scholarly journals Selective Intracellular Activation of a Novel Prodrug of the Human Immunodeficiency Virus Reverse Transcriptase Inhibitor Tenofovir Leads to Preferential Distribution and Accumulation in Lymphatic Tissue

2005 ◽  
Vol 49 (5) ◽  
pp. 1898-1906 ◽  
Author(s):  
William A. Lee ◽  
Gong-Xin He ◽  
Eugene Eisenberg ◽  
Tomas Cihlar ◽  
Swami Swaminathan ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Half Life ◽  
Mononuclear Cells ◽  
Parent Drug ◽  
Transcriptase Inhibitor ◽  
Peripheral Blood Mononuclear ◽  
Cell Extracts ◽  
Immunodeficiency Virus

ABSTRACT An isopropylalaninyl monoamidate phenyl monoester prodrug of tenofovir (GS 7340) was prepared, and its in vitro antiviral activity, metabolism, and pharmacokinetics in dogs were determined. The 50% effective concentration (EC50) of GS 7340 against human immunodeficiency virus type 1 in MT-2 cells was 0.005 μM compared to an EC50 of 5 μM for the parent drug, tenofovir. The (L)-alaninyl analog (GS 7340) was >1,000-fold more active than the (D)-alaninyl analog. GS 7340 has a half-life of 90 min in human plasma at 37°C and a half-life of 28.3 min in an MT-2 cell extract at 37°C. The antiviral activity (>10× the EC50) and the metabolic stability in MT-2 cell extracts (>35×) and plasma (>2.5×) were also sensitive to the stereochemistry at the phosphorus. After a single oral dose of GS 7340 (10 mg-eq/kg tenofovir) to male beagle dogs, the plasma bioavailability of tenofovir compared to an intravenous dose of tenofovir was 17%. The total intracellular concentration of all tenofovir species in isolated peripheral blood mononuclear cells at 24 h was 63 μg-eq/ml compared to 0.2 μg-eq/ml in plasma. A radiolabeled distribution study with dogs resulted in an increased distribution of tenofovir to tissues of lymphatic origin compared to the commercially available prodrug tenofovir DF (Viread).

Human serum alpha 1 acid glycoprotein reduces uptake, intracellular concentration, and antiviral activity of A-80987, an inhibitor of the human immunodeficiency virus type 1 protease.

1996 ◽  
Vol 40 (6) ◽  
pp. 1491-1497 ◽  
Author(s):  
J A Bilello ◽  
P A Bilello ◽  
K Stellrecht ◽  
J Leonard ◽  
D W Norbeck ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Peripheral Blood Mononuclear Cells ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Acid Glycoprotein ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
Hiv 1

The therapeutic utility of a human immunodeficiency virus type 1 (HIV-1) protease inhibitor may depend on its intracellular concentration, which is a property of its uptake, metabolism, and/or efflux. Previous studies in our laboratory indicated that the addition of alpha 1 acid glycoprotein (alpha 1 AGP) to the medium markedly increased the amount of the drug required to limit infection in vitro. In this study, physiologically relevant concentrations of alpha 1 AGP and a radiolabeled inhibitor, A-80987, were used to determine both the uptake and activity of the agent in HIV-1-infected human peripheral blood mononuclear cells and cell lines. Both the uptake and efflux of 14C-labeled A-80987 were rapid (t1/2, < 5 min). Uptake of the drug was linearly dependent on the concentration but insensitive to the metabolic inhibitors KF, sodium cyanide, or CCCP (carbonyl cyanide m-chlorophenyl hydrazone). The amount of A-80987 which entered the cells was inversely proportional to the concentration of alpha 1 AGP (r2, 0.99) and directly proportional to the amount of extracellular non-protein-bound drug (r2, 0.99). Most importantly, the antiviral activity of the drug in HIV-1-infected peripheral blood mononuclear cells and MT-2 cells was directly related to the amount of intracellular A-80987. This study demonstrates that A-80987 binds to alpha 1 AGP, resulting in a free fraction below 10%. Cellular uptake of A-80987 is proportionally decreased in the presence of alpha 1 AGP, which results in less-than-expected antiviral activity. Importantly, we demonstrate for the first time that the inhibition of HIV protease is highly correlated with the amount of intracellular inhibitor.

scholarly journals MDL 74,968, a new acyclonucleotide analog: activity against human immunodeficiency virus in vitro and in the hu-PBL-SCID.beige mouse model of infection.

1996 ◽  
Vol 40 (5) ◽  
pp. 1072-1077 ◽  
Author(s):  
C G Bridges ◽  
D L Taylor ◽  
P S Ahmed ◽  
T M Brennan ◽  
J M Hornsperger ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Hiv Infection ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Placebo Control ◽  
Intravenous Route ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Orally Bioavailable

The novel acyclonucleotide derivative of guanine, 9-[2-methylidene-3-(phosphonomethoxy)propyl] guanine (MDL 74,968), had antiviral activity comparable to those of 9-(2-phosphonomethoxyethyl) adenine (PMEA) and 2',3'-dideoxyinosine against laboratory strains of both human immunodeficiency virus (HIV) types 1 and 2 cultured in MT-4 cells and several clinical HIV isolates cultured in human peripheral blood mononuclear cells (PBMCs). MDL 74,968 was at least fourfold less toxic than PMEA to MT-4 cells or PBMCs, thereby producing a more favorable in vitro selectivity index for the former compound. Studies of acute toxicity in CD-1 mice showed that MDL 74,968 was not toxic at doses of 1,600 mg/kg of body weight via the intraperitoneal route or at doses of 500 mg/kg via the intravenous route. Furthermore, no adverse effects of MDL 74,968 were apparent when mice were treated at doses of 200 mg/kg twice daily for 5 days. Treatment by continuous subcutaneous infusion of MDL 74,968 or PMEA at the daily dose of 20 mg/kg in the hu-PBL-SCID.beige murine model of HIV infection significantly reduced the severity of infection compared with that in placebo-treated controls. Quantitation of virus recovery by endpoint titration of spleen cells in coculture with mitogen-activated PBMCs demonstrated that MDL 74,968 as well as PMEA significantly reduced the amount of virus (P < 0.02). Moreover, by using DNA extracted from spleens, the mean HIV:HLA PCR product ratio, which takes into account individual variation in immune system reconstitution, were 0.50 and 0.40 for MDL 74,968 and PMEA treatments, respectively, whereas animals receiving the placebo control had significantly higher levels of HIV proviral DNA (mean 0.78; P < 0.02). Taken together, these promising findings suggest that an orally bioavailable prodrug of MDL 74,968 should be developed for the treatment of HIV infection.

scholarly journals Design and Profiling of GS-9148, a Novel Nucleotide Analog Active against Nucleoside-Resistant Variants of Human Immunodeficiency Virus Type 1, and Its Orally Bioavailable Phosphonoamidate Prodrug, GS-9131

2007 ◽  
Vol 52 (2) ◽  
pp. 655-665 ◽  
Author(s):  
Tomas Cihlar ◽  
Adrian S. Ray ◽  
Constantine G. Boojamra ◽  
Lijun Zhang ◽  
Hon Hui ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mononuclear Cells ◽  
Biological Properties ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT GS-9148 [(5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonic acid] is a novel ribose-modified human immunodeficiency virus type 1 (HIV-1) nucleotide reverse transcriptase (RT) inhibitor (NRTI) selected from a series of nucleoside phosphonate analogs for its favorable in vitro biological properties including (i) a low potential for mitochondrial toxicity, (ii) a minimal cytotoxicity in renal proximal tubule cells and other cell types, (iii) synergy in combination with other antiretrovirals, and (iv) a unique resistance profile against multiple NRTI-resistant HIV-1 strains. Notably, antiviral resistance analysis indicated that neither the K65R, L74V, or M184V RT mutation nor their combinations had any effect on the antiretroviral activity of GS-9148. Viruses carrying four or more thymidine analog mutations showed a substantially smaller change in GS-9148 activity relative to that observed with most marketed NRTIs. GS-9131, an ethylalaninyl phosphonoamidate prodrug designed to maximize the intracellular delivery of GS-9148, is a potent inhibitor of multiple subtypes of HIV-1 clinical isolates, with a mean 50% effective concentration of 37 nM. Inside cells, GS-9131 is readily hydrolyzed to GS-9148, which is further phosphorylated to its active diphosphate metabolite (A. S. Ray, J. E. Vela, C. G. Boojamra, L. Zhang, H. Hui, C. Callebaut, K. Stray, K.-Y. Lin, Y. Gao, R. L. Mackman, and T. Cihlar, Antimicrob. Agents Chemother. 52:648-654, 2008). GS-9148 diphosphate acts as a competitive inhibitor of RT with respect to dATP (Ki = 0.8 μM) and exhibits low inhibitory potency against host polymerases including DNA polymerase γ. Oral administration of GS-9131 to beagle dogs at a dose of 3 mg/kg of body weight resulted in high and persistent levels of GS-9148 diphosphate in peripheral blood mononuclear cells (with a maximum intracellular concentration of >9 μM and a half-life of >24 h). This favorable preclinical profile makes GS-9131 an attractive clinical development candidate for the treatment of patients infected with NRTI-resistant HIV.

scholarly journals Induction of Murine Mucosal CCR5-Reactive Antibodies as an Anti-Human Immunodeficiency Virus Strategy

2005 ◽  
Vol 79 (11) ◽  
pp. 6848-6858 ◽  
Author(s):  
C. Barassi ◽  
E. Soprana ◽  
C. Pastori ◽  
R. Longhi ◽  
E. Buratti ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Flock House Virus ◽  
Peripheral Blood Mononuclear ◽  
Genital Mucosa ◽  
Conformationally Constrained ◽  
Main Site ◽  
Immunodeficiency Virus

ABSTRACT The genital mucosa is the main site of initial human immunodeficiency virus type 1 (HIV-1) contact with its host. In spite of repeated sexual exposure, some individuals remain seronegative, and a small fraction of them produce immunoglobulin G (IgG) and IgA autoantibodies directed against CCR5, which is probably the cause of the CCR5-minus phenotype observed in the peripheral blood mononuclear cells of these subjects. These antibodies recognize the 89-to-102 extracellular loop of CCR5 in its native conformation. The aim of this study was to induce infection-preventing mucosal anti-CCR5 autoantibodies in individuals at high risk of HIV infection. Thus, we generated chimeric immunogens containing the relevant CCR5 peptide in the context of the capsid protein of Flock House virus, a presentation system in which it is possible to engineer conformationally constrained peptide in a highly immunogenic form. Administered in mice via the systemic or mucosal route, the immunogens elicited anti-CCR5 IgG and IgA (in sera and vaginal fluids). Analogous to exposed seronegative individuals, mice producing anti-CCR5 autoantibodies express significantly reduced levels of CCR5 on the surfaces of CD4+ cells from peripheral blood and vaginal washes. In vitro studies have shown that murine IgG and IgA (i) specifically bind human and mouse CD4+ lymphocytes and the CCR5-transfected U87 cell line, (ii) down-regulate CCR5 expression of CD4+ cells from both humans and untreated mice, (iii) inhibit Mip-1β chemotaxis of CD4+ CCR5+ lymphocytes, and (iv) neutralize HIV R5 strains. These data suggest that immune strategies aimed at generating anti-CCR5 antibodies at the level of the genital mucosa might be feasible and represent a strategy to induce mucosal HIV-protective immunity.

scholarly journals Determination of ddATP Levels in Human Immunodeficiency Virus-Infected Patients Treated with Dideoxyinosine

2004 ◽  
Vol 48 (2) ◽  
pp. 589-595 ◽  
Author(s):  
Cecile Le Saint ◽  
Raphael Terreux ◽  
Daniele Duval ◽  
Jacques Durant ◽  
Helene Ettesse ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Highly Active Antiretroviral Therapy ◽  
Mononuclear Cells ◽  
Exchange Chromatography ◽  
Therapeutic Monitoring ◽  
Target Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus

ABSTRACT Clinical failures of the highly active antiretroviral therapy could result from inefficient intracellular concentrations of antiviral drugs. The determination of drug contents in target cells of each patient would be useful in clinical investigations and trials. The purpose of this work was to quantify the intracellular concentration of ddATP, the active metabolite of dideoxyinosine (ddI), in peripheral blood mononuclear cells (PBMCs) of human immunodeficiency virus (HIV)-infected patients treated with ddI. We have raised antibodies against ddA-citrate, a stable isostere of ddATP selected on the basis of its structural and electronic analogies with ddATP. The anti-ddA-citrate antibodies recognized ddATP and ddA with nanomolar affinities and cross-reacted neither with any of the nucleotide reverse transcriptase inhibitors used in HIV therapy nor with their phosphorylated metabolites. The three phosphorylated metabolites of ddI (ddAMP, ddADP, and ddATP) were purified by anion exchange chromatography and the amount of each metabolite was determined by radioimmunoassay with or without prior phosphatase treatment. The intracellular levels of the three ddI metabolites were measured both in an in vitro model and in PBMCs of HIV-infected patients under ddI treatment. The possibility to measure intracellular levels of ddATP from small blood samples of HIV-infected patients treated with ddI could be exploited to develop individual therapeutic monitoring.

scholarly journals Circulating Metabolites of the Human Immunodeficiency Virus Protease Inhibitor Nelfinavir in Humans: Structural Identification, Levels in Plasma, and Antiviral Activities

2001 ◽  
Vol 45 (4) ◽  
pp. 1086-1093 ◽  
Author(s):  
Kanyin E. Zhang ◽  
Ellen Wu ◽  
Amy K. Patick ◽  
Bradley Kerr ◽  
Mark Zorbas ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Protease Inhibitor ◽  
Active Metabolite ◽  
Parent Drug ◽  
Hiv Reverse Transcriptase ◽  
Immunodeficiency Virus ◽  
Antiviral Activities ◽  
Hiv 1

ABSTRACT Nelfinavir mesylate (Viracept, formally AG1343) is a potent and orally bioavailable human immunodeficiency virus (HIV) type 1 (HIV-1) protease inhibitor (K i = 2 nM) and is being widely prescribed in combination with HIV reverse transcriptase inhibitors for the treatment of HIV infection. The current studies evaluated the presence of metabolites circulating in plasma following the oral administration of nelfinavir to healthy volunteers and HIV-infected patients, as well as the levels in plasma and antiviral activities of these metabolites. The results showed that the parent drug was the major circulating chemical species, followed in decreasing abundance by its hydroxy-t-butylamide metabolite (M8) and 3′-methoxy-4′-hydroxynelfinavir (M1). Antiviral assays with HIV-1 strain RF-infected CEM-SS cells showed that the 50% effective concentrations (EC50) of nelfinavir, M8, and M1 were 30, 34, and 151 nM, respectively, and that the corresponding EC50 against another HIV-1 strain, IIIB, in MT-2 cells were 60, 86, and 653 nM. Therefore, apparently similar in vitro antiviral activities were demonstrated for nelfinavir and M8, whereas an approximately 5- to 11-fold-lower level of antiviral activity was observed for M1. The active metabolite, M8, showed a degree of binding to human plasma proteins similar to that of nelfinavir (ca. 98%). Concentrations in plasma of nelfinavir and its metabolites in 10 HIV-positive patients receiving nelfinavir therapy (750 mg three times per day) were determined by a liquid chromatography tandem mass spectrometry assay. At steady state (day 28), the mean plasma nelfinavir concentrations ranged from 1.73 to 4.96 μM and the M8 concentrations ranged from 0.55 to 1.96 μM, whereas the M1 concentrations were low and ranged from 0.09 to 0.19 μM. In conclusion, the findings from the current studies suggest that, in humans, nelfinavir forms an active metabolite circulating at appreciable levels in plasma. The active metabolite M8 may account for some of the antiviral activity associated with nelfinavir in the treatment of HIV disease.

Immunophenotypic Analysis of Peripheral Blood Mononuclear Cells Undergoing In Vitro Apoptosis After Isolation From Human Immunodeficiency Virus–Infected Children

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4230-4237 ◽  
Author(s):  
Thomas W. McCloskey ◽  
Saroj Bakshi ◽  
Soe Than ◽  
Parisa Arman ◽  
Savita Pahwa
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Mononuclear Cells ◽  
Apoptotic Cell ◽  
Costimulatory Molecule ◽  
T Cell Subsets ◽  
Live Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus

Abstract Lymphocytes of human immunodeficiency virus (HIV)-infected individuals undergo accelerated apoptosis in vitro, but the subsets of cells affected have not been clearly defined. This study examined the relationship between lymphocyte phenotype and apoptotic cell death in HIV-infected children by flow cytometry. Direct examination of the phenotype of apoptotic lymphocytes was accomplished using a combination of surface antigen labeling performed simultaneously with the Tdt mediated Utp nick end-labeling (TUNEL) assay. In comparison to live cells, apoptotic lymphocytes displayed an overrepresentation of CD45RO and HLA-DR expressing cells, while CD28 and CD95 expressing cells were underrepresented. Lymphocytes expressing CD4, CD8, and CD38 were equally represented in apoptotic and live populations. When percent lymphocyte apoptosis follow- ing culture was examined independently with lymphocyte subsets in fresh blood, apoptosis was negatively correlated with the percentage of CD4 cells, but not with specific CD4 T-cell subsets. Although not correlated with the percentage of total CD8 cells, apoptosis was positively correlated with specific CD8 T-cell subsets expressing CD45RO and CD95 and negatively correlated for CD8 T cells expressing CD45RA. These results provide direct evidence that a population of activated lymphocytes with the memory phenotype lacking the costimulatory molecule CD28 are especially prone to undergo apoptosis. The findings related to CD95 expression in fresh and apoptotic cells implicate Fas-dependent and Fas-independent pathways of apoptosis in HIV disease in children.

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