounds on viability of 293-EBNA cells.
  • Cell viability was determined with a colorimetric assay using the tetrazolium salt of MTS (Promega) to report cell proliferation, viability and cytotoxicity.
  • MTS indicates cell activity by serving as a substrate for mitochondria dehydrogenases for formation of soluble formazan dyes which can be quantitated by determining the absorbance at 490 nm using a plate reader. None of the three compounds tested in Figure 2 showed significant cytotoxic effect on 293-EBNA cells as the concentrations indicated.
  • Figures 4 and 5 show another example of assay results of three other compounds (see Table 1 for chemical names).
  • CT1501R (again) and CT2573 did not affect AP activity and cell viability significantly, whereas CT 1416 and CT2573 (structurally related to CT 1827 and CT1829) were able to inhibit AP expression by 50% at concentrations in the range of 1 to 4 ⁇ M.
  • some cytotoxicity was noted with CT1416 and CT2573 with cellular LD50 values at around 5 ⁇ M.
  • Figure 6 shows an example of the effect three amino-alcohol-substituted xanthine compounds (CT2575, CT3528, and CT2571) on AP reporter gene activity in 293-EBNA cells stably transfected with pCMV.AP.
  • CT2575 and CT2571 inhibited AP reporter gene expression in 293-EBNA cells with IC50 values of 3 ⁇ M and 8 ⁇ M, respectively. No appreciable inhibition of AP expression was detected when using the compound CT3528.
  • Figure 7 shows the effect of the three compounds shown in Figure 6 on the viability of 293-EBNA cells. Cell viability was measured by a colorimetric assay that uses Alamar Blue dye (Alamar, Inc.) to report cell proliferation, viability and cytotoxicity.
  • Alamar Blue dye Alamar, Inc.
  • This vital dye indicates cell activity by serving as substrate for mitochrondrial dehydrogenases for the formation of soluble dyes which can be quantitated by determining the difference in absorbance between A570 and A600 using a plate reader.
  • CT2571 was found to be cytotoxic to 293-EBNA cells while CT2575 and CT3528 showed no significant cytotoxic effect on 293-EBNA cells at the concentrations indicated.
  • Figures 8 and 9 show another example of the CMV assay results (as shown in Figures 6 and 7) of four other amino alcohol-substituted compounds. While all four compounds were able to inhibit AP activity by 50% in the range of 2 to 6 ⁇ M, CT1416 and CT1115 were found to be cytotoxic with an LD50 value of about 5 ⁇ M, whereas CT1827 and CT1829 showed no significant cytotoxic effect on 293-EBNA cells at the concentrations indicated. Therefore, a significant therapeutic window exists.
  • Figure 10 shows an example of assay results of three compounds (CT2576, CT1620, and CT3534, see Table 1 for chemical structures).
  • CT2576 inhibited AP reporter gene expression in 239-EBNA cells with ID50 values in the range of 4 to 6 ⁇ M using the HTV- LTR promoter construct transfected with a tat expression vector.
  • Figure 11 shows the results of the above-noted compounds in Figure 10 on viability of 293-EBNA cells.
  • Cell viability was determined with a colorimetric assay using the tetrazolium salt of MTS (Promega) to report cell proliferation, viability and cytotoxicity.
  • MTS indicates cell activity by serving as a substrate for mitochondria dehydrogenases for formation of soluble formazan dyes which can be quantitated by determining the absorbance at 490 nm using a plate reader. None of the three compounds tested in Figure 10 showed significant cytotoxic effect on 293-EBNA cells at the concentrations indicated.
  • Figure 12 shows the results of an assay with HTN-infected Ul cells where measurement of HIN-1 expression in the presence of various compounds was determined. Viral release was measured by determining the amount of HTV-lp24 antigen (Ag) expression in cell culture supernatants.
  • Compounds CT1501R, CT1829, CT1411 and CT2576 all suppressed constitutive HTV-l expression when plated at high density initially, but CT2576 was the most effective and CT1827 showed some cytotoxicity.
  • Figure 12 shows the effect of several compounds on T ⁇ F ⁇ -mediated upregulation of HTV-l in Ul cells.
  • T ⁇ F ⁇ is a potent stimulus of viral expression.
  • CT1501R, CT1829, CT1411 and CT2576 all suppressed HTV-l expression, but CT2576 was the most effective and CT1829 showed some cytotoxicity at a 25 ⁇ M concentration.
  • Figures 13 A and 13B illustrate the effect of CT2576 on PA and DG generation.
  • Figure 14 illustrates the effect of tat and T ⁇ F- ⁇ on HIV-LTR-mediated reporter gene expression in 293-EB ⁇ A cells.
  • the y-axis refers to the relative amount of AP reporter gene activity present in the culture media of 293-EB ⁇ A cells after transient transfection with various plasmids.
  • Freshly trypsinized 293-EB ⁇ A cells at 10 ⁇ cells per sample were transfected with 2 ⁇ g of either pHTN.AP, pHIN2.AP, or pHIN3.AP alone or in combination with pSN2tat72 or pHIV.tat using the cationic lipid DOTAP.
  • the plasmids pHTV.AP, pHIV2.AP, and pHTV3.AP contain nucleotides -453 to +80, -107 to +80, and -80 to +80 of the HTV-LTR sequence respectively.
  • Transfected cells were plated in 96-well microtiter plates at 2,000 cells per well. The cell culture media were changed 24 hr later into new medium. The samples were incubated for another 24 hr with or without T ⁇ F- ⁇ (20 ng/ml). 2 ⁇ l of medium from each well was used for AP assay. The height of each column represents the mean of triplicate samples with error bar indicating the standard deviation of the mean.
  • Figure 15 shows the effect of CT2576 on HTV-LTR expression activated by tat and
  • T ⁇ F- ⁇ in 293tat cells 293t ⁇ t cells, a cell line stably transfected with the expression plasmids pHIN. AP and pHTV.t ⁇ t, were plated onto 96-well microtiter plates at 2000 cells per well. The media were changed the next day and CT2576 was added at the indicated concentrations to the fresh media with or without TNF- ⁇ (20 ng ml). 2 ⁇ l of medium from each well was used for AP assay 24 hr later. After culture media were withdrawn for AP assay, cytotoxicity was measured for the same cell cultures using an indicator dye, Alamar BlueTM, that would change color in response to the reduction potential of cell medium due to cell growth.
  • Alamar BlueTM an indicator dye
  • the left y-axis refers to the absorbance value of the reduced form of Alamar BlueTM at 570 nm with the background absorbance at 600 nm from the oxidized form of Alamar BlueTM subtracted.
  • Each data point represents the mean of triplicate samples with error bar indicating the standard deviation of the mean.
  • Figure 16 shows the effect of CT2576 on NFKB activation and transcription.
  • Nuclear extracts (A) from 293tat cells were tested by EMS A for activation of NFKB.
  • a radiolabeled 40-mer covering the NFKB sequences in the HTV promoter was used as a probe.
  • Cells were incubated with CT2576 at 12 ⁇ M, human TNF- ⁇ at 40 ng/ml or LL-l ⁇ at 1 ng/ml, or pre-incubated for 30 min. with CT2576 and then stimulated with TNF- ⁇ or IL-l ⁇ for 18 hr prior to making extracts.
  • Northern blots analysis were performed using probes derived from AP cDNA and G3PDH cDNA as a control for normalization of mRNA level.
  • AP activity was determined in cell supernatants from the various samples.
  • Figure 17 illustrates a dose response curves of CT2576 on constitutive, TNF- ⁇ , and TL-6-mediated expression of HTV-l in a chronically infected Ul cell line (left Y-axis) and of CT3537 on HIV expression in PBL acutely infected with the JR-CSF primary isolate (right Y-axis). Ul cells were incubated for four days after the addition of CT2576 with or without TNF- ⁇ (20 pg/ml) or TL-6 (1 ng/ml). PBL were inoculated with HTV at 10 ng virus/10 6 cells/well and incubated with various concentrations of CT3537 for 7 days. Each data point represents the average expression levels of HTV-l p24-antigen in the cell supernatants measured by ELISA from triplicate samples with error bar indicating the SD.
  • Figure 18 shows a relationship between relative PA mass and activity of the compound CT3556.
  • Transformed and serum-stimulated cells were measured for total PA mass according to the methods described herein.
  • the t ⁇ t-transfected cells stimulated with serum (col. 3) showed a significant increase in PA mass when compared with non-t ⁇ t transfected cells stimulated with serum (col. 1).
  • This increase in PA was blocked by administration of CT3556 10 ⁇ M.
  • cells transfected with tar showed a significant drop in PA mass when treated with CT3556.
  • the columns in Figure 18 represent: (1) EB293 cells plus serum, (2) EB293 cells plus serum plus CT3556 (10 ⁇ M.), (3) 293tat cells plus serum, (4) 293t t cells plus serum plus CT3556 (10 ⁇ M.), (5) 293t ⁇ r cells plus serum, and (6) 293t ⁇ r cells plus serum plus CT3556 (10 ⁇ M.).
  • FIG 19 shows the same experiment as in Figure 18 only this time diacylglycerol was measured (DG).
  • DG was assayed by collecting the DG HPLC peak (Rf 2-4.5 min) and verifying the predominance of DG species by mass spectroscopy analysis. The DG peak was integrated for a quantitave analysis.
  • the six columns in Figure 19 correspond to the same columns in Figure 18.
  • Figure 20 shows a dose response relationship for CT2576 in inhibiting total PA mass in EB293 cells transfected with the tat expression plasmid and stimulated with serum.
  • the time points for PA determinations were from 0 to one minute.
  • Figure 21 shows two graphs for peak D (a measure of various PA species in an HPLC separation) and Peak A5 (a measure of lyso (bis) PA species) with EB293 cells (human embryonic origin) stimulated with human TNF (20 ng/ml, Genzyme) with or without treatment with CT2576 (10 ⁇ M.).
  • the left panel shows that drug treatment reduced PA mass across all time points measured, indicating inhibition of formation of PA species to acceleration of PA species metabolism.
  • CT2576 activity inhibits PA formation from lyso (bis) PA species.
  • Figure 22 shows an HPLC profile with the PA peak marked in stimulated but not treated cells.
  • Figure 23 illustrates the chemistry of a reaction to derivatize FFAs (free fatty acids) with 9-ADAM.
  • Figure 24 illustrates the chemistry of a reaction to make a 9-ADAM derivatizing reagent.
  • the present invention provides a method for treating viral infection by preventing viral replication in infected cells, comprising administering an effective amount of a compound that inhibits intracellular myrPA formation.
  • the compound is a small organic molecule that can mimic binding to a complex of enzymes that mediate signal amplification at the cellular plasma membrane. Specifically the compound acts by preventing formation of myrPA species in cells stimulated to signal viral replication and proliferation. Therefore, by attacking a host cell signaling mechanism, the compounds do not have resistant viral strains due to rapid viral mutation that is common in antiviral agents that act against viral proteins and viral mechanisms.
  • This procedure begins with those cells that would be normally infected with a virus, such as a retrovirus. However, it is not necessary to utilize a cell line containing a retroviral genome, such as Ul cells. In view of the necessity to utilize containment facilities when using an infected cell line, it is preferable to utilize a macrophage cell line that is not infected. Examples of appropriate macrophage cell lines include, U937, THP1, Ul, and P288s (available from standard sources, such as ATCC). The cells are stimulated with a stimulant with or without a candidate drug.
  • stimulants include 3-5% serum, such as fetal calf serum, or tumor necrosis factor (TNF) (e.g., 20 ng/ml) both of which are standard laboratory reagents. After a time course (within 5 minutes, preferably within 1 minute), the cells are immersed in ice cold methanol to stop any cellular signaling reactions.
  • serum such as fetal calf serum
  • TNF tumor necrosis factor
  • Bligh et al. Canadian J. Biochem. Physiol. 37:914-917, 1959) or that of Folch et al. (J. Biochem. 226:497-509, 1957). Briefly, the method of Bligh et al. involves an organic extraction of lipids from biological tissue homogenates or fluids. One volume of sample and three volumes of methano chloroform (2: 1) are vigorously shaken for 2 min. One volume of chloroform is added and then shaken vigorously for 30 sec.
  • the method of Folch et al. involves the extraction of lipids from biological tissue homogenates or body fluids.
  • One volume of sample plus 20 volumes of chlorofornrmethanol (2:1) are vigorously shaken for 2 min.
  • the mixture is filtered and an amount of 0J N KCl equal to 20% of the extraction mixture volume is added and the mixture is shaken vigorously for 2 min.
  • the aqueous and organic phases are allowed to separate.
  • the upper aqueous layer is discarded.
  • the lower organic layer contains a mixture of lipid classes.
  • Free fatty acids and neutral lipids can be separated from phospholipids by normal phase high performance liquid chromatography (HPLC) by modifying the method of Van Kessel et al. (Biochim et Biophys Acta 486:524-530, 1977). This method involves separation of lipids into their major classes by normal phase (silica) high performance liquid chromatography (HPLC).
  • HPLC normal phase high performance liquid chromatography
  • a 5 micron, 25 cm x 0.45 cm silica HPLC column is connected to a binary solvent delivery system followed with a UV detector. The lipid sample is injected on the column and a solvent gradient is run at 1.0 ml/min.
  • the solvent gradient is hexane:isopropanol:water in the proportions 3:4:0.75 run isocratically for 3 min, then ramped to hexane:isopropanol:water in the proportions 3:4:1.4 in 15 min, then run isocratically at the same proportions for 15 min.
  • Detection is at 206 nm.
  • the PAs run at about 6-8 min when run at 1 ml/min when run in hexane ⁇ sopropanol (3:4) according to the HPLC peaks shown in Figure 22 wherein the "PA peak" is listed as "D-2".
  • the PA peak is identified and isolated, it is subject to general alkaline hydrolysis or another method to isolate the FFAs (free fatty acids) from the PA species.
  • This assay is based upon identifying myrPA species in particular such that the identity of the acyl side chains of PA species in stimulated cells is critical for determining if a candidate compound is effective for inhibiting host cell signaling in infected cells to inhibit viral replication and prevent further progression of viral diseases.
  • the FFAs are isolated after hydrolysis.
  • the isolated FFAs are derivatized into fatty acid derivatives of 9-anthroyl diazomethane (9-ADAM), which absorbs light at 254 nm and fluoresces with emission at 410 nm.
  • 9-ADAM 9-anthroyl diazomethane
  • FFA derivatives were prepared essentially according to the method described in Nakaya et al. (Bull. Chem. Soc. Japan 40:691-692, 1967, and Yoshida et al., Analytical Biochem. 173:70-74, 1988). The derivatization is based on the reaction shown in Figure 23.
  • 9-anthraldehyde hydrazone for 9-anthroyl diazomethane derivatization was synthesized from 9-anthraldehyde and hydrazine monohydrate as follows: (a) 8.8 g 9-anthraldehyde (Aldrich Milwaukee, WI) was dissolved in 150 mL absolute ethanol and 8 mL hydrazine monohydrate (Aldrich Milwaukee, WI) was added dropwise with continual stirring, (b) The mixture cleared as hydrazine was added then turned opaque as the last drops were added, (c) The reaction was stirred at room temperature for 3 hr, then was filtered (Whatman #1 filter paper, Whatman Int. Maidstone UK) and dried, (d) The product was recrystalized twice with absolute ethanol. (e) The yield was 3J g of needle-like crystals.
  • the derivatizating reaction was carried out by diluting 50 ⁇ L of each FFA standard to 200 ⁇ L with methanol.
  • FFA standards (1.0 mg/mL) were made up in methanol using: Heptadecanoic acid 17:0 (Aldrich Chemical Milwaukee, WI); Arachidonic acid 20:4 (Matreya, Inc., Pleasant Gap, PA); Linoleic acid 18:2 (Matreya, Inc., Pleasant Gap, PA); Linolenic acid 18:3 (Matreya, Inc., Pleasant Gap, PA); Palmitic acid 16:0 (Matreya, Inc., Pleasant Gap, PA); Oleic acid 18:1 (Matreya, Inc., Pleasant Gap, PA); Stearic acid 18:0 (Matreya, Inc., Desi Gap, PA); Myristic acid 14:0 (Matreya, Inc., Desi Gap, PA); Why acid 12:0 (Matreya, Inc.,
  • a reverse phase HPLC procedure used to separate and quantitate the derivatized anthroyl FFAs A reverse phase "C8" column (4.6 cm x 25 cm, 5 micron Spherisorb® C8, AUtech Associates, Inc. Deerfield, IL) separated the saturated FFAs. A 3 micron, 15 cm “C18” column was connected to the HPLC followed by a 5 micron, 25 cm “C8” column.
  • the high performance liquid chromatograph was a model 517 from Gilson Medical Electronics, Inc., Middleton, WI. Two detectors were connected in tandem. The first was Model UVIS 200 from Linear Instruments, Reno, NV. The second was Model 121 Fluorometer from Gilson Medical Electronics.
  • the present invention provides a method for preventing the progression of viral diseases by inhibiting a host cell viral replication mechanism comprising administering an effective amount of a compound that can inhibit myrPA formation in stimulated cells.
  • the compounds described herein exert their antiviral activity by inhibiting one or a plurality of enzymes or an enzyme complex, resulting in a diminution of host (or infected) cell myrPA formation in response to an inflammatory stimulus (e.g., TNF or IL-1). Therefore, the inventive compounds describe a genus of compounds that share a common mechanism of action, on a cellular level, that results in decreased intracellular signaling within a virally-infected cell, that does not transmit an inflammatory signal that would otherwise (in the absence of drug) signal an infected cell to begin the process of viron replication, assembly and viron shedding.
  • an inflammatory stimulus e.g., TNF or IL-1
  • This cellular and biochemical mechanism of action provides evidence for a general antiviral activity of the genus of compounds described herein, or other compounds that exert a similar mechanism of action (that is, prevention of infected host cell PA accumulation in response to an inflammatory stimulus) by virtue of the ability of the drug to prevent normal viral replication within an infected host cell.
  • viruses that also replicated in an infected host cell in response to inflammatory stimuli (usually mediated by the cytokines TNF or IL-1) include, for example, cytomegalovirus (CMV), herpes family of viruses, including herpes simplex virus (HSV) 1, 2 and 6, hepatitis A, B, C and D, HTV 1 and 2, Epstein Barr virus (EBV), human T cell leukemia virus (HTLV), human papiloma virus, influenza, parainfluenza, respiratory syncytial virus, all adenoviruses, and rhinoviruses.
  • CMV cytomegalovirus
  • HSV herpes simplex virus
  • HSV herpes simplex virus
  • HTLV human T cell leukemia virus
  • human papiloma virus influenza, parainfluenza, respiratory syncytial virus, all adenoviruses, and rhinoviruses.
  • the inventive method results in prevention of progression and treatment of a viral disease, comprising administering an effective amount of a compound that can reduce intracellular myrPA concentrations in response to an inflammatory stimulus.
  • viral diseases include, for example, CMV retinitis, ADDS, systemic viral diseases affecting immunocompromised individuals
  • HTV-1 cold sores
  • HSV-2 genital herpes
  • A, B or C, or HSV-6 genital warts
  • EBV infectious mononucleosis and some lymphomas
  • shingles viralella zoster
  • pericarditis coxsackie virus
  • influenza influenza
  • cold and flu rhinoviruses and adenoviruses
  • HSV is its prolonged incubation period during which the virus is harbored in a quiescent state in (in the case of HTV-l) CD4 lymphocytes and mononuclear phagocytes.
  • inflammatory cofactors that can accelerate progression of the disease by upregulating expression of the virus, such as HTV-l, in chronically infected cells.
  • One such cofactor for HTV-l infection is human cytomegalovirus (HCMV), which is a cause of serious opportunistic infection in AIDS patients.
  • HCMV human cytomegalovirus
  • cytokines particularly TNF, IL-6 and IL-1 play an important role in upregulating viral replication in infected cells (Clouse et al., J. Immunol. 143:470, 1989). Therefore, blocking intracellular signaling of such inflammatory cytokines can serve to ablate the inflammatory signal and inhibit the upregulating event that normally triggers viral replication in infected cells.
  • the mammalian cell membrane associated enzyme lysophosphatidate acyl-CoA acyltransferase catalyzes the transfer of acyl CoA from lysophosphatidic acid (lyso-PA) to phosphatidic acid (PA) species.
  • lyso-PA lysophosphatidic acid
  • PA phosphatidic acid
  • Certain PA species function as lipid intermediates in cell activation and function directly as intracellular signaling molecules. PA is subsequently dephosphorylated to 1,2-sn diacylglycerol (DAG) via phosphatidate phosphohydrolase (PAH).
  • DAG 1,2-sn diacylglycerol
  • PAH phosphatidate phosphohydrolase
  • the compounds that inhibit viral replication in infected cells also have decreased amounts of intracellular PA species that are formed in response to inflammatory cellular activation.
  • CT1501R R-l(5-hydroxyhexyl)3,7- dimethylxanthine) or lisofylline (generic name) demonstrates antiviral activity by inhibiting HTV viron replication in infected monocytic cells stimulated to produce virons by TNF activation.
  • CT1501R is also and inhibitor of LPAAT in vitro with competitive inhibition in a Lineweaver-Burke graph and an IC50 for LPAAT inhibition in the range of 200 to 400 nM.
  • PA derived from lyso-PA is an important intracellular signaling intermediate for TNF or IL-1 induced viron replication in virally-infected cells
  • inhibitors of PA formation will act to prevent progression of viral diseases by blocking cell signaling to stimulate viron formation.
  • the compounds illustrated herein that are shown to inhibit viron formation or block promoter activity also inhibit PA formation. This invention further provides a method of delaying or preventing the occurrence of
  • AIDS which comprises administering to an HTV seropositive human an effective amount of a compound, or a pharmaceutically acceptable salt or hydrate or solvate thereof, wherein said compound can block intracellular myrPA formation from lyso-PA.
  • said compound is an amino alcohol-substituted or chiral primary or secondary alcohol-substituted heterocyclic compound wherein the heterocyclic moiety is a substituted or unsubstituted xanthine, a substituted or unsubstituted uracil or a substituted or unsubstituted thymine.
  • An amino alcohol or chiral primary or secondary alcohol compound, or a pharmaceutically acceptable salt or hydrate or solvate thereof can be administered to such human in a conventional dosage form prepared by combining the amino alcohol or chiral primary or secondary alcohol compound or a pharmaceutically acceptable salt or hydrate or solvate thereof, with a conventional pharmaceutically acceptable carrier or diluent according to known pharmaceutical formulation techniques.
  • the compound may be formulated into an opthalmic formulation for ocular administration to treat, for example, CMV retinitis.
  • Topical formulations of compound are appropriate for the treatment of herpes types I and JT and for papiloma virus.
  • the compound that can inhibit myristilated PA (myrPA) in either the sn-1 or sn-2 positions or both can be determined by following an assay procedure described herein.
  • the compound is a small organic molecule that can mimic binding to a complex of enzymes that mediate signal amplification.
  • the compounds include resolved enantiomers and/or diastereomers, hydrates, salts, solvates and mixtures thereof, the compounds having a straight or branched aliphatic hydrocarbon structure of formula I:
  • n is an integer from one to four and m is an integer from four to twenty.
  • R ⁇ and R2 are hydrogen, a straight or branched chain alkyl, alkenyl or alkynyl of up to twenty carbon atoms in length or -(CH2) ⁇ y 5- If ⁇ 1 or ⁇ 2 ls ⁇ (CH2) W R5, w may be an integer from one to twenty and R5 may be an hydroxyl, halo, Cj_g alkoxyl group or a substituted or unsubstituted carbocycle or heterocycle.
  • Ri and R2 may jointly form a substituted or unsubstituted, saturated or unsaturated heterocycle having from four to eight carbon atoms, N being a hetero atom of the resulting heterocycle.
  • R3 may be hydrogen, a hydroxy group, a Ci .3 straight or branched alkyl, or a C j _3 alkoxy.
  • Preferred compounds may have one of R ⁇ or R2 and R3 that form a substituted or unsubstituted linking carbon chain, having from one to four carbon atoms.
  • This R1/R3 or R2/R3 linking chain will join the O and N in a cyclic structure, an integer sum equal to n + a number of carbon atoms in the linking carbon chain being less than six.
  • a total sum of carbon atoms comprising Ri or R2, (CH2) n and
  • R4 is a terminal moiety comprising a substituted or unsubstituted, heterocyclic moiety, wherein the heterocyclic moiety consists essentially of one to three ring structures having 5-7 members each, a heteroatom, and a predominantly planar structure or essentially aromatic. However, if R4 is phthalimide, m of formula I is not less than five.
  • the compounds may include resolved enantiomers and/or diastereomers, hydrates, salts, solvates and mixtures thereof that have a straight or branched aliphatic hydrocarbon structure of formula II:
  • Rg and R7 are hydrogen, a straight or branched chain alkane, alkene or alkyne of up to twenty carbon atoms in length, or -(CH2) x Rg, at least one of Rg or R7 being -(CH2) x Rg.
  • x is an integer from zero to fourteen and Rg is a moiety having a general structure as provided in formula DI
  • R9 is H or a straight or branched chain alkane, alkene or alkyne of up to twenty carbon atoms in length.
  • R4 may be selected from the group consisting of substituted or unsubstituted acridinyl; acridonyl; alkylpyridinyl; anthraquinonyl; ascorbyl; azaazulenyl; azabenzanthracenyl; azabenzanthrenyl; azabenzophenanthrenyl; azachrysenyl; azacyclazinyl; azaindolyl; azanaphthacenyl; azanaphthalenyl; azapyrenyl; azatriphenylenyl; azepinyl; azinoindolyl; azinopyrrolyl; benzacridinyl; benzazapinyl; benzofuryl;
  • the most preferred ring systems (R 4 ) include, for example, dimethylxanthinyl, methylxanthinyl, phthalimidyl, homophthalimidyl, methylbenzoyleneureayl, quinazolinonyl, octylcarboxamidobenzenyl, methylbenzamidyl, methyldioxotetrahydropteridinyl, glutarimidyl, piperidonyl, succinimidyl, dimethoxybenzenyl, methyldihydrouracilyl, methyluracilyl, methylthyminyl, piperidinyl, dihydroxybenzenyl, or methylpurinyl, even more preferably, methylxanthinyl, dimethylxanthinyl or a derivative thereof.
  • Preferred and exemplary compounds are used throughout the specification and are designated by CT# according to Table 1 below.
  • CT1501R R- 1 -(5-hydroxyhexyl)-3,7-dimethylxanthine CT1115 N-( 11 -octylamino- 10-hydroxyundecyl)- homophthalimide
  • CT1827 3-( 11 -octylamino- 10-hydroxyundecyl)- 1 - methyluracil
  • CT1829 3-( 11 -octylamino- 10-hydroxyundecyl)- 1 - methyldihydrouracil
  • CT2573 l-(9-dodecylamino-8-hydroxynonyl)-3,7- dimethylxanthine CT2575 1 -( 1 -hexylamino-8-hydroxyundecyl)-3 ,7- dimethylxanthine CT3528 N-(l l-phenylamino-10-hydroxundecyl)-3,7- dimethylxanthine CT2576 1 -( 11 -octylamino- 10-hy droxyundecyl)-3 ,7- dimethylxanthine CT3556 1 -( 11 -N-octylaminoundecyl)-3,7- dimethylxanthine CT3537 1-[1 l-(N-octylacetamido)-10- acetoxyundecyl]-3,7-dimethylxanthine CT3534 l-(9-(2-hydroxydecyl-l-amino)non
  • An assay for measuring the anti-viral activity of the compounds involves testing the abilities of the compounds to inhibit gene expression directed by specific viral promoters in cell lines.
  • a plasmid construct, pCMV.AP using the human cytomegalovirus (CMV) enhancer and promoter to direct the expression of the secreted human placental alkaline phosphatase reporter gene were transformed into a tumor cell line (e.g., 293-EBNA cells). The cultured cells were treated with various concentrations of the compounds.
  • CMV human cytomegalovirus
  • alkaline phosphatase (AP) reporter gene in the individual cultures can then be measured by following the change in absorbance at A405 of the cell conditioned media in the presence of a suitable substrate (e.g., ortho-nitrophenol phosphate) (Berger et al., Gene 66JJ988).
  • a suitable substrate e.g., ortho-nitrophenol phosphate
  • expression vectors for secreted placental alkaline phosphatase was performed by obtaining a mammalian episomal expression vector pBL3, derived from the plasmids pMEP4 and pCEP4 (Invitrogen Corp). Specifically, a 600 bp Spe I - Kpn I fragment spanning the CMV promoter from pCEP4 and a 9500 bp Xba I — Kpn I vector fragment from pMEP4 were isolated and ligated together to form pBL3.
  • sPAP cDNA was amplified by PCR using the plasmid pGEM4Z/SEAP (ATCC) as template with primers 5'-GGATCCTCTAGACATGCTGGGGCCCTGCA-3' (SEQ ID NO. 1) and 5'- GGATCCGTCGACGTTAACCCGGGTGCGCG-3' (SEQ ID NO. 2).
  • the PCR product was then digested with Xba I and Sal I and inserted between the Nhe I site and the Xho I site within the multiple cloning region of pBL3.
  • the resulting plasmid was designated as pCMV.AP.
  • the plasmid construct, pHTV.AP using the human immunodeficiency virus (HTV) long terminal repeat (LTR) promoter (derived from pU3R-DI CAT, Sodroski et al., Science 227:171, 1985) to direct the expression of the sPAP reporter gene, was made by replacing the CMV promoter region from pCMV.AP with the HTV-LTR promoter.
  • pHTV.AP was co- transfected with pS V2tat72, an expression vector for the 72 amino acids tat protein from HTV (Frankel and Pabo, Cell 55:1189-1193, 1988), into 293-EBNA cells (Invitrogen). The cultured cells were treated with various concentrations of the compounds.
  • alkaline phosphatase (AP) reporter gene was measured by following change in absorbance at 405 nm of the cell conditioned media in the presence of a suitable substrate (e.g., ortho-nitrophenol phosphate) (Berger et al., Gene 66:1-10, 1988).
  • a suitable substrate e.g., ortho-nitrophenol phosphate
  • Another assay examined the effect of compounds on chronically HTV-l infected Ul cells. A relatively high density of Ul cells at 10 ⁇ cells/well were incubated in the absence or presence of compounds for 4 days.
  • HTV-l p24 antigen expression in the culture media was measured by using a ELISA kit (Abbott).
  • Ul cells were plated at a relatively low density of 2x10 ⁇ cell/well plus or minus TNF at 20 pg/ml and plus or minus compounds.
  • the expression of HTV-l p24 antigen in culture media was measured by ELISA four days later.
  • the mechanism of action of the compounds in inhibiting both HTV and CMV viron replication coupled with their biochemical mechanism of action in inhibiting specific intacellular signaling pathways, provides a conclusion that not only the illustrated compounds, but other compounds that have similar mechanisms of action in inhibiting intracellular signaling through myrPA, are therapeutically useful to treat or prevent progression of a large number of viral infections.
  • Plasmid pREP7 (Invitrogen) was converted to pREP7b by deleting 2,040 bp EBNA-1 coding region and by replacing the pBR322 origin of replication (ori) with the pBluescript KS ⁇ (Stratagene) ori region.
  • the cDNA for secreted placental alkaline phosphatase was generated by PCR with the primers (5'- GGATCCTCTAGACATGCTGGGGCCCTGCATGC-3') (SEQ ID NO. 3) and (5 - AAGCTTGTCGACGTTAACCCGGGTGCGCGGC-3') (SEQ TD NO. 4)using the plasmid pGEM-4Z/PLAP489 (American Type Culture Collection) as the template.
  • the 2,000 bp Xba I - Sai l fragment obtained was ligated into a Nhe I / Xho I vector derived from pREP7b to generate the plasmid pREP7b.
  • AP The 2,000 bp Xba I - Sai l fragment obtained was ligated into a Nhe I / Xho I vector derived from pREP7b to generate the plasmid pREP7b.
  • the 44 bp Xba I - Bgl IT fragment from plasmid pMEP4 (Invitrogen) and the 36 bp Ba R I - Kpn I fragment from pREP7 were ligated into the Xba I - Kpn I vector fragment from pREP7b.AP to generate the plasmid pMCS.AP.
  • the 720 bp Xho I - Hind IE fragment encoding the HIN-LTR promoter from the plasmid pU3R- m-CAT was ligated into the Xho I - Hind m vector fragment of pMCS.AP to generate the plasmid pHTV.AP.
  • the CMN promoter was derived from the 600 bp Spe I - Asp718 1 fragment from pCEP4 (Invitrogen).
  • the 360 bp SV40 early promoter fragments were produced from pSN2tat72 (Frankel et al., Cell 55: 1189-1193, 1988) by PCR using the primers (5'- GAGGCAGCTCTAGAATGTGTGTCAGTTA-3') (SEQ TD NO. 5) and (5'- GTCTACCGGTACCAAGCTTTTTGCAA-3 , ) (SEQ ID NO. 6).
  • the 480 bp phosphoglycerate kinase (PGK) promoter fragment was amplified from human placental genomic DNA (Promega, Madison, WI) based on published sequence (Pfeifer et al., Science 246:810-813, 1990) using the primers (5'-GGAATTCTAGAGGTTGGGGTTGCGCCTT-3') (SEQ ID NO. 7) and (5'-AACGAGGGAGCCGGGTACCGACGTGCGC-3') (SEQ ID NO. 8).
  • the above fragments were ligated into the Xba I - Aspl IS I vector fragment of pHTV.AP to generate pHIV2.AP, pHIN3.AP, pCMV.AP, pSVE.AP and pPGK.AP respectively.
  • the t t-expression plasmid directed by HTV-LTR promoter, pHTV.tat was generated by inserting the 400 bp EcoRN - Hindm fragment from pHIV.AP into the PVHII - Hindlll vector fragment from pSY2tat!2 (Frankel et al., Cell 55: 1189-1193, 1988). ⁇ FKB binding activity in nuclear extracts (Dignam et al., Nucleic Acids Res.
  • Transient transfections with various expression plasmids were performed on a human kidney embryonic cell line 293- ⁇ BNA (Invitrogen) using the cationic lipid DOTAP (Boehringer Mannheim).
  • the cell culture media were changed 24 hr after transfection into a serum-free medium AIM V (Life Technologies) before addition of compounds or TNF- ⁇ (PeproTech) at the appropriate concentrations.
  • AP activity measured by a colorimetric assay (Berger et al., Gene 66:1-10, 1988) was performed 19-24 hr later.
  • Stable cell lines 293tar, 293tat and 293tat2 were generated by transfections using conditions described in (Suresh et al., FASEB J. 4:227-231, 1990) with the plasmid pHTV.AP alone or along with either pHTV.t ⁇ t or pSN2t ⁇ t72, respectively.
  • the assay of HTV-l p24 antigen expression in the chronically HTV-l -infected promonocytic line Ul after 4 days and in acutely infected peripheral blood lymphocytes (PBL) after 7 days were described (Peterson et al., J. Clin. Invest. 89:574-580, 1992, and Brighty et al., Proc. Natl. Acad. Sci. USA 88:7802-7805, 1991).
  • the form and characterof the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
  • a therapeutic compound or a pharmaceutically acceptable salt or hydrate or solvate thereof is administered in a virally infected patient (e.g., a HIN seropositive human) in an amount sufficient to prevent or delay the occurrence of further viral infection and clinical symptoms of the virally-mediated disease (e.g., AIDS).
  • the route of administration of the compound is not critical but is usually oral or parenteral, preferably oral.
  • parenteral includes intravenous, intramuscular, subcutaneous, intranasal, intrarectal, transdermal, opthalmic, intravaginal or intraperitoneal administration.
  • the subcutaneous and intramuscular forms of parenteral administration are generally preferred.
  • the daily parenteral dosage regimen will preferably be from about 0.01 mg/kg to about 25 mg/kg of total body weight, most preferably from about 0.1 mg/kg to about 4 mg/kg.
  • each parenteral dosage unit will contain the active ingredient in an amount of from about 0.1 mg to about 400 mg.
  • the compounds are generally active when given orally and can be formulated as liquids, for example, syrups, suspensions or emulsions, tablets, capsules and lozenges.
  • a liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s), for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavoring or coloring agent.
  • a suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavoring or coloring agent.
  • a composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.
  • a composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule.
  • a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example, aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
  • suitable pharmaceutical carrier(s) for example, aqueous gums, celluloses, silicates or oils
  • the daily oral dosage regimen will preferably be from about 0.01 mg/kg to about 40 mg/kg of total body weight.
  • each oral dosage unit will contain the active ingredient in an amount of from about 0J mg to about 1000 mg.
  • the optimal quantity and spacing of individual dosages of a compound or a pharmaceutically acceptable salt or hydrate or solvate thereof will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment (i.e., the number of doses of a compound or a pharmaceutically acceptable salt or hydrate or solvate thereof given per day and duration of therapy) can be ascertained by those skilled in the art using conventional course of treatment determination tests.
  • the compounds of the present invention can be co-administered with further active ingredients, such as zidovudine, gancyclovir and other compounds with known anti-viral activity mediated by a different mechanism of action than prevention of viron production in infected cells.
  • further active ingredients such as zidovudine, gancyclovir and other compounds with known anti-viral activity mediated by a different mechanism of action than prevention of viron production in infected cells.
  • This example illustrates AP reporter gene activity in 293-EBNA cells transiently transfected with pHIV.AP alone or in combination with pSVtat72.
  • the expression level of AP increased by more than 50 fold in the presence of the tat expression vector, indicating transactivation of the HTV-LTR promoter by the 72 amino acid tat protein ( Figure 1).
  • CT1501R CT1827
  • CT1829 see Table 1 for chemical structures
  • both CT1827 and CT1829 inhibited AP reporter gene expression in 239-EBNA cells with D->50 values in the range of 4 to 6 ⁇ M using the HTV-LTR promoter construct transfected with a tat expression vector.
  • No appreciable inhibition of AP expression was observed with CT1501R ( Figure 2).
  • Figure 3 shows the results of the three compounds on viability of 293-EBNA cells.
  • Cell viability was determined with a calorimetric assay using the tetrazolium salt of MTS (Promega) to report cell proliferation, viability and cytotoxicity.
  • MTS indicates cell activity by serving as a substrate for mitochondria dehydrogenases for formation of soluble formazan dyes which can be quantitated by determining the absorbance at 490 nm using a plate reader. None of the three compounds tested showed significant cytotoxic effect on 293-EBNA cells as the concentrations indicated in Figure 3.
  • EXAMPLE 2 This example illustrates assay results of four compounds (CT1501R, CT1115, CT1416 and CT2573, see Table 1 for chemical names) for AP reporter gene activity in 293- EBNA cells transiently transfected with pHTV.AP alone or in combination with pSVt ⁇ t72.
  • CT 1501 R (again) and CT2573 did not affect AP activity and cell viability significantly, whereas CT1416 and CT2573 were able to inhibit AP expression by 50% at concentrations in the range of 1 to 4 ⁇ M ( Figure 4). However, some cytotoxicity was noted with CT1416 and CT2573 with cellular LD50 values at around 5 ⁇ M ( Figure 5).
  • EXAMPLE 3 EXAMPLE 3
  • This example illustrates assay results of three amino-alcohol-substituted xanthine compounds (CT2575, CT3528, and CT2571) on AP reporter gene activity in 293-EBNA cells stably transfected with pCMV.AP.
  • CT2575 and CT2571 inhibited AP reporter gene expression in 293-EBNA cells with IC50 values of 3 ⁇ M and 8 ⁇ M, respectively. No appreciable inhibition of AP expression was detected when using the compound CT3528 ( Figure 6).
  • Cell viability was measured by a colorometric assay that uses Alamar Blue dye (Alamar, Inc.) to report cell proliferation, viability and cytotoxicity.
  • Alamar Blue dye Alamar, Inc.
  • This vital dye indicates cell activity by serving as substrate for mitochrondrial dehydrogenases for the formation of soluble dyes which can be quantitated by determining the difference in absorbance between A570 and A600 using a plate reader.
  • CT2571 was found to be cytotoxic to 293-EBNA cells while CT2575 and CT3528 showed no significant cytotoxic effect on 293-EBNA cells at the concentrations indicated ( Figure 7).
  • This example illustrates assay results of the CMV assay results for four different amino alcohol-substituted heterocyclic compounds (CT1416, CT1115, CT1829 and CT1827). While all four compounds were able to inhibit AP activity by 50% in the range of 2 to 6 ⁇ M ( Figure 8), CT1416 and CT1115 were found to be cytotoxic with an LD50 value of about 5 ⁇ M ( Figure 9). CT1827 and CT1829 showed no significant cytotoxic effect on 293-EBNA cells at the concentrations indicated. Therefore, a significant therapeutic window exists.
  • EXAMPLE 5 This example illustrates assay results of three compounds (CT2576, CT1620, and CT3534, see Table 1 for chemical structures) in a tat activation of HTV-LTR promoter in EB293 cells.
  • CT2576 inhibited AP reporter gene expression in 239-EBNA cells with ID50 values in the range of 4 to 6 ⁇ M using the HTV-LTR promoter construct transfected with a tat expression vector ( Figure 10).
  • MTS tetrazolium salt of MTS (Promega) to report cell proliferation, viability and cytotoxicity.
  • MTS indicates cell activity by serving as a substrate for mitochondria dehydrogenases for formation of soluble formazan dyes which can be quantitated by determining the absorbance at 490 nm using a plate reader. None of the three compounds tested showed significant cytotoxic effect on 293-EBNA cells at the concentrations indicated ( Figure 11).
  • This example illustrates assay results of an assay with HJN-infected Ul cells where measurement of HIN-1 expression in response to an exogenous stimulant was determined. Niral release was measured by determining the amount of HTV-l p24 antigen (Ag) expression in cell culture supernatants. Compounds CT1501R, CT1829, CT1411 and CT2576 all suppressed HTV-l expression, but CT2576 was the most effective and CT1827 showed some cytotoxicity ( Figure 12, top panel).
  • FIG. 12 bottom panel shows the effect of several compounds on T ⁇ F ⁇ mediated upregulation of HTV-l in Ul cells.
  • T ⁇ F ⁇ is a potent stimulus of viral expression.
  • CT1501R, CT1829, CT1411 and CT2576 all suppressed HTV-l expression, but CT2576 was the most effective and CT1827 showed some cytotoxicity at a 25 ⁇ M concentration.
  • CT2576 suppressed both tat-dependent and T ⁇ F- ⁇ induced HTV-LTR transcription with an IC50 of approximately 10 ⁇ M.
  • Study of the effects of CT2576 on cell toxicity was performed using the Alamar BlueTM dye (Alamar Biosciences) according to the manufacturer's instructions.
  • CT2576 was not toxic to 293- EB ⁇ A cells at concentrations up to 35 ⁇ M.
  • To show CT2576 can inhibit HIN expression CT2576 was tested for the ability to block HTV p24 antigen expression in chronically or acutely infected cells.
  • CT2576 inhibited the T ⁇ F- ⁇ and JL-6 induced and the constitutive expression of HTV in the chronically infected Ul cells and in peripheral blood lymphocytes (PBL) freshly infected with a HTV strain from a clinical isolate with an IC50 of approximately 1 ⁇ M.
  • Pharmacological inhibition of synthesis of selected PL may therefore be a novel therapeutic approach to suppression of HTV replication.
  • CT2576 altered the pattern of PA composition in 293tat cells.
  • One of the steps after cell activation involves the hydrolysis of phosphatidylcholine (PC), the major phospholipid of cellular membranes, by phospholipases C and D, leading to the generation of second messengers, such as PA and DG, in the signal transduction cascade.
  • HPLC analysis of the total cellular phospholipid content (by the method described herein) of 293tat cells showed several difference in specific subspecies of PA After brief T ⁇ F- ⁇ stimulation (45 sec), a
  • the long chain unsaturated PA comprised 15% of the far UV absorbing lipids after preincubation with CT2576 and it increased to 20% after 2 minutes of stimulation with TNF- ⁇ .
  • CT2576 affected the turnover of PA subspecies in 293tat cells.
  • EXAMPLE 8 This example illustrates that CT2576 inhibited TNF- ⁇ and t ⁇ t-activated HTV-LTR promoter directed expression in 293tat cells.
  • Cell activation signals generated by interaction of ligands with receptors are eventually converted to changes in the pattern of gene expression.
  • a reporter gene expression system under the control of the HTV-LTR promoter was set up to investigate compounds that inhibit signaling pathway for HTV activation in infected cells.
  • AP reporter gene activity expressed in 293-EB ⁇ A cells transfected with either pHTV.AP alone or in combination with pSV2t ⁇ t72, pHIN.t ⁇ t or with T ⁇ F- ⁇ stimulation is shown in Figure 14.
  • the plasmid pHTV.AP contains the full-length HIN-LTR sequence that extends from nucleotides -453 to +80.
  • the expression level of AP was low using the pHTV.AP construct alone, indicating the presence of negative regulatory elements in the HIN-LTR sequence.
  • Induction with T ⁇ F- ⁇ increased the AP expression level by about 4 fold.
  • the AP expression level increased by 15 to 70 fold in the presence of the tat expression plasmids pHTV.t ⁇ t or pS V2t ⁇ t72.
  • T ⁇ F- ⁇ and t ⁇ t in combination enhanced the AP expression level by two fold when using the pSV2tot72 construct, and by ten fold when using the pHTV.t ⁇ t construct, but the final levels of AP activity were similar.
  • CT2576 inhibited AP reporter gene expression by 50% with an inhibition constant (IC50) in the range of 10 ⁇ M with or without T ⁇ F- ⁇ induction.
  • IC50 inhibition constant
  • the inhibition of AP expression is the result of two events: a decrease in AP expression and a decrease in tat expression and therefore lower tat protein levels for transactivation.
  • CT2576 did not affect the activation of ⁇ FKB in 293t ⁇ t cells.
  • ⁇ FKB is the major transcription factor involved in the activation of HTV-LTR promoter by T ⁇ F- ⁇ (Baeuerle et al., Annu. Rev. Immunol. 12:141-179, 1994)
  • the inhibition of TNF- ⁇ signaling by CT2576 shows that CT2576 interacted with a step in the signal transduction pathway leading to the activation of NFKB.
  • Electrophoretic mobility shift assay ( Figure 16) showed NFKB was activated in 293t ⁇ t cells upon induction with either TNF- ⁇ or IL-1 ⁇ , and CT2576 did not inhibit TNF- ⁇ dependent activation of NFKB.
  • This example illustrates that CT2576 inhibited both constitutive and cytokine-induced HTV expression in a chronically-infected Ul cell line.
  • a chronically HTV-l infected human promonocyte cell line, Ul (Poli et al., J. Exp. Med. 172:151-158, 1990), was used as a model to see if the inhibitory effect of CT2576 on HIV-LTR driven reporter gene expression could be extended to block HTV replication per se in infected cells.
  • TNF- ⁇ and IL-6 have been reported to upregulate the expression of HTV-l in Ul cells (Poli et al., Proc. Natl. Acad. Sci. USA 91:108-112, 1994).
  • Ul cells were plated onto 24- well microtiter plates to study the dose-response effect of CT2576 on the constitutive (10 ⁇ cells/well), TNF- ⁇ or IL- 6 mediated (2 x 10 ⁇ cells/well) expression of HTV-l.
  • CT2576 was capable of inhibiting the constitutive, the TNF- ⁇ mediated, as well as the IL-6 mediated expression of HTV in Ul cells with an IC50 value of less than 1 ⁇ M (Fig 17).
  • CT3537 also inhibited HTV expression in acutely infected cells.
  • CT2576 acetylated form of CT2576, CT-3537
  • PBL a human donor freshly infected with a clinical isolate HTV-l strain JR-CSF.
  • Analysis of p24 antigen release 7 days after infection at various concentrations of CT3537 showed that CT3537 inhibited HTV expression with an IC50 of less than 1 ⁇ M ( Figure 17).
  • CT2576 This example summarizes the data for the preferred embodiment, CT2576.
  • CT2576 which affects the generation of myrPA species in 293tat cells (Figure 13), inhibited tat and TNF- ⁇ induced activation of HIN-LTR directed AP expression with an IC50 of 10 ⁇ M ( Figure 15) with minimal cytotoxic effect.
  • Gel-shift assays and Northern blot analysis showed CT2576 did not block expression at the transcription stage ( Figure 16).
  • Various mechanisms of post-transcriptional regulation of gene expression have been described
  • CT2576 blocks activation of the HIN-LTR expression post-transcriptionally, CT2576 has a synergistic effect with other anti-HTV compounds that act by different mechanisms. CT2576 is also effective in suppressing HTV expression in cell types with constitutive ⁇ FKB activity, such as certain neurons involved in the etiology of AIDS dementia complex (Kaltschmidt et _ ., Mol. Cell. Biol 14: 3981-3992, 1994).
  • An inhibitor of viral reverse transcriptase is expected to only protect new cells from becoming infected but not to affect viral production from cells already infected with HTV.
  • Analysis by HTV-l p24 antigen production in Ul cell culture media showed that CT2576 inhibited T ⁇ F- ⁇ , IL-6 mediated and the constitutive expression in Ul cells ( Figure 17) with no cytotoxicity using the trypan-blue dye exclusion assay. While T ⁇ F- ⁇ induces HIN expression through activation of ⁇ FKB, EL-6 and IL-1 also stimulates HTV expression through other pathways independent of ⁇ FKB activation in Ul cells.
  • FIG. 18 shows that both compounds (at a clinically achievable concentration of 10 ⁇ M) can inhibit myrPA formation in tat-transfected cells stimulated with serum.
  • Figure 18 shows a relationship between relative PA mass and activity of the compound CT3556.
  • Transformed and serum-stimulated cells were measured for total PA mass according to the methods described herein.
  • the t ⁇ t-transfected cells stimulated with serum (col. 3) showed a significant increase in PA mass when compared with non-tat transfected cells stimulated with serum (col. 1). This increase in PA was blocked by administration of CT3556 10 ⁇ M.
  • cells transfected with tar showed a significant drop in PA mass when treated with CT3556.
  • the columns in Figure 18 represent: (1)
  • FIG. 19 shows the same experiment as in Figure 18 only this time diacylglycerol was measured (DG).
  • DG was assayed by collecting the DG HPLC peak (Rf 2-4.5 min) and verifying the predominance of DG species by mass spectroscopy analysis. The DG peak was integrated for a quantitave analysis.
  • the six columns in Figure 19 correspond to the same columns in Figure 18.
  • Figure 20 shows a dose response relationship for CT2576 in inhibiting total PA mass in EB293 cells transfected with the tat expression plasmid and stimulated with serum.
  • the time points for PA determinations were from 0 to one minute.
  • Figure 21 shows two graphs for peak D (a measure of various PA species in an HPLC separation) and Peak A5 (a measure of lyso (bis) PA species) with EB293 cells (human embryonic origin) stimulated with human TNF- ⁇ (20 ng/ml, Genzyme) with or without treatment with CT2576 (10 ⁇ M.).
  • the left panel shows that drug treatment reduced PA mass across all time points measured, indicating inhibition of formation of PA species to acceleration of PA species metabolism.
  • the CT2576 activity inhibits PA formation from lyso (bis) PA species.
  • ADDRESSEE Cell Therapeutics, Inc.
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL no
  • ANTI-SENSE no
  • SEQUENCE DESCRIPTION SEQ ID NO: 5: GAG GCA GCT CTA GAA TGT GTG TCA GTT A 31

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Abstract

There is disclosed a method of preventing or delaying the occurrence of acquired immunodeficiency syndrome (AIDS) in human immunodeficiency virus (HIV) seropositive humans by administering an effective amount of a compound that inhibits cellular signaling through a specific phospholipid-based cellular signaling and signal amplification pathway. The invention further provides a method for preventing or delaying clinical symptoms of a group of viral diseases wherein the viral disease is mediated by host cell viral replication. The invention provides an advantage by attacking host cellular signaling mechanisms to prevent the development of drug resistance from rapidly mutating viruses.

Description

METHOD FOR INHIBITING INTRACELLULAR VIRAL REPLICATION
Technical Field of the Invention The present invention provides a method of preventing or delaying the occurrence of acquired immunodeficiency syndrome (AIDS) in human immunodeficiency virus (HTV) seropositive humans by administering an effective amount of a compound that inhibits cellular signaling through a specific phospholipid-based cellular signaling and signal amplification pathway. The invention further provides a method for preventing or delaying clinical symptoms of a group of viral diseases wherein the viral disease is mediated by host cell viral replication. The invention provides an advantage by attacking host cellular signaling mechanisms to prevent the development of drug resistance from rapidly mutating viruses.
Background of the Invention
Drugs currently approved for AIDS therapy belong to classes of compounds that directly attack viral proteins, such as reverse transcriptase (e.g., AZT or zidovudine) or HTV protease. Such compounds have the problem of rapidly developing drug resistance due to rapid transformation of the infecting viral genome. However, despite the problem of rapid drug resistance, such drugs have been used to treat HTV seropositive individuals to help prevent the spread and progression of the infection into the clinical symptoms of AIDS. This problem is exemplified by a recent clinical study (Aboulker et al., Lancet 341:889-890, 1993) showing that AZT treatment of asymptomatic patients in early stages of HIV infection showed no delay in the progression of AIDS disease, although a consistently higher number of CD4+ T cells was observed in the treatment group when compared to the placebo group, suggesting that CD4 may not be a good marker for AIDS drug efficacy and the need for other approaches for the treatment of AIDS.
HIV infection leads to progressive depletion of CD4+ T lymphocytes and eventual development of clinical symptoms associated with AIDS. After the primary HTV infection, there is a prolonged incubation period of clinical latency that can last as long as twelve years (Weiss, Science 260:1273-1279, 1993). Recent studies indicate viral replication occurs continuously (Pantaleo et al., Nature 362:355-358, 1993) and multiple components of the immune system are chronically activated (Fauci, Science 262:1011-1018, 1993; Bass et al., Clin Immunol Immunopathol. 64:63-70, 1992) during this asymptomatic phase of disease. Persistent activation of the immune system may lead to overproduction of a number of cytokines (Fauci, Science 262:1011-1018, 1993), induction of HIV expression in latently infected cells (Rosenberg et al., AIDS Res. Hum. Retroviruses 5:1-4, 1989), and apoptosis of T cells (Gougeon, Science 260:1269-1270, 1993). Like other retroviruses, all the genes of HTV are expressed under the control of the long terminal repeat (LTR) promoter through multiply spliced mRNAs and precursor proteins that are processed into individual products (Zeichner, Clin. Perinatol. 21:39-73, 1994). Unique DNA and RNA elements in the HIV LTR make this region a target for regulation by many cellular transcription factors as well as the HIV trans-activator protein tat (Gaynor, AIDS 6:347-363, 1992; and Jones et al., Annu. Rev. Biochem. 63:111-143, 1994). The LTR promoter contains cw-acting repressive sequences that inhibit HIV transcription initiation in resting T cells (Lu et al., J. Virol. 64:5226-5229, 1990). Mitogenic stimulation of T cells can activate the LTR promoter to synthesize large amounts of viral RNA. Cytokines, such as tumor necrosis factor (TNF-α) and interleukin-6 (IL-6), are known to induce HTV synthesis in infected cells (Poli et al., Semin. Immunol. 5: 165-173, 1993).
HTV infection leads to progressive depletion of specific populations of T lymphocytes and progression of the infection to clinical symptoms associated with AIDS (acquired immunodeficiency syndrome), including HJN-related dementia. Currently, there is no effective treatment for HIV-related dementia, although current therapy, AZT, may have some minor short-term effectiveness.
Microglial cells and TΝFα production have been implicated in the neuropathogenesis of HIV-related dementia. Moreover, microglia and TΝFα release have been suggested to play a role in other neurodegenerative disorders, such as Alzheimer's disease and multiple sclerosis.
The use of immunosuppressive and immunomodulatory agents have been shown to suppress viral replication. Specifically, immunomodulating CD8 lymphocytes have been shown to suppress replication of HIV in peripheral blood mononuclear cells (Waler et al., Science 234:1563, 1986) and activated CD8+ T cells have been shown to inhibit replication of HTV in cultures of CD4+ cells from asymptomatic HIV seropositive individuals (Brinchmann et al., /. Immunol. 144:2691, 1990). Further, the immunosuppressive compound, cyclosporin A (CyA) has a protective effect in several animal models of viral infection. Specifically, chronic treatment with CyA before and after infection with LP-BM5 murine leukemia virus was effective against the development of immunodeficiency disease (Cerny et al., Ewr. J. Immunol. 21:1747, 1991). There is also evidence that CyA increases T4 cells and inhibits lymphadenopathy in AIDS and HlV-seropositive, non-ATDS patients (Andrieu et al., Immunol. Immunopath. 46:181, 1988).
The HIV genome encodes at least seven groups of viral proteins (Haseltine, FASEB 5:2349, 1991). These groups of proteins include the three classes of polypeptides present in the majority of animal retroviruses: (1) structural, nonenvelope polypeptides encoded by the gag gene; (2) enzymes required for viron replication (reverse transcriptase/rt) and for cleavage of viral precursor proteins (protease/pr), both encoded by the pol gene; and (3) envelope polypeptides encoded by the env genes. In addition, HIV's possess genes that encode four sets of polypeptides not found in the majority of typical retroviruses. These include a transactivating regulator of RNA translation (tat), a cw-acting downregulator of RNA transcription (nef), a regulator that modulates the expression of structural proteins (rev), a protein that modulates viral infectivity i if), and two proteins (vpr, vpu) whose functions are still unclear. The genomic order of viral genes encoding the initial portion of each of these proteins (5' to 3') is gag-pr-pol-vif-tat-rev-env-nef.
There are a number of models for predicting the effectiveness of a particular therapy for preventing the progression of HTV infection into an ADDS syndrome and related HIV- related dementia. One such key indicator is the tat protein of HIV type 1 (HIN-1), which is a potent trans-activator of expression of genes from the viral long term repeat (LTR) in vitro, and is essential for viral replication and virus-mediated cytopathicity (Narmus, Genes Dev. 2:1055, 1988 and Cullen, FASEB J. 5:2361, 1991). Tat appears to exert its effect through novel mechanisms that depend upon the recognition of specific, structured, ex¬ acting viral DΝA sequences. The tat gene encodes a 14 kD protein that transactivates and increases transcription of DΝA in HTN-infected cells. The tat gene consists of two separate segments that have been mapped to a location in the HTN genome between the vif and nef regions. It encodes for a 14 kD protein that exists as a metal-linked dimer in infected cells. Products of the tat gene have been shown to greatly augment the rate of viral protein synthesis in HTN-infected cells, and thus to increase the production of HIN virons.
Viral replication of a large number of classes of viruses occurs in a host cell and is often accelerated by primary inflammatory mediators, such as tumor necrosis factor (TΝF) (Poli et al., Proc. Natl. Acad. Sci. USA 87:782, 1990). Therefore, if a drug were able to ablate or significantly diminish the signal to replicate in a virally infected host cell, such a drug could conceivably block the progression of a virus-based disease in a large number of indications. The present invention was made in an effort to find such a drug that acts by such a mechanism of action to have broad spectrum antiviral activity directed toward blocking propagation of the virus but not directly cytotoxic to the virus.
Therefore, there is also a need in the art to develop therapeutic compounds for HTN infection that can block the progression of the disease by acting intracellularly to prevent formation of the tat protein and thereby prevent viron assembly within an infected cell. This invention follows a discovery concerning a functional class of compounds that inhibit a specific group of phospholipid-based second messenger signal amplification pathways also inhibit transactivation of HIV-LTR promoter by tαt protein with minimal cytotoxic effects.
Summary of the Invention
The present invention provides a method for treating viral infection by preventing viral replication in infected cells by inhibiting host cell viral replication signaling, comprising administering an effective amount of a compound that inhibits formation of myristilated PA (Phosphatidic acid) in response to an inflammatory stimulus. An inflammatory stimulus can be mediated by, for example, TNF or TL-1. The compound that can inhibit myristilated PA (myrPA) in either the sn-1 or sn-2 positions or both can be determined by following an assay procedure described herein. Preferably, the compound is a small organic molecule that can mimic binding to a complex of enzymes that mediate signal amplification. The compounds include resolved enantiomers and/or diastereomers, hydrates, salts, solvates and mixtures thereof, the compounds having a straight or branched aliphatic hydrocarbon structure of formula I:
In formula I, n is an integer from one to four and m is an integer from four to twenty. Independently, R\ and R2 are hydrogen, a straight or branched chain alkyl, alkenyl or alkynyl of up to twenty carbon atoms in length or -(CH2)W 5- If Ri or R2 is -(CH2)WR5, w may be an integer from one to twenty and R5 may be an hydroxyl, halo, Ci _g alkoxyl group or a substituted or unsubstituted carbocycle or heterocycle. Alternatively, Ri and R2 may jointly form a substituted or unsubstituted, saturated or unsaturated heterocycle having from four to eight carbon atoms, N being a hetero atom of the resulting heterocycle. R3 may be hydrogen, a hydroxy group, a Ci .3 straight or branched alkyl, or a C 1.3 alkoxy. Preferred compounds may have one of R\ or R2 and R3 that form a substituted or unsubstituted linking carbon chain, having from one to four carbon atoms. This R1/R3 or R2 R3 linking chain will join the O and N in a cyclic structure, an integer sum equal to n + a number of carbon atoms in the linking carbon chain being less than six.
In the compounds, a total sum of carbon atoms comprising Ri or R2, (CH2)n and (CH2) does not exceed forty. R4 is a terminal moiety comprising a substituted or unsubstituted, heterocyclic moiety, wherein the heterocyclic moiety consists essentially of one to three ring structures having 5-7 members each, a heteroatom, and a predominantly planar structure or essentially aromatic. However, if R4 is phthalimide, m of formula I is not less than five. The compounds may include resolved enantiomers and/or diastereomers, hydrates, salts, solvates and mixtures thereof that have a straight or branched aliphatic hydrocarbon structure of formula II: R
In the above formula TI, n, m, R3, and R4 are defined as provided in formula I above. Rg and R7 are hydrogen, a straight or branched chain alkane, alkene or alkyne of up to twenty carbon atoms in length, or -(CH2)XR8» at least one of Rg or R7 being -(CH2)χRs- In formula II, x is an integer from zero to fourteen and Rg is a moiety having a general structure as provided in formula HI
R R o
3
R 4 (CH2) m (CH2) p — z — m
H
In formula HI above, m, R3, and R4 are defined as provided in formula I above. Z is N or CH and p is an integer from zero to four. R9 is H or a straight or branched chain alkane, alkene or alkyne of up to twenty carbon atoms in length.
The present invention provides a method for treating viral infections by attacking host cell mechanisms that contribute to intracellular viral replication without attacking directly any viral proteins involved in viral replication. By targeting a host cell signaling mechanism, the illustrated compounds provide an unexpected advantage of avoiding drug resistance from rapid viral mutation that has plagued current AIDS therapies that attack viral reverse transcriptase and protease enzymes. Moreover, the compounds are active against a very wide range of retroviruses because there is no need for viral specificity when targeting common host cell mechanisms. However, the method of treatment is designed to prevent further progression of the virally-mediated disease but not to eradicate the virus. Therefore, the present inventive method includes combination therapeutic regimens that combine an agent designed to prevent host cell viral replication by targeting host cell signaling together with an agent or group of agents that are directly antiviral.
The compounds described herein exert their antiviral activity by inhibiting one or a plurality of enzymes or an enzyme complex, resulting in a diminution of host (or infected) cell PA (phosphatidic acid) formation in response to an inflammatory stimulus (e.g., TNF or IL-1) mediated through a Type I TNF or IL-1 receptor on the host cell surface. Therefore, the inventive compounds describe a genus of compounds that share a common mechanism of action, on a cellular level, that results in decreased intracellular signaling within a virally- infected cell, that does not transmit an inflammatory signal that would otherwise (in the absence of drug) signal an infected cell to begin the process of viron replication, assembly and viron shedding. This cellular and biochemical mechanism of action, as illustrated by cells infected by the HTN virus as described herein, provides evidence for a general antiviral activity of the genus of compounds described herein, or other compounds that exert a similar mechanism of action (that is, prevention of infected host cell PA accumulation in response to an inflammatory stimulus) by virtue of the ability of the drug to prevent normal viral replication within an infected host cell.
Examples of other viruses that also replicated in an infected host cell in response to inflammatory stimuli (usually mediated by the cytokines TΝF or IL-1) include, for example, cytomegalovirus (CMN), herpes family of viruses, including herpes simplex virus (HSN) 1, 2 and 6, hepatitis A, B, C and D, HTV 1 and 2, Epstein Barr virus (EBV), human T cell leukemia virus (HTLV), human papiloma virus, influenza, parainfluenza, respiratory syncytial virus, all adenoviruses, and rhinoviruses. Therefore, the inventive method results in prevention of progression and treatment of a viral disease, comprising administering an effective amount of a compound that can reduce intracellular myrPA concentrations in response to an inflammatory stimulus. Examples of viral diseases include, for example, CMV retinitis, AIDS, ARDS, systemic viral diseases affecting immunocompromised individuals (e.g., AIDS or transplant recipients), cold sores (HSV-1), genital herpes (HSV- 2), hepatitis (A, B or C, or HSV-6), genital warts (human papiloma virus), infectious mononucleosis and some lymphomas (EBV), shingles (Varicella zoster), pericarditis (coxsackie virus), influenza, and cold and flu (rhinoviruses and adenoviruses). As a result of the inventive method to inhibit the progression of a viral disease caused by retroviruses and related families of DΝA viruses, the inventive method further provides a method for treating a consequence of viral infection of the retroviral variety, wherein consequences of retroviral infections include, but are not limited to, cachexia associated with HTV infection, cachexia associated with EBV infection, HTV, EBV and HTLV related malignancies such as Kaposi's sarcoma and lymphoma, ATDS-related opportunistic infections (PCP, MAI, etc.) and for prophylaxis of opportunistic infection in AIDS patients.
Brief Description of the Drawings
Figure 1 shows AP reporter gene activity in 293-EBΝA cells transiently transfected with pHIV. AP alone or in combination with pSVt t72. The expression level of AP increased by more than 50 fold in the presence of the tat expression vector, indicating transactivation of the HTV-LTR promoter by the 72 amino acid tat protein. Figure 2 shows an example of assay results of three compounds (CT1501R, CT1827, and CT1829, see Table 2 for chemical structures). Both CT1827 and CT1829 inhibited AP reporter gene expression in 239-EBNA cells with ID50 values in the range of 4 to 6 μM using the HTV-LTR promoter construct transfected with a tat expression vector. No appreciable inhibition of AP expression was observed with CT1501R.
Figure 3 shows the results of the above-noted compounds on viability of 293-EBNA cells. Cell viability was determined with a colorimetric assay using the tetrazolium salt of MTS (Promega) to report cell proliferation, viability and cytotoxicity. MTS indicates cell activity by serving as a substrate for mitochondria dehydrogenases for formation of soluble formazan dyes which can be quantitated by determining the absorbance at 490 nm using a plate reader. None of the three compounds tested in Figure 2 showed significant cytotoxic effect on 293-EBNA cells as the concentrations indicated.
Figures 4 and 5 show another example of assay results of three other compounds (see Table 1 for chemical names). CT1501R (again) and CT2573 did not affect AP activity and cell viability significantly, whereas CT 1416 and CT2573 (structurally related to CT 1827 and CT1829) were able to inhibit AP expression by 50% at concentrations in the range of 1 to 4 μM. However, some cytotoxicity was noted with CT1416 and CT2573 with cellular LD50 values at around 5 μM.
Figure 6 shows an example of the effect three amino-alcohol-substituted xanthine compounds (CT2575, CT3528, and CT2571) on AP reporter gene activity in 293-EBNA cells stably transfected with pCMV.AP. CT2575 and CT2571 inhibited AP reporter gene expression in 293-EBNA cells with IC50 values of 3 μM and 8 μM, respectively. No appreciable inhibition of AP expression was detected when using the compound CT3528. Figure 7 shows the effect of the three compounds shown in Figure 6 on the viability of 293-EBNA cells. Cell viability was measured by a colorimetric assay that uses Alamar Blue dye (Alamar, Inc.) to report cell proliferation, viability and cytotoxicity. This vital dye indicates cell activity by serving as substrate for mitochrondrial dehydrogenases for the formation of soluble dyes which can be quantitated by determining the difference in absorbance between A570 and A600 using a plate reader. CT2571 was found to be cytotoxic to 293-EBNA cells while CT2575 and CT3528 showed no significant cytotoxic effect on 293-EBNA cells at the concentrations indicated.
Figures 8 and 9 show another example of the CMV assay results (as shown in Figures 6 and 7) of four other amino alcohol-substituted compounds. While all four compounds were able to inhibit AP activity by 50% in the range of 2 to 6 μM, CT1416 and CT1115 were found to be cytotoxic with an LD50 value of about 5 μM, whereas CT1827 and CT1829 showed no significant cytotoxic effect on 293-EBNA cells at the concentrations indicated. Therefore, a significant therapeutic window exists. Figure 10 shows an example of assay results of three compounds (CT2576, CT1620, and CT3534, see Table 1 for chemical structures). CT2576 inhibited AP reporter gene expression in 239-EBNA cells with ID50 values in the range of 4 to 6 μM using the HTV- LTR promoter construct transfected with a tat expression vector. Figure 11 shows the results of the above-noted compounds in Figure 10 on viability of 293-EBNA cells. Cell viability was determined with a colorimetric assay using the tetrazolium salt of MTS (Promega) to report cell proliferation, viability and cytotoxicity. MTS indicates cell activity by serving as a substrate for mitochondria dehydrogenases for formation of soluble formazan dyes which can be quantitated by determining the absorbance at 490 nm using a plate reader. None of the three compounds tested in Figure 10 showed significant cytotoxic effect on 293-EBNA cells at the concentrations indicated.
Figure 12 (top panel) shows the results of an assay with HTN-infected Ul cells where measurement of HIN-1 expression in the presence of various compounds was determined. Viral release was measured by determining the amount of HTV-lp24 antigen (Ag) expression in cell culture supernatants. Compounds CT1501R, CT1829, CT1411 and CT2576 all suppressed constitutive HTV-l expression when plated at high density initially, but CT2576 was the most effective and CT1827 showed some cytotoxicity.
Figure 12 (bottom panel) shows the effect of several compounds on TΝFα-mediated upregulation of HTV-l in Ul cells. TΝFα is a potent stimulus of viral expression. Again, CT1501R, CT1829, CT1411 and CT2576 all suppressed HTV-l expression, but CT2576 was the most effective and CT1829 showed some cytotoxicity at a 25 μM concentration.
Figures 13 A and 13B illustrate the effect of CT2576 on PA and DG generation.
Figure 14 illustrates the effect of tat and TΝF-α on HIV-LTR-mediated reporter gene expression in 293-EBΝA cells. The y-axis refers to the relative amount of AP reporter gene activity present in the culture media of 293-EBΝA cells after transient transfection with various plasmids. Freshly trypsinized 293-EBΝA cells at 10^ cells per sample were transfected with 2 μg of either pHTN.AP, pHIN2.AP, or pHIN3.AP alone or in combination with pSN2tat72 or pHIV.tat using the cationic lipid DOTAP. The plasmids pHTV.AP, pHIV2.AP, and pHTV3.AP contain nucleotides -453 to +80, -107 to +80, and -80 to +80 of the HTV-LTR sequence respectively. Transfected cells were plated in 96-well microtiter plates at 2,000 cells per well. The cell culture media were changed 24 hr later into new medium. The samples were incubated for another 24 hr with or without TΝF-α (20 ng/ml). 2 μl of medium from each well was used for AP assay. The height of each column represents the mean of triplicate samples with error bar indicating the standard deviation of the mean. Figure 15 shows the effect of CT2576 on HTV-LTR expression activated by tat and
TΝF-α in 293tat cells. 293tαt cells, a cell line stably transfected with the expression plasmids pHIN. AP and pHTV.tαt, were plated onto 96-well microtiter plates at 2000 cells per well. The media were changed the next day and CT2576 was added at the indicated concentrations to the fresh media with or without TNF-α (20 ng ml). 2 μl of medium from each well was used for AP assay 24 hr later. After culture media were withdrawn for AP assay, cytotoxicity was measured for the same cell cultures using an indicator dye, Alamar Blue™, that would change color in response to the reduction potential of cell medium due to cell growth. The left y-axis refers to the absorbance value of the reduced form of Alamar Blue™ at 570 nm with the background absorbance at 600 nm from the oxidized form of Alamar Blue™ subtracted. Each data point represents the mean of triplicate samples with error bar indicating the standard deviation of the mean.
Figure 16 shows the effect of CT2576 on NFKB activation and transcription. Nuclear extracts (A) from 293tat cells were tested by EMS A for activation of NFKB. A radiolabeled 40-mer covering the NFKB sequences in the HTV promoter was used as a probe. Cells were incubated with CT2576 at 12 μM, human TNF-α at 40 ng/ml or LL-lβ at 1 ng/ml, or pre-incubated for 30 min. with CT2576 and then stimulated with TNF-α or IL-lβ for 18 hr prior to making extracts. RNA was extracted from 293tat cells (B) after treatment with CT2576 without or with TNF-α or TL- lβ for 18 hr. Northern blots analysis were performed using probes derived from AP cDNA and G3PDH cDNA as a control for normalization of mRNA level. AP activity was determined in cell supernatants from the various samples.
Figure 17 illustrates a dose response curves of CT2576 on constitutive, TNF-α, and TL-6-mediated expression of HTV-l in a chronically infected Ul cell line (left Y-axis) and of CT3537 on HIV expression in PBL acutely infected with the JR-CSF primary isolate (right Y-axis). Ul cells were incubated for four days after the addition of CT2576 with or without TNF-α (20 pg/ml) or TL-6 (1 ng/ml). PBL were inoculated with HTV at 10 ng virus/106 cells/well and incubated with various concentrations of CT3537 for 7 days. Each data point represents the average expression levels of HTV-l p24-antigen in the cell supernatants measured by ELISA from triplicate samples with error bar indicating the SD.
Figure 18 shows a relationship between relative PA mass and activity of the compound CT3556. Transformed and serum-stimulated cells were measured for total PA mass according to the methods described herein. The tαt-transfected cells stimulated with serum (col. 3) showed a significant increase in PA mass when compared with non-tαt transfected cells stimulated with serum (col. 1). This increase in PA was blocked by administration of CT3556 10 μM. Similarly cells transfected with tar showed a significant drop in PA mass when treated with CT3556. The columns in Figure 18 represent: (1) EB293 cells plus serum, (2) EB293 cells plus serum plus CT3556 (10 μM.), (3) 293tat cells plus serum, (4) 293t t cells plus serum plus CT3556 (10 μM.), (5) 293tαr cells plus serum, and (6) 293tαr cells plus serum plus CT3556 (10 μM.).
Figure 19 shows the same experiment as in Figure 18 only this time diacylglycerol was measured (DG). DG was assayed by collecting the DG HPLC peak (Rf 2-4.5 min) and verifying the predominance of DG species by mass spectroscopy analysis. The DG peak was integrated for a quantitave analysis. The six columns in Figure 19 correspond to the same columns in Figure 18.
Figure 20 shows a dose response relationship for CT2576 in inhibiting total PA mass in EB293 cells transfected with the tat expression plasmid and stimulated with serum. The time points for PA determinations were from 0 to one minute.
Figure 21 shows two graphs for peak D (a measure of various PA species in an HPLC separation) and Peak A5 (a measure of lyso (bis) PA species) with EB293 cells (human embryonic origin) stimulated with human TNF (20 ng/ml, Genzyme) with or without treatment with CT2576 (10 μM.). The left panel shows that drug treatment reduced PA mass across all time points measured, indicating inhibition of formation of PA species to acceleration of PA species metabolism. However, when considering the right hand graph showing increased lyso (bis) PA species across all time points, the CT2576 activity inhibits PA formation from lyso (bis) PA species.
Figure 22 shows an HPLC profile with the PA peak marked in stimulated but not treated cells.
Figure 23 illustrates the chemistry of a reaction to derivatize FFAs (free fatty acids) with 9-ADAM.
Figure 24 illustrates the chemistry of a reaction to make a 9-ADAM derivatizing reagent.
Detailed Description of the Invention
The present invention provides a method for treating viral infection by preventing viral replication in infected cells, comprising administering an effective amount of a compound that inhibits intracellular myrPA formation. The compound is a small organic molecule that can mimic binding to a complex of enzymes that mediate signal amplification at the cellular plasma membrane. Specifically the compound acts by preventing formation of myrPA species in cells stimulated to signal viral replication and proliferation. Therefore, by attacking a host cell signaling mechanism, the compounds do not have resistant viral strains due to rapid viral mutation that is common in antiviral agents that act against viral proteins and viral mechanisms. Moreover, we have found this intracellular signaling mechanism, several exemplary compounds that can be used to prevent further progression of viral diseases by inhibiting host cell viral replication, and have correlated the mechanism of drug action on a cellular basis with the disease to provide a definitive test for myrPA is stimulated host cells as a means for determining whether or not a candidate compound will be effective in preventing the further progression of retroviral-mediated diseases, such as ADDS or CMV diseases. MyrPA Assay
This procedure begins with those cells that would be normally infected with a virus, such as a retrovirus. However, it is not necessary to utilize a cell line containing a retroviral genome, such as Ul cells. In view of the necessity to utilize containment facilities when using an infected cell line, it is preferable to utilize a macrophage cell line that is not infected. Examples of appropriate macrophage cell lines include, U937, THP1, Ul, and P288s (available from standard sources, such as ATCC). The cells are stimulated with a stimulant with or without a candidate drug. Examples of stimulants include 3-5% serum, such as fetal calf serum, or tumor necrosis factor (TNF) (e.g., 20 ng/ml) both of which are standard laboratory reagents. After a time course (within 5 minutes, preferably within 1 minute), the cells are immersed in ice cold methanol to stop any cellular signaling reactions.
One must first quantitatively and qualitatively separate PAs from the other lipids found in serum by a chemical extraction of lipids and high performance liquid chromatography (HPLC) to separate and detect PAs. Chemical extraction can be accomplished, for example, by the method of Bligh et al. (Canadian J. Biochem. Physiol. 37:914-917, 1959) or that of Folch et al. (J. Biochem. 226:497-509, 1957). Briefly, the method of Bligh et al. involves an organic extraction of lipids from biological tissue homogenates or fluids. One volume of sample and three volumes of methano chloroform (2: 1) are vigorously shaken for 2 min. One volume of chloroform is added and then shaken vigorously for 30 sec. One volume of water is added and then shaken vigorously for 30 sec. The mixture is filtered and the upper aqueous layer is discarded. The lower organic layer contains a mixture of lipid classes. The method of Folch et al. involves the extraction of lipids from biological tissue homogenates or body fluids. One volume of sample plus 20 volumes of chlorofornrmethanol (2:1) are vigorously shaken for 2 min. The mixture is filtered and an amount of 0J N KCl equal to 20% of the extraction mixture volume is added and the mixture is shaken vigorously for 2 min. The aqueous and organic phases are allowed to separate. The upper aqueous layer is discarded. The lower organic layer contains a mixture of lipid classes. Free fatty acids and neutral lipids can be separated from phospholipids by normal phase high performance liquid chromatography (HPLC) by modifying the method of Van Kessel et al. (Biochim et Biophys Acta 486:524-530, 1977). This method involves separation of lipids into their major classes by normal phase (silica) high performance liquid chromatography (HPLC). A 5 micron, 25 cm x 0.45 cm silica HPLC column is connected to a binary solvent delivery system followed with a UV detector. The lipid sample is injected on the column and a solvent gradient is run at 1.0 ml/min. The solvent gradient is hexane:isopropanol:water in the proportions 3:4:0.75 run isocratically for 3 min, then ramped to hexane:isopropanol:water in the proportions 3:4:1.4 in 15 min, then run isocratically at the same proportions for 15 min. Detection is at 206 nm. The PAs run at about 6-8 min when run at 1 ml/min when run in hexaneύsopropanol (3:4) according to the HPLC peaks shown in Figure 22 wherein the "PA peak" is listed as "D-2".
Once the PA peak is identified and isolated, it is subject to general alkaline hydrolysis or another method to isolate the FFAs (free fatty acids) from the PA species. This assay is based upon identifying myrPA species in particular such that the identity of the acyl side chains of PA species in stimulated cells is critical for determining if a candidate compound is effective for inhibiting host cell signaling in infected cells to inhibit viral replication and prevent further progression of viral diseases. The FFAs are isolated after hydrolysis. The isolated FFAs are derivatized into fatty acid derivatives of 9-anthroyl diazomethane (9-ADAM), which absorbs light at 254 nm and fluoresces with emission at 410 nm. FFA derivatives were prepared essentially according to the method described in Nakaya et al. (Bull. Chem. Soc. Japan 40:691-692, 1967, and Yoshida et al., Analytical Biochem. 173:70-74, 1988). The derivatization is based on the reaction shown in Figure 23. Briefly, 9-anthraldehyde hydrazone for 9-anthroyl diazomethane derivatization was synthesized from 9-anthraldehyde and hydrazine monohydrate as follows: (a) 8.8 g 9-anthraldehyde (Aldrich Milwaukee, WI) was dissolved in 150 mL absolute ethanol and 8 mL hydrazine monohydrate (Aldrich Milwaukee, WI) was added dropwise with continual stirring, (b) The mixture cleared as hydrazine was added then turned opaque as the last drops were added, (c) The reaction was stirred at room temperature for 3 hr, then was filtered (Whatman #1 filter paper, Whatman Int. Maidstone UK) and dried, (d) The product was recrystalized twice with absolute ethanol. (e) The yield was 3J g of needle-like crystals.
The following solutions were made in ethyl acetate: 9-anthraldehyde hydrazone (0.0276 M, 0.0304 g/5 mL), Quinuclidine (0.2760 M, 0J534 g/5 mL (oxidizing reagent)), and N-chlorosuccinimide (0.0276M, 0.0184 g/5 mL (catalyst)). Equal volumes of these solutions were mixed to react at room temperature for 30 min. The resulting 9-anthryl diazomethane (9-ADAM) was unstable and was made fresh daily. This reaction is shown in Figure 24.
The derivatizating reaction was carried out by diluting 50 μL of each FFA standard to 200 μL with methanol. FFA standards (1.0 mg/mL) were made up in methanol using: Heptadecanoic acid 17:0 (Aldrich Chemical Milwaukee, WI); Arachidonic acid 20:4 (Matreya, Inc., Pleasant Gap, PA); Linoleic acid 18:2 (Matreya, Inc., Pleasant Gap, PA); Linolenic acid 18:3 (Matreya, Inc., Pleasant Gap, PA); Palmitic acid 16:0 (Matreya, Inc., Pleasant Gap, PA); Oleic acid 18:1 (Matreya, Inc., Pleasant Gap, PA); Stearic acid 18:0 (Matreya, Inc., Pleasant Gap, PA); Myristic acid 14:0 (Matreya, Inc., Pleasant Gap, PA); Laurie acid 12:0 (Matreya, Inc., Pleasant Gap, PA); Arachidic acid 20:0 (Matreya, Inc., Pleasant Gap, PA); and n-Docosanoic acid 22:0 (Matreya, Inc., Pleasant Gap, PA). Derivatizing solution (200 μL) was added. The mixture was reacted for 1 hr at room temperature to form each derivatized standard. 20 μL was injected into an HPLC and run by a reverse phase method described below.
A reverse phase HPLC procedure used to separate and quantitate the derivatized anthroyl FFAs. A reverse phase "C8" column (4.6 cm x 25 cm, 5 micron Spherisorb® C8, AUtech Associates, Inc. Deerfield, IL) separated the saturated FFAs. A 3 micron, 15 cm "C18" column was connected to the HPLC followed by a 5 micron, 25 cm "C8" column. The high performance liquid chromatograph was a model 517 from Gilson Medical Electronics, Inc., Middleton, WI. Two detectors were connected in tandem. The first was Model UVIS 200 from Linear Instruments, Reno, NV. The second was Model 121 Fluorometer from Gilson Medical Electronics.
Table 2 below shows the chromatographic conditions used.
Table 2 UV Detection: 254 nm
Fluorescent Detection: Excitation: 305-395 nm bandpass filter Emission: 430-470 nm bandpass filter
Buffer A: 70% Acetonitrile: 30% H2O
Buffer B : 100% Acetonitrile
Flow: 1.0 mL per min
Gradient: 40% B for 2 min from 40% to 45% B in 18 min from 45% to 54% B in 10 min from 54% to 70% B in 5 min from 70% to 94% B in 19 min from 94% to 99% B in 1 min 99% B for 29 min from 99% to 40% B in 1 min 40% B for 5 min The foregoing method was used to correlate the intracellular mechanisms of cell signaling inhibition of the exemplary compounds with the predictive antiviral data in general and the ability to prevent HTV replication in particular. Therefore, the present invention provides a method for preventing the progression of viral diseases by inhibiting a host cell viral replication mechanism comprising administering an effective amount of a compound that can inhibit myrPA formation in stimulated cells.
Mechanism of Action
The compounds described herein exert their antiviral activity by inhibiting one or a plurality of enzymes or an enzyme complex, resulting in a diminution of host (or infected) cell myrPA formation in response to an inflammatory stimulus (e.g., TNF or IL-1). Therefore, the inventive compounds describe a genus of compounds that share a common mechanism of action, on a cellular level, that results in decreased intracellular signaling within a virally-infected cell, that does not transmit an inflammatory signal that would otherwise (in the absence of drug) signal an infected cell to begin the process of viron replication, assembly and viron shedding. This cellular and biochemical mechanism of action, as illustrated by cells infected by the HTV virus as described herein, provides evidence for a general antiviral activity of the genus of compounds described herein, or other compounds that exert a similar mechanism of action (that is, prevention of infected host cell PA accumulation in response to an inflammatory stimulus) by virtue of the ability of the drug to prevent normal viral replication within an infected host cell.
Examples of other viruses that also replicated in an infected host cell in response to inflammatory stimuli (usually mediated by the cytokines TNF or IL-1) include, for example, cytomegalovirus (CMV), herpes family of viruses, including herpes simplex virus (HSV) 1, 2 and 6, hepatitis A, B, C and D, HTV 1 and 2, Epstein Barr virus (EBV), human T cell leukemia virus (HTLV), human papiloma virus, influenza, parainfluenza, respiratory syncytial virus, all adenoviruses, and rhinoviruses. Therefore, the inventive method results in prevention of progression and treatment of a viral disease, comprising administering an effective amount of a compound that can reduce intracellular myrPA concentrations in response to an inflammatory stimulus. Examples of viral diseases include, for example, CMV retinitis, ADDS, systemic viral diseases affecting immunocompromised individuals
(e.g., ADDS or transplant recipients), cold sores (HSV-1), genital herpes (HSV-2), hepatitis (A, B or C, or HSV-6), genital warts (human papiloma virus), infectious mononucleosis and some lymphomas (EBV), shingles (Varicella zoster), pericarditis (coxsackie virus), influenza, and cold and flu (rhinoviruses and adenoviruses). One notable feature of HTV-l infection and many other viral infections (such as
HSV) is its prolonged incubation period during which the virus is harbored in a quiescent state in (in the case of HTV-l) CD4 lymphocytes and mononuclear phagocytes. Often, there are inflammatory cofactors that can accelerate progression of the disease by upregulating expression of the virus, such as HTV-l, in chronically infected cells. One such cofactor for HTV-l infection is human cytomegalovirus (HCMV), which is a cause of serious opportunistic infection in AIDS patients. It has been proposed that cytokines, particularly TNF, IL-6 and IL-1 play an important role in upregulating viral replication in infected cells (Clouse et al., J. Immunol. 143:470, 1989). Therefore, blocking intracellular signaling of such inflammatory cytokines can serve to ablate the inflammatory signal and inhibit the upregulating event that normally triggers viral replication in infected cells.
The mammalian cell membrane associated enzyme lysophosphatidate acyl-CoA acyltransferase (LPAAT) catalyzes the transfer of acyl CoA from lysophosphatidic acid (lyso-PA) to phosphatidic acid (PA) species. Certain PA species function as lipid intermediates in cell activation and function directly as intracellular signaling molecules. PA is subsequently dephosphorylated to 1,2-sn diacylglycerol (DAG) via phosphatidate phosphohydrolase (PAH). The compounds that inhibit viral replication in infected cells also have decreased amounts of intracellular PA species that are formed in response to inflammatory cellular activation. For example, CT1501R (R-l(5-hydroxyhexyl)3,7- dimethylxanthine) or lisofylline (generic name) demonstrates antiviral activity by inhibiting HTV viron replication in infected monocytic cells stimulated to produce virons by TNF activation. CT1501R is also and inhibitor of LPAAT in vitro with competitive inhibition in a Lineweaver-Burke graph and an IC50 for LPAAT inhibition in the range of 200 to 400 nM. Therefore, if PA derived from lyso-PA is an important intracellular signaling intermediate for TNF or IL-1 induced viron replication in virally-infected cells, then inhibitors of PA formation will act to prevent progression of viral diseases by blocking cell signaling to stimulate viron formation. The compounds illustrated herein that are shown to inhibit viron formation or block promoter activity also inhibit PA formation. This invention further provides a method of delaying or preventing the occurrence of
AIDS, which comprises administering to an HTV seropositive human an effective amount of a compound, or a pharmaceutically acceptable salt or hydrate or solvate thereof, wherein said compound can block intracellular myrPA formation from lyso-PA. Preferably, said compound is an amino alcohol-substituted or chiral primary or secondary alcohol-substituted heterocyclic compound wherein the heterocyclic moiety is a substituted or unsubstituted xanthine, a substituted or unsubstituted uracil or a substituted or unsubstituted thymine. An amino alcohol or chiral primary or secondary alcohol compound, or a pharmaceutically acceptable salt or hydrate or solvate thereof, can be administered to such human in a conventional dosage form prepared by combining the amino alcohol or chiral primary or secondary alcohol compound or a pharmaceutically acceptable salt or hydrate or solvate thereof, with a conventional pharmaceutically acceptable carrier or diluent according to known pharmaceutical formulation techniques. Moreover, the compound may be formulated into an opthalmic formulation for ocular administration to treat, for example, CMV retinitis. Topical formulations of compound are appropriate for the treatment of herpes types I and JT and for papiloma virus.
Illustrative Compounds
The compound that can inhibit myristilated PA (myrPA) in either the sn-1 or sn-2 positions or both can be determined by following an assay procedure described herein. Preferably, the compound is a small organic molecule that can mimic binding to a complex of enzymes that mediate signal amplification. The compounds include resolved enantiomers and/or diastereomers, hydrates, salts, solvates and mixtures thereof, the compounds having a straight or branched aliphatic hydrocarbon structure of formula I:
In formula I, n is an integer from one to four and m is an integer from four to twenty. Independently, R\ and R2 are hydrogen, a straight or branched chain alkyl, alkenyl or alkynyl of up to twenty carbon atoms in length or -(CH2)\y 5- If ^1 or ^2 ls ~(CH2)WR5, w may be an integer from one to twenty and R5 may be an hydroxyl, halo, Cj_g alkoxyl group or a substituted or unsubstituted carbocycle or heterocycle. Alternatively, Ri and R2 may jointly form a substituted or unsubstituted, saturated or unsaturated heterocycle having from four to eight carbon atoms, N being a hetero atom of the resulting heterocycle. R3 may be hydrogen, a hydroxy group, a Ci .3 straight or branched alkyl, or a Cj_3 alkoxy.
Preferred compounds may have one of R\ or R2 and R3 that form a substituted or unsubstituted linking carbon chain, having from one to four carbon atoms. This R1/R3 or R2/R3 linking chain will join the O and N in a cyclic structure, an integer sum equal to n + a number of carbon atoms in the linking carbon chain being less than six. In the compounds, a total sum of carbon atoms comprising Ri or R2, (CH2)n and
(CH2)m does not exceed forty. R4 is a terminal moiety comprising a substituted or unsubstituted, heterocyclic moiety, wherein the heterocyclic moiety consists essentially of one to three ring structures having 5-7 members each, a heteroatom, and a predominantly planar structure or essentially aromatic. However, if R4 is phthalimide, m of formula I is not less than five.
The compounds may include resolved enantiomers and/or diastereomers, hydrates, salts, solvates and mixtures thereof that have a straight or branched aliphatic hydrocarbon structure of formula II:
R
In the above formula π, n, m, R3, and R4 are defined as provided in formula I above. Rg and R7 are hydrogen, a straight or branched chain alkane, alkene or alkyne of up to twenty carbon atoms in length, or -(CH2)xRg, at least one of Rg or R7 being -(CH2)xRg. In formula π, x is an integer from zero to fourteen and Rg is a moiety having a general structure as provided in formula DI
R
R
R 4— «*2>m (CH2) p— m
H
In formula HI above, m, R3, and R4 are defined as provided in formula I above. Z is
N or CH and p is an integer from zero to four. R9 is H or a straight or branched chain alkane, alkene or alkyne of up to twenty carbon atoms in length. For example, R4 may be selected from the group consisting of substituted or unsubstituted acridinyl; acridonyl; alkylpyridinyl; anthraquinonyl; ascorbyl; azaazulenyl; azabenzanthracenyl; azabenzanthrenyl; azabenzophenanthrenyl; azachrysenyl; azacyclazinyl; azaindolyl; azanaphthacenyl; azanaphthalenyl; azapyrenyl; azatriphenylenyl; azepinyl; azinoindolyl; azinopyrrolyl; benzacridinyl; benzazapinyl; benzofuryl; benzonaphthyridinyl; benzopyranonyl; benzopyranyl; benzopyronyl; benzoquinolinyl; benzoquinolizinyl; benzothiepinyl; benzothiophenyl; benzylisoquinolinyl; biotinyl; bipyridinyl; butenolidyl; butyrolactonyl; caprolactamyl; carbazolyl; carbolinyl; catechinyl; chromenopyronyl; chromonopyranyl; coumarinyl; coumaronyl; decahydroquinolinyl; decahydroquinolonyl; diazaanthracenyl; diazaphenanthrenyl; dibenzazepinyl; dibenzofuranyl; dibenzothiophenyl; dichromylenyl; dihydrofuranyl; dihydroisocoumarinyl; dihydroisoquinolinyl; dihydropyranyl; dihydropyridinyl; dihydropyridonyl; dihydropyronyl; dihydrothiopyranyl; diprylenyl; dioxanthylenyl; enantholactamyl; flavanyl; flavonyl; fluoranyl; fluorescienyl; furandionyl; furanochromanyl; furanonyl; furanoquinolinyl; furanyl; furopyranyl; furopyronyl; heteroazulenyl; hexahydropyrazinoisoquinolinyl; hydrofuranyl; hydrofurnanonyl; hydroindolyl; hydropyranyl; hydropyridinyl; hydropyrrolyl; hydroquinolinyl; hydrothiochromenyl; hydrothiophenyl; indolizidinyl; indolizinyl; indolonyl; isatinyl; isatogenyl; isobenzofurandionyl; isobenzofuranyl; isochromanyl; isoflavonyl; isoindolinyl; isoindolobenzazepinyl; isoindolyl; isoquinolinyl; isoquinuclidinyl; lactamyl; lactonyl; maleimidyl; monoazabenzonaphthenyl; naphthalenyl; naphthimidazopyridinedionyl; naphthindolizinedionyl; naphthodihydropyranyl; naphthofuranyl; naphthothiophenyl; naphthyridinyl; oxepinyl; oxindolyl; oxolenyl; perhydroazolopyridinyl; perhydroindolyl; phenanthraquinonyl; phenanthridinyl; phenanthrolinyl; phthalideisoquinolinyl; phthalimidyl; phthalonyl; piperidinyl; piperidonyl; prolinyl; pyradinyl; pyranoazinyl; pyranoazolyl; pyranopyrandionyl; pyranopyridinyl; pyranoquinolinyl; pyranopyradinyl; pyranyl; pyrazolopyridinyl; pyridinethionyl; pyridinonaphthalenyl; pyridinopyridinyl; pyridinyl; pyridocolinyl; pyridoindolyl; pyridopyridinyl; pyridopyrimidinyl; pyridopyrrolyl; pyridoquinolinyl; pyronyl; pyrrocolinyl; pyrrolidi'nyl; pyrrolizidinyl; pyrrolizinyl; pyrrolodiazinyl; pyrrolonyl; pyrrolopyrimidinyl; pyrroloquinolonyl; pyrrolyl; quinacridonyl; quinolinyl; quinolizidinyl; quinolizinyl; quinolonyl; quinuclidinyl; rhodaminyl; spirocoumaranyl; succinimidyl; sulfolanyl; sulfolenyl; tetrahydrofuranyl; tetrahydroisoquinolinyl; tetrahydropyranyl; tetrahydropyridinyl; tetrahydrothiapyranyl; tetrahydrothiophenyl; tetrahydrothiopyranonyl; tetrahydrothiopyranyl; tetronyl; thiabenzenyl; thiachromanyl; thiadecalinyl; thianaphthenyl; thiapyranyl; thiapyronyl; thiazolopyridinyl; thienopryidinyl; thienopyrrolyl; thienothiophenyl; thiepinyl; thiochromenyl; thiocoumarinyl; thiophenyl; thiopyranyl; triazaanthracenyl; triazinoindolyl; triazolopyridinyl; tropanyl; xanthenyl; xanthonyl, xanthydrolyl, adeninyl; alloxanyl; alloxazinyl; anthranilyl; azabenzanthrenyl; azabenzonaphthenyl; azanaphthacenyl; azaphenoxazinyl; azapurinyl; azinyl; azoloazinyl; azolyl; barbituric acid; benzazinyl; benzimidazolethionyl; benzimidazolonyl; benzimidazolyl; benzisothiazolyl; benzisoxazolyl; benzocinnolinyl; benzodiazocinyl; benzodioxanyl; benzodioxolanyl; benzodioxolyl; benzopyridazinyl; benzothiazepinyl; benzothiazinyl; benzothiazolyl; benzoxazinyl; benzoxazolinonyl; benzoxazolyl; cinnolinyl; depsidinyl; diazaphenanthrenyl; diazepinyl; diazinyl; dibenzoxazepinyl; dihydrobenzimidazolyl; dihydrobenzothiazinyl; dihydrooxazolyl; dihydropyridazinyl; dihydropyrimidinyl; dihydrothiazinyl; dioxanyl; dioxenyl; dioxepinyl; dioxinonyl; dioxolanyl; dioxolonyl; dioxopiperazinyl; dipyrimidopyrazinyl; dithiolanyl; dithiolenyl; dithiolyl; flavinyl; furopyrimidinyl; glycocyamidinyl; guaninyl; hexahydropyrazinoisoquinolinyl; hexahydropyridazinyl; hydantoinyl; hydroimidazolyl; hydropyrazinyl; hydropyrazolyl; hydropyridazinyl; hydropyrimidinyl; imidazolinyl; imidazolyl; imidazoquinazolinyl; imidazothiazolyl; indazolebenzopyrazolyl; indoxazenyl; inosinyl; isoalloxazinyl; isothiazolyl; isoxazolidinyl; isoxazolinonyl; isoxazolinyl; isoxazolonyl; isoxazolyl; lumazinyl; methylthyminyl; methyluracilyl; morpholinyl; naphthimidazolyl; oroticyl; oxathianyl; oxathiolanyl; oxazinonyl; oxazolidinonyl; oxazolidinyl; oxazolidonyl; oxazolinonyl; oxazolinyl; oxazolonyl; oxazolopyrimidinyl; oxazolyl; perhydrocinnolinyl; perhydropyrroloazinyl; perhydropyrrolooxazinyl; perhydropyrrolothiazinyl; perhydrothiazinonyl; perimidinyl; phenazinyl; phenothiazinyl; phenoxathiinyl; phenoxazinyl; phenoxazonyl; phthalazinyl; piperazindionyl; piperazinodionyl; polyquinoxalinyl; pteridinyl; pterinyl; purinyl; pyrazinyl; pyrazolidinyl; pyrazolidonyl; pyrazolinonyl; pyrazolinyl; pyrazolobenzodiazepinyl; pyrazolonyl; pyrazolopyridinyl; pyrazolopyrimidinyl; pyrazolotriazinyl; pyrazolyl; pyridazinyl; pyridazonyl; pyridopyrazinyl; pyridopyrimidinyl; pyrimidinethionyl; pyrimidinyl; pyrimidionyl; pyrimidoazepinyl; pyrimidopteridinyl; pyrrolobenzodiazepinyl; pyrrolodiazinyl; pyrrolopyrimidinyl; quinazolidinyl; quinazolinonyl; quinazolinyl; quinoxalinyl; sultamyl; sultinyl; sultonyl; tetrahydrooxazolyl; tetrahydropyrazinyl; tetrahydropyridazinyl; tetrahydroquinoxalinyl; tetrahydrothiazolyl; thiazepinyl; thiazinyl; thiazolidinonyl; thiazoUdinyl; thiazolinonyl; thiazolinyl; thiazolobenzimidazolyl; thiazolyl; thienopyrimidinyl; thiazolidinonyl; thyminyl; triazolopyrimidinyl; uracilyl; xanthinyl; xylitolyl, azabenzonaphthenyl; benzofuroxanyl; benzothiadiazinyl; benzotriazepinonyl; benzotriazolyl; benzoxadizinyl; dioxadiazinyl; dithiadazolyl; dithiazolyl; furazanyl; furoxanyl; hydrotriazolyl; hydroxytrizinyl; oxadiazinyl; oxadiazolyl; oxathiazinonyl; oxatriazolyl; pentazinyl; pentazolyl; petrazinyl; polyoxadiazolyl; sydononyl; tetraoxanyl; tetrazepinyl; tetrazinyl; tetrazolyl; thiadiazinyl; thiadiazolinyl; thiadiazolyl; thiadioxazinyl; thiatriazinyl; thiatriazolyl; thiatriazolyl; triazepinyl; triazinoindolyl; triazinyl; triazolinedionyl; triazolinyl; triazolyl; trioxanyl; triphenodioxazinyl; triphenodithiazinyl; trithiadiazepinyl; trithianyl; trixolanyl.
In these compounds, the most preferred ring systems (R4) include, for example, dimethylxanthinyl, methylxanthinyl, phthalimidyl, homophthalimidyl, methylbenzoyleneureayl, quinazolinonyl, octylcarboxamidobenzenyl, methylbenzamidyl, methyldioxotetrahydropteridinyl, glutarimidyl, piperidonyl, succinimidyl, dimethoxybenzenyl, methyldihydrouracilyl, methyluracilyl, methylthyminyl, piperidinyl, dihydroxybenzenyl, or methylpurinyl, even more preferably, methylxanthinyl, dimethylxanthinyl or a derivative thereof. Preferred and exemplary compounds are used throughout the specification and are designated by CT# according to Table 1 below.
Table 1
CT1501R R- 1 -(5-hydroxyhexyl)-3,7-dimethylxanthine CT1115 N-( 11 -octylamino- 10-hydroxyundecyl)- homophthalimide
CT1416 N-( 11 -octylamino- 10-hy droxyundecyl)-3- methylxanthine
CT1620 N-( 11 -octylamino- 10-hydroxyundecyl)-2- piperdone
CT1827 3-( 11 -octylamino- 10-hydroxyundecyl)- 1 - methyluracil
CT1829 3-( 11 -octylamino- 10-hydroxyundecyl)- 1 - methyldihydrouracil
CT2571 l-(9-decylamino-8-hydroxynonyl)-3,7- dimethylxanthine
CT2573 l-(9-dodecylamino-8-hydroxynonyl)-3,7- dimethylxanthine CT2575 1 -( 1 -hexylamino-8-hydroxyundecyl)-3 ,7- dimethylxanthine CT3528 N-(l l-phenylamino-10-hydroxundecyl)-3,7- dimethylxanthine CT2576 1 -( 11 -octylamino- 10-hy droxyundecyl)-3 ,7- dimethylxanthine CT3556 1 -( 11 -N-octylaminoundecyl)-3,7- dimethylxanthine CT3537 1-[1 l-(N-octylacetamido)-10- acetoxyundecyl]-3,7-dimethylxanthine CT3534 l-(9-(2-hydroxydecyl-l-amino)nonyl)-3,7- dimethylxanthine Antiviral Assays
An assay for measuring the anti-viral activity of the compounds involves testing the abilities of the compounds to inhibit gene expression directed by specific viral promoters in cell lines. Specifically, a plasmid construct, pCMV.AP, using the human cytomegalovirus (CMV) enhancer and promoter to direct the expression of the secreted human placental alkaline phosphatase reporter gene were transformed into a tumor cell line (e.g., 293-EBNA cells). The cultured cells were treated with various concentrations of the compounds. The expression of the alkaline phosphatase (AP) reporter gene in the individual cultures can then be measured by following the change in absorbance at A405 of the cell conditioned media in the presence of a suitable substrate (e.g., ortho-nitrophenol phosphate) (Berger et al., Gene 66JJ988).
The construction of expression vectors for secreted placental alkaline phosphatase (sPAP) was performed by obtaining a mammalian episomal expression vector pBL3, derived from the plasmids pMEP4 and pCEP4 (Invitrogen Corp). Specifically, a 600 bp Spe I - Kpn I fragment spanning the CMV promoter from pCEP4 and a 9500 bp Xba I — Kpn I vector fragment from pMEP4 were isolated and ligated together to form pBL3. Full length sPAP cDNA was amplified by PCR using the plasmid pGEM4Z/SEAP (ATCC) as template with primers 5'-GGATCCTCTAGACATGCTGGGGCCCTGCA-3' (SEQ ID NO. 1) and 5'- GGATCCGTCGACGTTAACCCGGGTGCGCG-3' (SEQ ID NO. 2). The PCR product was then digested with Xba I and Sal I and inserted between the Nhe I site and the Xho I site within the multiple cloning region of pBL3. The resulting plasmid was designated as pCMV.AP.
The plasmid construct, pHTV.AP, using the human immunodeficiency virus (HTV) long terminal repeat (LTR) promoter (derived from pU3R-DI CAT, Sodroski et al., Science 227:171, 1985) to direct the expression of the sPAP reporter gene, was made by replacing the CMV promoter region from pCMV.AP with the HTV-LTR promoter. pHTV.AP was co- transfected with pS V2tat72, an expression vector for the 72 amino acids tat protein from HTV (Frankel and Pabo, Cell 55:1189-1193, 1988), into 293-EBNA cells (Invitrogen). The cultured cells were treated with various concentrations of the compounds. The expression of the alkaline phosphatase (AP) reporter gene in the individual cultures was measured by following change in absorbance at 405 nm of the cell conditioned media in the presence of a suitable substrate (e.g., ortho-nitrophenol phosphate) (Berger et al., Gene 66:1-10, 1988). Another assay examined the effect of compounds on chronically HTV-l infected Ul cells. A relatively high density of Ul cells at 10^ cells/well were incubated in the absence or presence of compounds for 4 days. HTV-l p24 antigen expression in the culture media was measured by using a ELISA kit (Abbott). Alternatively, Ul cells were plated at a relatively low density of 2x10^ cell/well plus or minus TNF at 20 pg/ml and plus or minus compounds. The expression of HTV-l p24 antigen in culture media was measured by ELISA four days later.
Several compounds have been identified that inhibit CMV promoter driven expression with minimal cytotoxic effects. These compounds, (e.g., CT2575, CT1827 and CT1829) were also able to inhibit HTV promoter driven expression with minimal cytotoxic effects. As CMV is known to be a cause of serious opportunistic infection in AIDS patients (Schooley, Rev. Infect. Dis. 12 (Suppl.): 811J990) as well as a cofactor for HIV expression in infected cells (Peterson et al., J. Clin. Invest. 89: 574J992), compounds that are capable of inhibiting both the HTV promoter and the CMV promoter are of therapeutic interest in AIDS treatment. Moreover, the mechanism of action of the compounds in inhibiting both HTV and CMV viron replication, coupled with their biochemical mechanism of action in inhibiting specific intacellular signaling pathways, provides a conclusion that not only the illustrated compounds, but other compounds that have similar mechanisms of action in inhibiting intracellular signaling through myrPA, are therapeutically useful to treat or prevent progression of a large number of viral infections.
We set up a cell-based system with a cell line cotransfected with an expression plasmid using HTV-l -LTR promoter to direct synthesis of AP reporter gene and an expression plasmid to direct synthesis of the first 72 amino acids of tat protein. Plasmid pREP7 (Invitrogen) was converted to pREP7b by deleting 2,040 bp EBNA-1 coding region and by replacing the pBR322 origin of replication (ori) with the pBluescript KS~ (Stratagene) ori region. The cDNA for secreted placental alkaline phosphatase (AP) was generated by PCR with the primers (5'- GGATCCTCTAGACATGCTGGGGCCCTGCATGC-3') (SEQ ID NO. 3) and (5 - AAGCTTGTCGACGTTAACCCGGGTGCGCGGC-3') (SEQ TD NO. 4)using the plasmid pGEM-4Z/PLAP489 (American Type Culture Collection) as the template. The 2,000 bp Xba I - Sai l fragment obtained was ligated into a Nhe I / Xho I vector derived from pREP7b to generate the plasmid pREP7b. AP. The 44 bp Xba I - Bgl IT fragment from plasmid pMEP4 (Invitrogen) and the 36 bp Ba R I - Kpn I fragment from pREP7 were ligated into the Xba I - Kpn I vector fragment from pREP7b.AP to generate the plasmid pMCS.AP. The 720 bp Xho I - Hind IE fragment encoding the HIN-LTR promoter from the plasmid pU3R- m-CAT (Sodroski et al., Science 227:171-173, 1985) was ligated into the Xho I - Hind m vector fragment of pMCS.AP to generate the plasmid pHTV.AP. The CMN promoter was derived from the 600 bp Spe I - Asp718 1 fragment from pCEP4 (Invitrogen). The 360 bp SV40 early promoter fragments were produced from pSN2tat72 (Frankel et al., Cell 55: 1189-1193, 1988) by PCR using the primers (5'- GAGGCAGCTCTAGAATGTGTGTCAGTTA-3') (SEQ TD NO. 5) and (5'- GTCTACCGGTACCAAGCTTTTTGCAA-3,) (SEQ ID NO. 6). The 480 bp phosphoglycerate kinase (PGK) promoter fragment was amplified from human placental genomic DNA (Promega, Madison, WI) based on published sequence (Pfeifer et al., Science 246:810-813, 1990) using the primers (5'-GGAATTCTAGAGGTTGGGGTTGCGCCTT-3') (SEQ ID NO. 7) and (5'-AACGAGGGAGCCGGGTACCGACGTGCGC-3') (SEQ ID NO. 8). The above fragments were ligated into the Xba I - Aspl IS I vector fragment of pHTV.AP to generate pHIV2.AP, pHIN3.AP, pCMV.AP, pSVE.AP and pPGK.AP respectively. The t t-expression plasmid directed by HTV-LTR promoter, pHTV.tat, was generated by inserting the 400 bp EcoRN - Hindm fragment from pHIV.AP into the PVHII - Hindlll vector fragment from pSY2tat!2 (Frankel et al., Cell 55: 1189-1193, 1988). ΝFKB binding activity in nuclear extracts (Dignam et al., Nucleic Acids Res.
11:1475-1489, 1983) of cells was measured by electrophoretic mobility-shift assay (ΕMSA) (Carthew et al., Cell 43:439-448, 1985) using 5'-labeled double-stranded synthetic DΝA with two ΝF-κB motifs. Northern blot analysis was performed with total RNA run on 1% agarose/formaldhyde gel and probed with 3 p_ιabeled cDNA of AP and glyceraldehyde-3- phosphate dehydrogenase (G3PDH).
Transient transfections with various expression plasmids were performed on a human kidney embryonic cell line 293-ΕBNA (Invitrogen) using the cationic lipid DOTAP (Boehringer Mannheim). The cell culture media were changed 24 hr after transfection into a serum-free medium AIM V (Life Technologies) before addition of compounds or TNF-α (PeproTech) at the appropriate concentrations. AP activity measured by a colorimetric assay (Berger et al., Gene 66:1-10, 1988) was performed 19-24 hr later.
Stable cell lines 293tar, 293tat and 293tat2 were generated by transfections using conditions described in (Suresh et al., FASEB J. 4:227-231, 1990) with the plasmid pHTV.AP alone or along with either pHTV.tαt or pSN2tαt72, respectively. The assay of HTV-l p24 antigen expression in the chronically HTV-l -infected promonocytic line Ul after 4 days and in acutely infected peripheral blood lymphocytes (PBL) after 7 days were described (Peterson et al., J. Clin. Invest. 89:574-580, 1992, and Brighty et al., Proc. Natl. Acad. Sci. USA 88:7802-7805, 1991). Formulation and Dosage
It will be recognized by one of skill in the art that the form and characterof the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. A therapeutic compound or a pharmaceutically acceptable salt or hydrate or solvate thereof is administered in a virally infected patient (e.g., a HIN seropositive human) in an amount sufficient to prevent or delay the occurrence of further viral infection and clinical symptoms of the virally-mediated disease (e.g., AIDS). The route of administration of the compound is not critical but is usually oral or parenteral, preferably oral. The term parenteral, as used herein, includes intravenous, intramuscular, subcutaneous, intranasal, intrarectal, transdermal, opthalmic, intravaginal or intraperitoneal administration. The subcutaneous and intramuscular forms of parenteral administration are generally preferred. The daily parenteral dosage regimen will preferably be from about 0.01 mg/kg to about 25 mg/kg of total body weight, most preferably from about 0.1 mg/kg to about 4 mg/kg. Preferably, each parenteral dosage unit will contain the active ingredient in an amount of from about 0.1 mg to about 400 mg. The compounds are generally active when given orally and can be formulated as liquids, for example, syrups, suspensions or emulsions, tablets, capsules and lozenges. A liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s), for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavoring or coloring agent. A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose. A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule. Alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example, aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule. The daily oral dosage regimen will preferably be from about 0.01 mg/kg to about 40 mg/kg of total body weight. Preferably, each oral dosage unit will contain the active ingredient in an amount of from about 0J mg to about 1000 mg.
It will be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of a compound or a pharmaceutically acceptable salt or hydrate or solvate thereof will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment (i.e., the number of doses of a compound or a pharmaceutically acceptable salt or hydrate or solvate thereof given per day and duration of therapy) can be ascertained by those skilled in the art using conventional course of treatment determination tests. In addition, the compounds of the present invention can be co-administered with further active ingredients, such as zidovudine, gancyclovir and other compounds with known anti-viral activity mediated by a different mechanism of action than prevention of viron production in infected cells.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following examples are, therefore, to be construed as merely illustrative and not a limitation of the scope of the present invention in any way.
EXAMPLE 1
This example illustrates AP reporter gene activity in 293-EBNA cells transiently transfected with pHIV.AP alone or in combination with pSVtat72. The expression level of AP increased by more than 50 fold in the presence of the tat expression vector, indicating transactivation of the HTV-LTR promoter by the 72 amino acid tat protein (Figure 1).
When three compounds (CT1501R, CT1827, and CT1829, see Table 1 for chemical structures) were added at various concentrations that are likely to be clinically achievable, both CT1827 and CT1829 inhibited AP reporter gene expression in 239-EBNA cells with D->50 values in the range of 4 to 6 μM using the HTV-LTR promoter construct transfected with a tat expression vector. No appreciable inhibition of AP expression was observed with CT1501R (Figure 2).
Figure 3 shows the results of the three compounds on viability of 293-EBNA cells. Cell viability was determined with a calorimetric assay using the tetrazolium salt of MTS (Promega) to report cell proliferation, viability and cytotoxicity. MTS indicates cell activity by serving as a substrate for mitochondria dehydrogenases for formation of soluble formazan dyes which can be quantitated by determining the absorbance at 490 nm using a plate reader. None of the three compounds tested showed significant cytotoxic effect on 293-EBNA cells as the concentrations indicated in Figure 3.
EXAMPLE 2 This example illustrates assay results of four compounds (CT1501R, CT1115, CT1416 and CT2573, see Table 1 for chemical names) for AP reporter gene activity in 293- EBNA cells transiently transfected with pHTV.AP alone or in combination with pSVtαt72. CT 1501 R (again) and CT2573 did not affect AP activity and cell viability significantly, whereas CT1416 and CT2573 were able to inhibit AP expression by 50% at concentrations in the range of 1 to 4 μM (Figure 4). However, some cytotoxicity was noted with CT1416 and CT2573 with cellular LD50 values at around 5 μM (Figure 5). EXAMPLE 3
This example illustrates assay results of three amino-alcohol-substituted xanthine compounds (CT2575, CT3528, and CT2571) on AP reporter gene activity in 293-EBNA cells stably transfected with pCMV.AP. CT2575 and CT2571 inhibited AP reporter gene expression in 293-EBNA cells with IC50 values of 3 μM and 8 μM, respectively. No appreciable inhibition of AP expression was detected when using the compound CT3528 (Figure 6).
Cell viability was measured by a colorometric assay that uses Alamar Blue dye (Alamar, Inc.) to report cell proliferation, viability and cytotoxicity. This vital dye indicates cell activity by serving as substrate for mitochrondrial dehydrogenases for the formation of soluble dyes which can be quantitated by determining the difference in absorbance between A570 and A600 using a plate reader. CT2571 was found to be cytotoxic to 293-EBNA cells while CT2575 and CT3528 showed no significant cytotoxic effect on 293-EBNA cells at the concentrations indicated (Figure 7).
EXAMPLE 4
This example illustrates assay results of the CMV assay results for four different amino alcohol-substituted heterocyclic compounds (CT1416, CT1115, CT1829 and CT1827). While all four compounds were able to inhibit AP activity by 50% in the range of 2 to 6 μM (Figure 8), CT1416 and CT1115 were found to be cytotoxic with an LD50 value of about 5 μM (Figure 9). CT1827 and CT1829 showed no significant cytotoxic effect on 293-EBNA cells at the concentrations indicated. Therefore, a significant therapeutic window exists.
EXAMPLE 5 This example illustrates assay results of three compounds (CT2576, CT1620, and CT3534, see Table 1 for chemical structures) in a tat activation of HTV-LTR promoter in EB293 cells. CT2576 inhibited AP reporter gene expression in 239-EBNA cells with ID50 values in the range of 4 to 6 μM using the HTV-LTR promoter construct transfected with a tat expression vector (Figure 10).
Cell viability was determined with a colorimetric assay using the tetrazolium salt of MTS (Promega) to report cell proliferation, viability and cytotoxicity. MTS indicates cell activity by serving as a substrate for mitochondria dehydrogenases for formation of soluble formazan dyes which can be quantitated by determining the absorbance at 490 nm using a plate reader. None of the three compounds tested showed significant cytotoxic effect on 293-EBNA cells at the concentrations indicated (Figure 11). EXAMPLE 6
This example illustrates assay results of an assay with HJN-infected Ul cells where measurement of HIN-1 expression in response to an exogenous stimulant was determined. Niral release was measured by determining the amount of HTV-l p24 antigen (Ag) expression in cell culture supernatants. Compounds CT1501R, CT1829, CT1411 and CT2576 all suppressed HTV-l expression, but CT2576 was the most effective and CT1827 showed some cytotoxicity (Figure 12, top panel).
Figure 12 bottom panel shows the effect of several compounds on TΝFα mediated upregulation of HTV-l in Ul cells. TΝFα is a potent stimulus of viral expression. Again, CT1501R, CT1829, CT1411 and CT2576 all suppressed HTV-l expression, but CT2576 was the most effective and CT1827 showed some cytotoxicity at a 25 μM concentration.
EXAMPLE 7
This example illustrates that CT2576 suppressed both tat-dependent and TΝF-α induced HTV-LTR transcription with an IC50 of approximately 10 μM. Study of the effects of CT2576 on cell toxicity was performed using the Alamar Blue™ dye (Alamar Biosciences) according to the manufacturer's instructions. CT2576 was not toxic to 293- EBΝA cells at concentrations up to 35 μM. To show CT2576 can inhibit HIN expression, CT2576 was tested for the ability to block HTV p24 antigen expression in chronically or acutely infected cells. Specifically, CT2576 inhibited the TΝF-α and JL-6 induced and the constitutive expression of HTV in the chronically infected Ul cells and in peripheral blood lymphocytes (PBL) freshly infected with a HTV strain from a clinical isolate with an IC50 of approximately 1 μM. Pharmacological inhibition of synthesis of selected PL may therefore be a novel therapeutic approach to suppression of HTV replication. CT2576 altered the pattern of PA composition in 293tat cells. One of the steps after cell activation involves the hydrolysis of phosphatidylcholine (PC), the major phospholipid of cellular membranes, by phospholipases C and D, leading to the generation of second messengers, such as PA and DG, in the signal transduction cascade. HPLC analysis of the total cellular phospholipid content (by the method described herein) of 293tat cells showed several difference in specific subspecies of PA After brief TΝF-α stimulation (45 sec), a
Cl4-16 highly saturated acyl chain peak of PA (Rf 6-7 min.) increased up to 80%. This peak changed from 5.4% to 9% of the total far UV absorbing lipids at this time. In contrast, a PA peak comprised mainly of Cjg and C20 unsaturated acyl chains (Rf 9.5-10.5 min.) was absent in 293tat cells. Preincubation of these cells with 8 μM CT2576 altered the PA composition of these cells. The short chained saturated PA subspecies was less after
CT2576 preincubation than without treatment and showed very little change with addition of TΝF-α. The long chain unsaturated PA comprised 15% of the far UV absorbing lipids after preincubation with CT2576 and it increased to 20% after 2 minutes of stimulation with TNF- α. CT2576 affected the turnover of PA subspecies in 293tat cells.
EXAMPLE 8 This example illustrates that CT2576 inhibited TNF-α and tαt-activated HTV-LTR promoter directed expression in 293tat cells. Cell activation signals generated by interaction of ligands with receptors are eventually converted to changes in the pattern of gene expression. A reporter gene expression system under the control of the HTV-LTR promoter was set up to investigate compounds that inhibit signaling pathway for HTV activation in infected cells. Stably transfected cell lines containing the plasmid constructs, pHTV.AP, using the HTV-LTR promoter to direct the expression of AP reporter gene and pSV2t t72 or pHIN.tαt, expression vectors for the first exon of tat protein from HIN were used to study the effects of various concentrations of CT-2576 on AP expression.
AP reporter gene activity expressed in 293-EBΝA cells transfected with either pHTV.AP alone or in combination with pSV2tαt72, pHIN.tαt or with TΝF-α stimulation is shown in Figure 14. The plasmid pHTV.AP contains the full-length HIN-LTR sequence that extends from nucleotides -453 to +80. The expression level of AP was low using the pHTV.AP construct alone, indicating the presence of negative regulatory elements in the HIN-LTR sequence. Induction with TΝF-α increased the AP expression level by about 4 fold. The AP expression level increased by 15 to 70 fold in the presence of the tat expression plasmids pHTV.tαt or pS V2tαt72. The tαt-expression plasmid regulated by the S V40 early promoter has been reported to be more potent than the one directed by HTV- LTR in cotransfection assays in MDA468 cells (Νabell et al., Cell Growth & Differentiation 5:87-93, 1994). TΝF-α and tαt in combination enhanced the AP expression level by two fold when using the pSV2tot72 construct, and by ten fold when using the pHTV.tαt construct, but the final levels of AP activity were similar.
The effect of CT2576 on HIN promoter activity in 293tαt cells, with the HIN.AP construct cotransfected with pHTV.tat, is shown in Figure 15. CT2576 inhibited AP reporter gene expression by 50% with an inhibition constant (IC50) in the range of 10 μM with or without TΝF-α induction. The inhibition of AP expression is the result of two events: a decrease in AP expression and a decrease in tat expression and therefore lower tat protein levels for transactivation. The measurement of 293tαt cells viability using Alamar Blue™ indicated CT2576 was not cytotoxic at concentrations up to 32 μM.
EXAMPLE 9
This example illustrates that CT2576 did not affect the activation of ΝFKB in 293tαt cells. As ΝFKB is the major transcription factor involved in the activation of HTV-LTR promoter by TΝF-α (Baeuerle et al., Annu. Rev. Immunol. 12:141-179, 1994), the inhibition of TNF-α signaling by CT2576 shows that CT2576 interacted with a step in the signal transduction pathway leading to the activation of NFKB. Electrophoretic mobility shift assay (Figure 16) showed NFKB was activated in 293tαt cells upon induction with either TNF-α or IL-1 β, and CT2576 did not inhibit TNF-α dependent activation of NFKB. Northern blot analysis (Figure 16) again showed induction of AP transcripts with either TNF-α or EL-lβ. However, not only was there no decrease in AP mRNA steady-state levels in the presence of CT2576, there was an increase in mRNA level despite an 83-90% decrease in AP activity after TNF-α or E -lβ induction in the presence of CT2576. Therefore, CT2576 blocked expression of AP as a post-transcriptional step.
EXAMPLE 9
This example illustrates that CT2576 inhibited both constitutive and cytokine-induced HTV expression in a chronically-infected Ul cell line. A chronically HTV-l infected human promonocyte cell line, Ul (Poli et al., J. Exp. Med. 172:151-158, 1990), was used as a model to see if the inhibitory effect of CT2576 on HIV-LTR driven reporter gene expression could be extended to block HTV replication per se in infected cells. TNF-α and IL-6 have been reported to upregulate the expression of HTV-l in Ul cells (Poli et al., Proc. Natl. Acad. Sci. USA 91:108-112, 1994). Ul cells were plated onto 24- well microtiter plates to study the dose-response effect of CT2576 on the constitutive (10^ cells/well), TNF-α or IL- 6 mediated (2 x 10^ cells/well) expression of HTV-l. CT2576 was capable of inhibiting the constitutive, the TNF-α mediated, as well as the IL-6 mediated expression of HTV in Ul cells with an IC50 value of less than 1 μM (Fig 17).
CT3537 also inhibited HTV expression in acutely infected cells. For acute infection, the effect of an acetylated form of CT2576, CT-3537, was tested on PBL from a human donor freshly infected with a clinical isolate HTV-l strain JR-CSF. Analysis of p24 antigen release 7 days after infection at various concentrations of CT3537 showed that CT3537 inhibited HTV expression with an IC50 of less than 1 μM (Figure 17).
EXAMPLE 10 This example summarizes the data for the preferred embodiment, CT2576. CT2576, which affects the generation of myrPA species in 293tat cells (Figure 13), inhibited tat and TNF-α induced activation of HIN-LTR directed AP expression with an IC50 of 10 μM (Figure 15) with minimal cytotoxic effect. Gel-shift assays and Northern blot analysis showed CT2576 did not block expression at the transcription stage (Figure 16). Various mechanisms of post-transcriptional regulation of gene expression have been described
(Rhoads, /. Biol. Chem. 268:3017-3020, 1993; Hawa et al., J. Mol Endocrinology 10:43- " 49, 1993; Morandi et al., J. Cell. Physiol. 160:367-377, 1994; and Takahito et al., Proc. Natl. Acad. Sci. USA 91:7455-7459, 1994). The mechanism of action of CT2576 is therefore different from that of Ro 24-7429 (Hsu et al., Proc. Natl. Acad. Sci. USA 90:6395- 6399, 1993), a compound that blocks HIN-LTR promoter transcription through interaction with the tat protein, though both compounds were selected initially on their potential to inhibit tat activation of the HTV-LTR promoter using a reporter gene expression assay. As CT2576 blocks activation of the HIN-LTR expression post-transcriptionally, CT2576 has a synergistic effect with other anti-HTV compounds that act by different mechanisms. CT2576 is also effective in suppressing HTV expression in cell types with constitutive ΝFKB activity, such as certain neurons involved in the etiology of AIDS dementia complex (Kaltschmidt et _ ., Mol. Cell. Biol 14: 3981-3992, 1994). We studied the effect of CT-2576 on HIN expression in a chronically HIN-1 infected promonocytic Ul cell line (Folks et al., Science 238:800-802, 1987). Chronically infected monocytes or macrophages have been found to be a major source for HIN dissemination in the pathogenesis of AIDS. TΝF-α and other cytokines can induce HTV expression in Ul cells. Ul cells also express HIV constitutively when grown to a relatively high density. AZT has been found not to be active in affecting HTV replication in chronically infected cell lines (Hsu et al., Science 254:1799-1802, 1991). An inhibitor of viral reverse transcriptase is expected to only protect new cells from becoming infected but not to affect viral production from cells already infected with HTV. Analysis by HTV-l p24 antigen production in Ul cell culture media showed that CT2576 inhibited TΝF-α, IL-6 mediated and the constitutive expression in Ul cells (Figure 17) with no cytotoxicity using the trypan-blue dye exclusion assay. While TΝF-α induces HIN expression through activation of ΝFKB, EL-6 and IL-1 also stimulates HTV expression through other pathways independent of ΝFKB activation in Ul cells.
EXAMPLE 11
This example illustrates that CT3556 and CT2576 inhibited myrPA formation in stimulated cells. Figures 18-21 show that both compounds (at a clinically achievable concentration of 10 μM) can inhibit myrPA formation in tat-transfected cells stimulated with serum. Specifically, Figure 18 shows a relationship between relative PA mass and activity of the compound CT3556. Transformed and serum-stimulated cells were measured for total PA mass according to the methods described herein. The tαt-transfected cells stimulated with serum (col. 3) showed a significant increase in PA mass when compared with non-tat transfected cells stimulated with serum (col. 1). This increase in PA was blocked by administration of CT3556 10 μM. Similarly cells transfected with tar showed a significant drop in PA mass when treated with CT3556. The columns in Figure 18 represent: (1)
EB293 cells plus serum, (2) EB293 cells plus serum plus CT3556 (10 μM.), (3) 293tat cells plus serum, (4) 293tαt cells plus serum plus CT3556 (10 μM.), (5) 293tαr cells plus serum, and (6) 293tαr cells plus serum plus CT3556 (10 μM.). Figure 19 shows the same experiment as in Figure 18 only this time diacylglycerol was measured (DG). DG was assayed by collecting the DG HPLC peak (Rf 2-4.5 min) and verifying the predominance of DG species by mass spectroscopy analysis. The DG peak was integrated for a quantitave analysis. The six columns in Figure 19 correspond to the same columns in Figure 18. Figure 20 shows a dose response relationship for CT2576 in inhibiting total PA mass in EB293 cells transfected with the tat expression plasmid and stimulated with serum. The time points for PA determinations were from 0 to one minute. Figure 21 shows two graphs for peak D (a measure of various PA species in an HPLC separation) and Peak A5 (a measure of lyso (bis) PA species) with EB293 cells (human embryonic origin) stimulated with human TNF-α (20 ng/ml, Genzyme) with or without treatment with CT2576 (10 μM.). The left panel shows that drug treatment reduced PA mass across all time points measured, indicating inhibition of formation of PA species to acceleration of PA species metabolism. However, when considering the right hand graph showing increased lyso (bis) PA species across all time points, the CT2576 activity inhibits PA formation from lyso (bis) PA species.
SEQUENCE LISTING
(1) GENERAL INFORMATION: (i) APPLICANT: Leung, David W.
Underiner, Gail E. Singer, Jack W. (ii) TITLE OF INVENTION: METHOD FOR INHIBITING INTRACELLULAR VIRAL REPLICATION (iϋ) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Cell Therapeutics, Inc.
(B) STREET: 201 Elliott Avenue West
(C) CITY: Seattle (D) STATE: Washington
(E) COUNTRY: U.S.A.
(F) ZIP: 98119
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5" disk, 1.44Mb, high density (B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: MS-DOS Version 6
(D) SOFTWARE: WORD for WINDOWS (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: (B) FILING DATE: Ol-Nov-1994
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/147,255
(B) FILING DATE: Ol-Nov-1993 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Oster; Jeffrey B.
(B) REGISTRATION NUMBER: 32,585
(C) REFERENCE/DOCKET NUMBER: 0802A (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (206)282-7100 (B) TELEFAX: (206)284-6206 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GGA TCC TCT AGA CAT GCT GGG GCC CTG CA 29
(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA
(iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GGA TCC GTC GAC GTT AAC CCG GGT GCG CG 29
(2) INFORMATION FOR SEQ ID NO: 3 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GGA TCC TCT AGA CAT GCT GGG GCC CTG CAT GC 32
(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: AAG CTT GTC GAC GTT AAC CCG GGT GCG CGG C 31 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GAG GCA GCT CTA GAA TGT GTG TCA GTT A 31
(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GTC TAC CGG TAC CAA GCT TTT TGC AA 26
(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GGA ATT CTA GAG GTT GGG GTT GCG CCT T 28
(2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: AAC GAG GGA GCC GGG TAC CGA CGT GCG C 28

Claims

We claim:
1. A method of preventing or delaying the occurrence of acquired immunodeficiency syndrome (ATDS) in human immunodeficiency virus (HIN) seropositive humans, comprising administering an effective amount of a compound that inhibits cellular accumulation of myrPA (myristilated phosphatidic acid) in stimulated monocytic cells.
2. A method for treating viral infection by preventing viral replication in infected cells by inhibiting host cell viral replication signaling, comprising administering an effective amount of a compound that inhibits cellular accumulation of myrPA in stimulated monocytic cells.
3. The method of claims 1 or 2 wherein the compound is a small organic molecule, including resolved enantiomers and/or diastereomers, hydrates, salts, solvates and mixtures thereof, wherein the compounds having a straight or branched aliphatic hydrocarbon structure of formula I:
wherein n is an integer from one to four and m is an integer from four to twenty, Ri and R2 are hydrogen, a straight or branched chain alkyl, alkenyl or alkynyl of up to twenty carbon atoms in length or -(CH2)WR5, with the proviso that if R\ or R2 is -(CH2)WR5, w is an integer from one to twenty and R5 is an hydroxyl, halo, Ci.g alkoxyl group or a substituted or unsubstituted carbocycle or heterocycle, or R\ and R2 may jointly form a substituted or unsubstituted, saturated or unsaturated heterocycle having from four to eight carbon atoms, Ν being a hetero atom of the resulting heterocycle, R3 is hydrogen, a hydroxy group, a Cj_3 straight or branched alkyl, or a Ci .3 alkoxy, and R4 is a terminal moiety comprising a substituted or unsubstituted, heterocyclic moiety, wherein the heterocyclic moiety consists essentially of one to three ring structures having 5-7 members each, a heteroatom, and a predominantly planar structure or essentially aromatic, with the proviso that if R4 is phthalimide, m of formula I is not less than five.
4. The method of claim 3 wherein a total sum of carbon atoms comprising R or R2, (CH2)n and (CH2)m does not exceed forty.
5. The method of claims 1 or 2 wherein the compound is a resolved enantiomer and/or diastereomer, hydrate, salt, solvate or mixture thereof that have a straight or branched aliphatic hydrocarbon structure of formula IT:
R
wherein n, m, R3, and R4 are defined as provided in formula I, R5 and R7 are hydrogen, a straight or branched chain alkane, alkene or alkyne of up to twenty carbon atoms in length, or -(CH2) R8> at least one of R6 or R7 being -(CH2)xRg, x is an integer from zero to fourteen and Rg is a moiety having a general structure as provided in formula DT
R R
R 4 (CH2) m (CH2) — m
H wherein m, R3, and R4 are defined as provided in formula I, Z is N or CH and p is an integer from zero to four, R9 is H or a straight or branched chain alkane, alkene or alkyne of up to twenty carbon atoms in length.
6. The method of claims 1-5 wherein R4 is selected from the group consisting of substituted or unsubstituted acridinyl; acridonyl; alkylpyridinyl; anthraquinonyl; ascorbyl; azaazulenyl; azabenzanthracenyl; azabenzanthrenyl; azabenzophenanthrenyl; azachrysenyl; azacyclazinyl; azaindolyl; azanaphthacenyl; azanaphthalenyl; azapyrenyl; azatriphenylenyl; azepinyl; azinoindolyl; azinopyrrolyl; benzacridinyl; benzazapinyl; benzofuryl; benzonaphthyridinyl; benzopyranonyl; benzopyranyl; benzopyronyl; benzoquinolinyl; benzoquinolizinyl; benzothiepinyl; benzothiophenyl; benzylisoquinolinyl; biotinyl; bipyridinyl; butenolidyl; butyrolactonyl; caprolactamyl; carbazolyl; carbolinyl; catechinyl; chromenopyronyl; chromonopyranyl; coumarinyl; coumaronyl; decahydroquinolinyl; decahydroquinolonyl; diazaanthracenyl; diazaphenanthrenyl; dibenzazepinyl; dibenzofuranyl; dibenzothiophenyl; dichromylenyl; dihydrofuranyl; dihydroisocoumarinyl; dihydroisoquinolinyl; dihydropyranyl; dihydropyridinyl; dihydropyridonyl; dihydropyronyl; dihydrothiopyranyl; diprylenyl; dioxanthylenyl; enantholactamyl; flavanyl; flavonyl; fluoranyl; fluorescienyl; furandionyl; furanochromanyl; furanonyl; furanoquinolinyl; furanyl; furopyranyl; furopyronyl; heteroazulenyl; hexahydropyrazinoisoquinolinyl; hydrofuranyl; hydrofumanonyl; hydroindolyl; hydropyranyl; hydropyridinyl; hydropyirolyl; hydroquinolinyl; hydrothiochromenyl; hydrothiophenyl; indolizidinyl; indolizinyl; indolonyl; isatinyl; isatogenyl; isobenzofurandionyl; isobenzofuranyl; isochromanyl; isoflavonyl; isoindolinyl; isoindolobenzazepinyl; isoindolyl; isoquinolinyl; isoquinuclidinyl; lactamyl; lactonyl; maleimidyl; monoazabenzonaphthenyl; naphthalenyl; naphthimidazopyridinedionyl; naphthindolizinedionyl; naphthodihydropyranyl; naphthofuranyl; naphthothiophenyl; naphthyridinyl; oxepinyl; oxindolyl; oxolenyl; perhydroazolopyridinyl; perhydroindolyl; phenanthraquinonyl; phenanthridinyl; phenanthrolinyl; phthalideisoquinolinyl; phthalimidyl; phthalonyl; piperidinyl; piperidonyl; prolinyl; pyradinyl; pyranoazinyl; pyranoazolyl; pyranopyrandionyl; pyranopyridinyl; pyranoquinolinyl; pyranopyradinyl; pyranyl; pyrazolopyridinyl; pyridinethionyl; pyridinonaphthalenyl; pyridinopyridinyl; pyridinyl; pyridocolinyl; pyridoindolyl; pyridopyridinyl; pyridopyrimidinyl; pyridopyrrolyl; pyridoquinolinyl; pyronyl; pyrrocolinyl; pyrrolidinyl; pyrrolizidinyl; pyrrolizinyl; pyrrolodiazinyl; pyrrolonyl; pyrrolopyrimidinyl; pyrroloquinolonyl; pyrrolyl; quinacridonyl; quinolinyl; quinolizidinyl; quinolizinyl; quinolonyl; quinuclidinyl; rhodaminyl; spirocoumaranyl; succinimidyl; sulfolanyl; sulfolenyl; tetrahydrofuranyl; tetrahydroisoquinolinyl; tetrahydropyranyl; tetrahydropyridinyl; tetrahydrothiapyranyl; tetrahydrothiophenyl; tetrahydrothiopyranonyl; tetrahydrothiopyranyl; tetronyl; thiabenzenyl; thiachromanyl; thiadecalinyl; thianaphthenyl; thiapyranyl; thiapyronyl; thiazolopyridinyl; thienopryidinyl; thienopyrrolyl; thienothiophenyl; thiepinyl; thiochromenyl; thiocoumarinyl; thiophenyl; thiopyranyl; triazaanthracenyl; triazinoindolyl; triazolopyridinyl; tropanyl; xanthenyl; xanthonyl, xanthydrolyl, adeninyl; alloxanyl; alloxazinyl; anthranilyl; azabenzanthrenyl; azabenzonaphthenyl; azanaphthacenyl; azaphenoxazinyl; azapurinyl; azinyl; azoloazinyl; azolyl; barbituric acid; benzazinyl; benzimidazolethionyl; benzimidazolonyl; benzimidazolyl; benzisothiazolyl; benzisoxazolyl; benzocinnolinyl; benzodiazocinyl; benzodioxanyl; benzodioxolanyl; benzodioxolyl; benzopyridazinyl; benzothiazepinyl; benzothiazinyl; benzothiazolyl; benzoxazinyl; benzoxazolinonyl; benzoxazolyl; cinnolinyl; depsidinyl; diazaphenanthrenyl; diazepinyl; diazinyl; dibenzoxazepinyl; dihydrobenzimidazolyl; dihydrobenzothiazinyl; dihydrooxazolyl; dihydropyridazinyl; dihydropyrimidinyl; dihydrothiazmyl; dioxanyl; dioxenyl; dioxepinyl; dioxinonyl; dioxolanyl; dioxolonyl; dioxopiperazinyl; dipyrimidopyrazinyl; dithiolanyl; dithiolenyl; dithiolyl; flavinyl; furopyrimidinyl; glycocyamidinyl; guaninyl; hexahydropyrazinoisoquinolinyl; hexahydropyridazinyl; hydantoinyl; hydroimidazolyl; hydropyrazinyl; hydropyrazolyl; hydropyridazinyl; hydropyrimidinyl; imidazolinyl; imidazolyl; imidazoquinazolinyl; imidazothiazolyl; indazolebenzopyrazolyl; indoxazenyl; inosinyl; isoalloxazinyl; isothiazolyl; isoxazolidinyl; isoxazolinonyl; isoxazolinyl; isoxazolonyl; isoxazolyl; lumazinyl; methylthyminyl; methyluracilyl; mo holinyl; naphthimidazolyl; oroticyl; oxathianyl; oxathiolanyl; oxazinonyl; oxazolidinonyl; oxazolidinyl; oxazolidonyl; oxazolinonyl; oxazolinyl; oxazolonyl; oxazolopyrimidinyl; oxazolyl; perhydrocinnolinyl; perhydropyrroloazinyl; perhydropyπOlooxazinyl; perhydropyrrolothiazinyl; perhydrothiazinonyl; perimidinyl; phenazinyl; phenothiazinyl; phenoxathiinyl; phenoxazinyl; phenoxazonyl; phthalazinyl; piperazindionyl; piperazinodionyl; polyquinoxalinyl; pteridinyl; pterinyl; purinyl; pyrazinyl; pyrazolidinyl; pyrazolidonyl; pyrazolinonyl; pyrazolinyl; pyrazolobenzodiazepinyl; pyrazolonyl; pyrazolopyridinyl; pyrazolopyrimidinyl; pyrazolotriazinyl; pyrazolyl; pyridazinyl; pyridazonyl; pyridopyrazinyl; pyridopyrimidinyl; pyrimidinethionyl; pyrimidinyl; pyrimidionyl; pyrimidoazepinyl; pyrimidopteridinyl; pyrrolobenzodiazepinyl; pyrrolodiazinyl; pyrrolopyrimidinyl; quinazolidinyl; quinazolinonyl; quinazolinyl; quinoxalinyl; sultamyl; sultinyl; sultonyl; tetrahydrooxazolyl; tetrahydropyrazinyl; tetrahydropyridazinyl; tetrahydroquinoxalinyl; tetrahydrothiazolyl; thiazepinyl; thiazinyl; thiazolidinonyl; thiazolidinyl; thiazolinonyl; thiazolinyl; thiazolobenzimidazolyl; thiazolyl; thienopyrimidinyl; thiazolidinonyl; thyminyl; triazolopyrimidinyl; uracilyl; xanthinyl; xylitolyl, azabenzonaphthenyl; benzofuroxanyl; benzothiadiazinyl; benzotriazepinonyl; benzotriazolyl; benzoxadizinyl; dioxadiazinyl; dithiadazolyl; dithiazolyl; furazanyl; furoxanyl; hydrotriazolyl; hydroxytrizinyl; oxadiazinyl; oxadiazolyl; oxathiazinonyl; oxatriazolyl; pentazinyl; pentazolyl; petrazinyl; polyoxadiazolyl; sydononyl; tetraoxanyl; tetrazepinyl; tetrazinyl; tetrazolyl; thiadiazinyl; thiadiazolinyl; thiadiazolyl; thiadioxazinyl; thiatriazinyl; thiatriazolyl; thiatriazolyl; triazepinyl; triazinoindolyl; triazinyl; triazolinedionyl; triazolinyl; triazolyl; trioxanyl; triphenodioxazinyl; triphenodithiazinyl; trithiadiazepinyl; trithianyl; trixolanyl.
7. The method of claim 6 wherein R4 is selected from the group consisting of dimethylxanthinyl, methylxanthinyl, phthalimidyl, homophthalimidyl, methylbenzoyleneureayl, quinazolinonyl, octylcarboxamidobenzenyl, methylbenzamidyl, methyldioxotetrahydropteridinyl, glutarimidyl, piperidonyl, succinimidyl, dimethoxybenzenyl, methyldihydrouracilyl, methyluracilyl, methylthyminyl, piperidinyl, dihydroxybenzenyl, and methylpurinyl
8. The method of claims 1 or 2 wherein the compound is selected from the group consisting of 1 -( 11 -hexylamino-8-hydroxyundecyl)-3 ,7-dimethylxanthine, N-( 11 - phenylamino- 10-hydroxundecyl)-3 ,7-dimethylxanthine, 1 -( 11 -octylamino- 10- hydroxyundecyl)-3 ,7-dimethylxanthine, 1 -( 11 -N-octy laminoundecyl)-3 ,7-dimethylxanthine, 1-[1 l-(N-octylacetamido)-10-acetoxyundecyl]-3,7-dimethylxanthine, and l-(9-(2- hydroxydecyl- 1 -amino)nonyl)-3,7-dimethylxanthine.
9. The method of claim 2 wherein the viral infections and viral diseases are selected from the group consisting of cytomegalovirus (CMV), herpes family of viruses, herpes simplex virus (HSV) 1, 2 and 6, hepatitis A, B, C and D, HTV 1 and 2, Epstein Barr virus (EBV), human T cell leukemia virus (HTLV), human papiloma virus, influenza, parainfluenza, respiratory syncytial virus, all adenoviruses, and rhinoviruses, CMV retinitis, AIDS, ARDS, systemic viral diseases affecting immunocompromised individuals (AIDS or transplant recipients), cold sores (HSV-1), genital heφes (HSV-2), hepatitis (A, B or C, or HSN-6), genital warts (human papiloma virus), infectious mononucleosis and some lymphomas (EBN), shingles (Varicella zoster), pericarditis (coxsackie virus), influenza, cold and flu (rhinoviruses and adenoviruses), cachexia associated with HIN infection, cachexia associated with EBV infection, HTV, EBV and HTLV related malignancies (Kaposi's sarcoma and lymphoma), AIDS-related opportunistic infections (PCP, MAI) for prophylaxis of opportunistic infection in AIDS patients, and combinations thereof.
EP95901093A 1993-11-01 1994-11-01 Method for inhibiting intracellular viral replication Withdrawn EP0676958A1 (en)

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US14725593A 1993-11-01 1993-11-01
US147255 1993-11-01
PCT/US1994/012512 WO1995012396A1 (en) 1993-11-01 1994-11-01 Method for inhibiting intracellular viral replication

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US5985926A (en) * 1993-11-01 1999-11-16 Cell Therapeutics, Inc. Method for inhibiting intracellular viral replication

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US5288721A (en) * 1992-09-22 1994-02-22 Cell Therapeutics, Inc. Substituted epoxyalkyl xanthines
US5340813A (en) * 1992-11-09 1994-08-23 Cell Therapeutics, Inc. Substituted aminoalkyl xanthine compounds
US5354756A (en) * 1993-01-12 1994-10-11 Cell Therapeutics, Inc. Olefin-substituted long chain xanthine compounds
US5521315A (en) * 1993-01-12 1996-05-28 Cell Therapeutics, Inc. Olefin substituted long chain compounds
US5670506A (en) * 1993-04-05 1997-09-23 Cell Therapeutics, Inc. Halogen, isothiocyanate or azide substituted xanthines
AU6702894A (en) * 1993-04-09 1994-11-08 Cell Therapeutics, Inc. Ring-substituted cell signaling inhibitors

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WO1995012396A1 (en) 1995-05-11

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