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WO1998031375A1 - Isolation en tranchee pour dispositifs micromecamiques - Google Patents

Isolation en tranchee pour dispositifs micromecamiques Download PDF

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Publication number
WO1998031375A1
WO1998031375A1 PCT/US1998/000784 US9800784W WO9831375A1 WO 1998031375 A1 WO1998031375 A1 WO 1998031375A1 US 9800784 W US9800784 W US 9800784W WO 9831375 A1 WO9831375 A1 WO 9831375A1
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Prior art keywords
hiv
retrovirus
virus
cytidine
ctp synthase
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PCT/US1998/000784
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English (en)
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WO1998031375A9 (fr
Inventor
Wen-Yi Gao
David G. Johns
Hiroaki Mitsuya
Victor E. Marquez
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The United States Of America As Represented By Thesecretary Of The Department Of Health And Human Sevices
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Priority to AU58255/98A priority Critical patent/AU5825598A/en
Publication of WO1998031375A1 publication Critical patent/WO1998031375A1/fr
Publication of WO1998031375A9 publication Critical patent/WO1998031375A9/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present invention relates to a method to increase activity of cytidine-based anti-HIV drugs and to overcome resistance of human immunodeficiency virus (HIV) to cytidine-based anti-HIV drugs by administration to a patient in need thereof a cytidine-based anti-HIV drug in combination with CTP synthase inhibitors . More specifically, the invention relates to a method where 2' ,3'-dideoxycytidine (ddC) or ⁇ -L-2 ' , 3 ' -dideoxy-3 ' - thiacytidine (3-TC) are administered in combination with the CTP synthase inhibitors 3-deazauridine and/or cyclopentenyl cytosine.
  • ddC 2' ,3'-dideoxycytidine
  • 3-TC ⁇ -L-2 '
  • AIDS immune deficiency syndrome
  • T4 lymphocyte a white blood cell that marshals the immune defenses.
  • T4 lymphocyte a white blood cell that marshals the immune defenses.
  • This depletion of T4 cells in AIDS causes a severe depression of the immune response, so that a compound which is to be effective against AIDS must modify virus effect without much help from host immunity.
  • the virus also affects cells in the central nervous system, where it is protected by the blood-brain barrier from compounds that might otherwise be effective against the virus. In infecting its host, the HIV binds to specific cell-surface receptor molecules .
  • the virus penetrates the cell cytoplasm and sheds its protein coat, thereby baring its genetic material, a single strand of RNA.
  • a viral enzyme, reverse transcriptase accompanies the RNA.
  • the virus which is a retrovirus, reverse transcribes the RNA into DNA.
  • DNA copies of the HIV genome become integrated into the chromosomes of the host cell .
  • This integrated viral genome may remain latent until the host cell is stimulated, such as by another infection.
  • the proviral DNA is then transcribed into mRNA, which directs the synthesis of viral proteins.
  • the provirus also gives rise to other RNA copies that will serve as the genetic material of viral progeny.
  • the proteins and the genomic RNA congregate at the cell membrane and assemble to form new HIV particles, which then break off from the cell.
  • Two HIV genes, tat and trt/art appear to control this burst of replication, which destroys the cell. These genes code for small proteins that boost the transcription of proviral DNA and the synthesis of viral proteins .
  • Several compounds have been shown to reduce the activity of reverse transcriptase in vitro.
  • the reverse transcription is the step that is essential to viral replication and irrelevant to host cells. It has been found that HIV replication is considerably slower in the presence of compounds such as suramin, antimoniotungstate, phosphonoformate, and a class of nucleoside analogues known as dideoxynucleosides .
  • Nucleoside analogues are a class of synthetic compounds that resemble the naturally occurring nucleosides, which are chemical precursors of DNA and RNA.
  • a nucleoside comprises a single-or double-ring base linked to a five-carbon sugar molecule.
  • An analogue differs from the naturally-occurring nucleoside in large or small features of the base or the sugar.
  • An enzyme that normally acts on a nucleoside in the course of viral replication can also bind to the nucleoside analogue. Because the nucleoside and the analogue differ, however, binding to the analogue can incapacitate the enzyme, thereby disrupting a molecular process crucial to viral replication.
  • zidovudine ZT
  • didanosine ddl
  • ddC zalcitabine
  • FDA has approved the marketing of five new drugs for the treatment of HIV infection. These include stavudine (D4T) and lamivudine (3-TC) , which are nucleoside analogues similar to AZT, ddl and ddC. The other three are protease inhibitors, a new class of anti-HIV drugs (Med Lett Drugs Ther, 1996, 38(972): 35-37).
  • 3-TC also called L-2 ' , 3 ' -dideoxy-3 ' -thiacytidine, and ddC, also called 2 ' , 3 ' -dideoxycytidine
  • 3-TC in particular, a beta-L(-) nucleoside analog, was shown to synergistically inhibit replication of HIV in vitro when combined with 3'-azido-3'- deoxythymidine (AZT) without added toxicity (Bridges et al . , 1996, Bichem Pharmacol 51(6): 731-736).
  • Virus resistance encountered in multiple drug therapies is indicative that a stronger combination of drugs is required for the long-term treatment of patients infected with HIV.
  • CTP synthase inhibitors such as 3-deazauridine (3-DU) and cyclopentenyl cytosine (CPE-C) .
  • CTP synthase inhibitors such as 3-deazauridine (3-DU) and cyclopentenyl cytosine (CPE-C) .
  • CTP synthase inhibitors such as 3-deazauridine (3-DU) and cyclopentenyl cytosine (CPE-C)
  • the potency of 3-TC against the replication of a recombinant HIV mutant multiply cross-resistant to AZT, ddl, ddC, D4T, ddG and protease inhibitors such as KNI, was increased approximately 50-fold by combination of 3-TC with a low level of 3-DU.
  • Consequences of using such combinations would be increased therapeutic effectiveness of 3-TC or ddC, including significant dose-reduction and inhibition of growth of HIV mutants and hence of clinical drug resistance.
  • the present invention relates to a composition comprising a cytidine-based nucleoside analogue and a CTP synthase inhibitor wherein the combination provides increased prevention or inhibition of the replication and spread of retroviruses including HIV relative to the effects of the nucleoside analogue alone.
  • Another object of the present invention relates to a method of preventing and/or inhibiting the replication and spread of a retrovirus, by exposing a cell population, including cells infected by the retrovirus, to a composition comprising a combination of a nucleoside analogue and a CTP synthase inhibitor.
  • the term "retrovirus” is inclusive of any virus that utilizes reverse transcriptase in its life cycle and would therefore be susceptible to the antiviral activity of nucleoside analogues, including, for example, HIV (HIV-1 and HIV-2), HTLV-1, HTLV-2 or SIV. Also encompassed are viruses such as HBV that, although not technically classified as a "retrovirus", utilize a reverse transcriptase and are therefore susceptible to the antiviral activity of nucleoside analogues.
  • the present invention also encompasses methods of treating HIV-infected and AIDS patients with a composition comprising a nucleoside analogue and a CTP synthase inhibitor in order to prevent and/or inhibit the replication and spread of HIV in these patients . Since the administration of a CTP synthase inhibitor could benefit patients already receiving therapy with a nucleoside analogue drug alone, improvements to such a therapeutic regiment are also claimed.
  • the nucleoside analogue is a cytidine analogue such as ddC or 3-TC.
  • the CTP synthase inhibitor is preferably 3- deazauridine (3-DU) or cyclopentenyl cytosine (CPE-C) .
  • 3-DU 3- deazauridine
  • CPE-C cyclopentenyl cytosine
  • the preferred embodiments of the invention include pharmaceutical compositions comprising the combination of either ddC or 3-TC and either 3-DU or CPE-C.
  • the pharmaceutical compositions can optionally contain a pharmaceutically acceptable carrier and/or vehicle.
  • the preferred method of the invention comprises preventing and/or inhibiting retroviral or HIV replication and spread by treating a cell population, including cells infected with HIV, with such a composition. Additionally, the preferred method comprises treating an HIV infected or AIDS patients with such a composition so as to prevent and/or inhibit HIV replication and spread in the patient.
  • treatment encompasses administration of compounds propylactically to prevent or suppress an undesired condition, and therapeutic administration to eliminate or reduce the extent or symptoms of the condition.
  • Treatment according to the invention may be for a human or an animal infected with a retrovirus, or it may include application in vitro to a cell culture or extracellular media. Treatment may be by systemic administration to a patient or locally to an affected site.
  • the compositions of the present invention i.e., compositions comprising a cytidine-based nucleoside analog and a CTP synthase inhibitor, may be made into pharmaceutical compositions with appropriate pharmaceutically acceptable carriers or diluents.
  • compositions included in the composition include the monosodium salt and the following 5' esters: monophosphate; disodium; monophosphate; diphosphate; triphosphate; acetate; 3-methyl-butyrate; octanoate; palmitate; 3-chloro benzoate; benzoate; 4-methyl benzoate; hydrogen succinate; pivalate; and mesylate.
  • esters also included within the scope of this invention are the pharmaceutically acceptable salts, esters, salts of such esters, nitrile oxides, or any other covalently linked or non-linked compound which, upon administration to the recipient, is capable of providing (directly or indirectly) a nucleoside analog as described above, or an anti-virally active metabolite or residue thereof.
  • the nucleoside and the synthase inhibitor of the present invention may be administered alone in solution.
  • the active ingredient (s) may be used or administered in a pharmaceutical formulation.
  • These formulations comprise at least one active ingredient (the nucleoside or the synthase inhibitor or both) , together with one or more pharmaceutically acceptable carriers and/or other therapeutic agents.
  • pharmaceutically acceptable carriers include those well known to practitioners in the art as suitable for oral, rectal, nasal, topical, buccal, sublingual, vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration.
  • the compounds according to the invention may also be used in the manufacture of pharmaceuticals for the treatment or prophylaxis of viral infections.
  • the administration of the composition to humans suffering from AIDS, under conditions which effectively interrupt or suppress activity of the HIV virus, can be accomplished by one or more of several means of administration.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid; in an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electurary or paste. Tablets may, if desired, be enteric coated.
  • Formulations suitable for topical administration include lozenges comprising the active ingredient in a flavor, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing in addition to the active ingredients such carriers as are known in the art to be appropriate.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.
  • sterile liquid carrier for example, water for injections
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • the administered ingredients may also be used in therapy in conjunction with other anti-viral drugs and biologicals, or in conjunction with other immune modulating therapy including bone marrow or lymphocyte transplants or medications . In the preferred embodiment, whatever administrative method is chosen should result in effective circulating levels of each compound.
  • an "effective amount" of the composition is such as to produce the desired effect in a patient which can be monitored using several end-points known to those skilled in the art.
  • effects could be monitored in terms of a therapeutic effect, e.g., alleviation of some symptom associated with the disease being treated, or further evidence involving decrease in detectable virus or increase in CD4+ T cell count.
  • oral administration in the form of a tablet or capsule is preferred.
  • the preliminary dosage range for oral administration is broad, since it is expected that dose modifications might need to be made in individual patients to ameliorate or forestall toxic side effects.
  • the preliminary dosage ranges are lower than what is currently known as the clinical standard for each of the compounds of the invention. Since the CTP synthase inhibitor will potentiate the activity of the nucleoside analogue, comparatively less analogue should be required than what is currently excepted.
  • the standard dosage for ddC alone currently averages a 0.75 mg tablet three times a day.
  • the dose range for ddC when coadministered with either CPE-C or 3-DU is between 0.05 to 1.0 mg three times a day depending on the individual patient.
  • the standard dosage for 3-TC currently averages 150 mg twice a day.
  • the dose range for 3-TC when coadministered with either CPE-C or 3-DU is between 10 to 200 mg twice a day depending on the patient.
  • CTP synthase inhibitors have not been used for the treatment of HIV, both CPE-C and 3-DU have been applied as anti-tumor drugs.
  • the quantity of these drugs that is needed to potentiate the activity of the nucleoside analogue is significantly less than the quantity required for antitumor -In ⁇
  • the dosage range for CPE-C according to the present invention is 1 to 25 mg twice a day, and the dosage range for 3-DU according to the present invention is 5 to 250 mg twice a day. Again, the exact dosage within each range will depend on the individual. In addition, dosages will be otpimized to achieve circulating plasma concentrations within the following ranges for each preferred drug: 3-TC, 0.002 to 1.000 ⁇ M; 3-DU, 0.5 to 10.0 ⁇ M; ddC, 0.01 to 1.00 ⁇ M; and CPE-C, 0.001 to 0.020 ⁇ M.
  • Fig. 1 is a graph illustrating the potentiating effect of 3-DU at three concentrations (0.5, 1 and 2 ⁇ M) on 3-TC activity (also at three concentrations) against an HIV-1 clinical strain.
  • Drug susceptibility was determined using PHA-stimulated PBM cells from an HIV-seronegative blood donor.
  • a 10 2 50% tissue culture infectious dose (TCID 50 ) of virus stock was used to infect one million cells. Concentration of the HIV p24 protein was determined on day 7 by radioimmunoassay .
  • Fig. 2 is a graph illustrating the potentiating effect of 3-DU at three concentrations on ddC activity (also at three concentrations) against an HIV-1 clinical strain using the same cells and detection method at day 7 as for Fig. 1.
  • Fig. 3 is a graph illustrating the effect of 3-DU alone on PHA-treated PBM cells at day 6. Cell viability was determined by trypan blue-exclusion.
  • Fig. 4 is a graph illustrating the effect of 3-DU on 3-TC activity against an HIV-1 mutant (HIV-1 184 ) resistant to AZT, ddl, ddC, D4T, ddG and protease inhibitor KNI272, using the same cells and detection method at day 7 as for HIV-1 mutant (HIV-1 184 ) resistant to AZT, ddl, ddC, D4T, ddG and protease inhibitor KNI272, using the same cells and detection method at day 7 as for HIV-1 mutant (HIV-1 184 ) resistant to AZT, ddl, ddC, D4T, ddG and protease inhibitor KNI272, using the same cells and detection method at day 7 as for HIV-1 mutant (HIV-1 184 ) resistant to AZT, ddl, ddC, D4T, ddG and protease inhibitor KNI272, using the same cells and detection method at day 7 as for HIV-1 mutant (HI
  • Fig. 5 is a graph illustrating the effect of 3-DU on ddC activity against the HIV mutant, using the same cells and detection method at day 7 as for Fig. 1.
  • Fig. 6 is a graph demonstrating the effect of CPE-C at two concentrations (40 nM and 100 nM) on 3-TC activity against an HIV-1 clinical strain using the same cells and detection method at day 7 as for Fig. 1.
  • Fig. 7 is a graph demonstrating the effect of CPE-C alone on PHA-treated PMB cells at concentrations ranging from 0 to 100 nM) .
  • PHA-stimulated PBM cells (3 X 10 7 ) were incubated with CPE-C at various concentrations in 15 ml culture medium. Cell viability was determined at day 6 with trypan blue-exclusion.
  • Fig. 8 is a graph showing the effect of 2 ⁇ M 3-DU on cellular dNTP pools in PHA-treated PBM cells after up to 50 hours in culture. The concentration of dNTPs was determined using a known enzymatic assay (Sherman et al, 1989, Anal Biochem 180: 222-226).
  • Fig. 9 is a graph demonstrating the effect of various concentrations of 3-DU (0 to 2.0 ⁇ M) after 20 hours incubation on the activity of deoxycytidine kinase (dCK) activity in PHA-treated PMB cells.
  • dCK activity was determined using a known assay (Gao et al, 1995, Proc. Natl. Acad. Sci. USA 92: 8333-8337).
  • Fig. 10 is a graph showing the time-dependent activation of dCK by 2 ⁇ M 3-DU in PHA-treated PMB cells. dCK activity was determined after 10, 20, 30, 40 and 50 hours of culture.
  • Fig. 11 is a diagram summarizing the potentiating effect of the CTP synthase inhibitor. As shown in the figure, inhibition of CTP synthase causes a fall in dCTP levels. Such a decrease causes a compensatory increase in deoxycytidine kinase, thereby leading to increased levels of the phosphorylated cytidine-based nucleotides .
  • the HIV-1 clinical strain used for in vitro studies was isolated from a patient with advanced HIV-1 infection prior to antiviral therapy.
  • the multi-resistant HIV-1 mutant was produced by recombination of HIV-l 62/75r77rll6(151
  • HIV-1 431 which is resistant to the protease inhibitor KNI .
  • the reverse transcriptase mutations at codons A62V, V75I, F77L, F116Y, and Q151M originated during AZT/ddC combination therapy.
  • Mutant HIV-1 184 resistant to 3-TC, contains a mutation widely known in the art (Gao et al, 1993, Antimicrob Agents Chemother 37: 1390-1392). Both the 431 and 184 mutants were constructed by site-directed mutagenesis in our lab using a plasmid vector encoding the HIV-1 genome, which was then used to transfect COS cells for the production of virus .
  • PBM Peripheral blood mononuclear
  • 3-TC has been licensed to Glaxo-Wellcome and is sold under the trade name Epivir.
  • ddC has been licensed to
  • both 3-DU and CPE-C may be obtained from the pharmaceutical resources branch at the National Cancer Inst. in Bethesda, MD.
  • the concentration of p24 in the cell cultures was decreased from approximately 140 ng/ml in untreated cultures to about 100 ng/ml in cultures treated with 0.1 ⁇ M 3-TC alone.
  • the addition of 3-DU at a concentration of 0.5 ⁇ M resulted in a 25% decrease of detectable HIV-1 p24 in the presence of 0.1 ⁇ M 3-TC, while at concentrations of 1 and 2 ⁇ M 3-DU, respectively, the amount of HIV-1 p24 was reduced to less than 10 ng/ml in the presence of 0.1 ⁇ M 3-TC.
  • the potentiating effect of 3-DU on the cytidine analogue ddC was also tested using the clinical HIV-1 isolate.
  • the experimental conditions were the same as for Example 1 except 3-DU was tested at concentrations 0.1, 0.5 and 1 ⁇ M.
  • ddC alone reduced detectable viral p24 protein from approximately 105 ng/ml in untreated control cultures to about 75 ng/ml at day 7 when supplied at a concentration of 0.04 ⁇ M.
  • a concentration of 0.01 ⁇ M of ddC alone had a negligible antiviral effect.
  • detectable viral protein in cultures with 0.01 ⁇ M ddC was reduced by about 20% to 40% in the presence of varying concentrations of 3-DU (0.1 to 1.0 ⁇ M) .
  • 3-DU 0.1 to 1.0 ⁇ M
  • 3-DU The potentiating effect of 3-DU on 3-TC activity was also tested using an HIV mutant strain resistant to AZT, ddl, ddC, D4T, ddG and KNI272. Experimental conditions were the same as described in Example 1 (Radioimmunoassay Lot No. 189179). 3-DU was tested at concentrations ranging from 0 to 2 ⁇ M, each in the presence of 0 to 100 nM of 3- TC. As shown in Fig. 4, 3-TC alone was mildly effective at reducing detectable p24 protein from the multi-resistant virus. Based on the data, we have estimated that a critical dose of 586 nM 3-TC would reduce virus production by 50%.
  • Example 4 Effect of 3-DU on ddC activity against a 3-TC- resistant HIV-1 mutant (HIV-1 184 )
  • the potentiating effect of CPE-C was also tested using the assay described in Example 1. Again, one million PHA- stimulated PBM cells were infected with 10 2 TCID 50 infectious doses in the presence of either 0, 40 nM, or 100 nM CPE-C and various concentrations of 3-TC at day 0. On day 7, the concentration of viral p24 protein was measured using the radioimmunoassay as described above (Assay Lot No. 189179) .
  • Example 6 Effect of 3-DU on dNTP pools
  • PHA-stimulated PBM cells were incubated with 3-DU at 2 ⁇ M in 20 ml of culture medium for 20 hours.
  • Cellular dNTPs were then extracted and the concentration was determined using an enzymatic assay originally developed at Burroughs Wellcome (Sherman et al, 1989, Anal Biochem 180: 222-226) .
  • a modification of this assay has also been published that corrects for the presence of dideoxy nucleotides (Gao et al, 1994, Anal Biochem 222: 116-122).
  • Fig. 8 only dCTP pools decline over a period of 50 hours, as would be expected as a consequence of the inhibition of CTP synthase.
  • Example 7 Effect of 3-DU on deoxycytidine kinase (dCK) activity As dCTP levels rise, a concomitant increase in dCK activity would be expected due to feedback stimulation of the salvage pathway.
  • 3-DU deoxycytidine kinase
  • Fig. 10 demonstrates the time-dependent activation of dCK and indicates that, although levels of dCK rise without the presence of 3-DU, the concentration of dCK peaks at approximately 30 hours in untreated cells, while the concentration of dCK in cells treated with 2 ⁇ M 3-DU continues to rise even at 50 hours. Additionally, the concentration of dCK in treated cells after 30 hours of culture is indeed higher than in untreated cells, indicating that, as expected, 3-DU treatment leads to higher levels of dCK by decreasing cellular pools of dCTP.
  • Fig. 11 summarizes how CTP synthase inhibition acts to potentiate the activity of the anti-viral nucleoside analogue. Since deoxycytidine kinase is responsible for the initial phosphorylation step of ddC and 3-TC, the effect of increased levels of dCK is a concomitant increase in mono-phosphate and thus in tri-phosphate derivatives of the antiviral drugs, thereby increasing the potency and speed with which the nucleoside analogues are recognized by the viral reverse transcriptase.

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Abstract

Cette invention concerne un procédé d'isolation qui améliore les caractéristiques de fonctionnement de dispositifs micromécaniques au silicium. Ce procédé consiste à intégrer des segments d'isolation diélectriques dans une microstructure au silicium constituée par une grille de barrettes montées en porte-à-faux. Une couche métallique appliquée à la partie supérieure des barrettes de silicium, qui assure le contact avec lesdites barrettes et relie celles-ci entre elles, commande électriquement le dispositif en vue de son déplacement ou d'une action de transducteur. Des chemins de connexion multiples sont incorporés au cours d'une opération de marquage du métal avant définition de la structure. La présente invention offre l'avantage de faciliter la mise en oeuvre de procédés existants en rendant possible l'exécution de toutes les opérations de marquage lithographique sur des topographies plates et en rendant superflues les opérations compliquées de métallisation par pulvérisation cathodique requises pour la plupart des procédés de fabrication de structures à rapport dimensionnel élevé. Cette invention peut être exploitée au prix de quelques modifications seulement dans le cadre de processus de fabrication de circuits intégrés pour dispositifs complètement intégrés.
PCT/US1998/000784 1997-01-21 1998-01-20 Isolation en tranchee pour dispositifs micromecamiques WO1998031375A1 (fr)

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AU58255/98A AU5825598A (en) 1997-01-21 1998-01-20 Enhanced suppression of hiv-1 by the combination of cytidine nucleoside analogues and ctp synthase inhibitors

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US3391897P 1997-01-21 1997-01-21
US60/033,918 1997-01-21

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WO1998031375A1 true WO1998031375A1 (fr) 1998-07-23
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WO2001080809A3 (fr) * 2000-04-27 2002-02-28 Anders Hofer Medicament permettant de traiter les maladies induites par un protozoaire parasitique
WO2014186435A3 (fr) * 2013-05-14 2015-02-19 University Of Georgia Research Foundation, Inc. Compositions et procédés de réduction de la formation de néo-intima

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001080809A3 (fr) * 2000-04-27 2002-02-28 Anders Hofer Medicament permettant de traiter les maladies induites par un protozoaire parasitique
US7157449B2 (en) 2000-04-27 2007-01-02 Anders Hofer Medicament for the treatment of diseases caused by parasitic protozoa
WO2014186435A3 (fr) * 2013-05-14 2015-02-19 University Of Georgia Research Foundation, Inc. Compositions et procédés de réduction de la formation de néo-intima
US10328182B2 (en) 2013-05-14 2019-06-25 University Of Georgia Research Foundation, Inc. Compositions and methods for reducing neointima formation
US11246965B2 (en) 2013-05-14 2022-02-15 University Of Georgia Research Foundation, Inc. Compositions and methods for reducing neointima formation

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