Anti-human Immunodeficiency Virus Drug Research Articles

Cytochrome P450 3A5 Plays a Prominent Role in the Oxidative Metabolism of the Anti-Human Immunodeficiency Virus Drug Maraviroc

Maraviroc is an anti-human immunodeficiency virus drug that acts by blocking viral entry into target cells. With use of ultra-performance liquid chromatography-mass spectrometry several monooxygenated, dioxygenated, and glucuronidated metabolites of maraviroc were identified both in vitro and in vivo. Characterization of the enzymes involved in the production of these metabolites determined that cytochrome P450 3A5 was the principal enzyme responsible for the formation of an abundant metabolite of maraviroc that resulted from oxygenation of the dichlorocyclohexane ring. For the formation of this metabolite, the V(max) values for CYP3A4 and CYP3A5 were 0.04 and 0.93 pmol · min⁻¹ · pmol P450⁻¹, and the K(m) values were 11.1 and 48.9 μM, respectively. Furthermore, human liver microsomes isolated from donors homozygous for the loss-of-function CYP3A5*3 allele exhibited a 79% decrease in formation of this metabolite compared with those homozygous for the wild-type CYP3A5*1 allele. To probe which divergent residues between CYP3A4 and CYP3A5 might play a role in the differential activities of these enzymes toward maraviroc, mutations were introduced into both enzymes and metabolism of maraviroc was measured. A CYP3A5 L57F mutant exhibited a 61% decrease in the formation of this metabolite, whereas formation by a CYP3A4 F57L mutant was increased by 337% compared with that of the wild type. Taken together, these data provide novel insights into the biotransformation of maraviroc as well as the potential role of CYP3A4 and CYP3A5 divergent residues in the enzymatic activities of these two highly homologous enzymes.

Lamivudine Salts with Improved Solubilities

To optimize solubility of drugs, current strategies mainly focus on engineering and screening of smart crystal phases. Two salts of the anti-human immunodeficiency virus (HIV) drug lamivudine--namely, lamivudine hydrochloride and lamivudine hydrochloride monohydrate, were prepared in the course of screening the crystallization conditions of lamivudine duplex, an uncommon DNA-mimic, double-stranded helical structure made up of partially protonated drug pairs. Here, water solubilities of lamivudine hydrochloride, lamivudine hydrochloride monohydrate, and lamivudine duplex are reported. The aqueous solubility of this anti-HIV drug was significantly increased in both salts and also in lamivudine duplex in relation to the water solubility of lamivudine form II. In comparison with the lamivudine form II incorporated into therapeutic formulations, the drug solubility was increased at a temperature of 299 ± 2 K by factors of 1.2, 3.3, and 4.5 in lamivudine hydrochloride, lamivudine hydrochloride monohydrate, and lamivudine duplex, respectively, demonstrating that this solid-state property of lamivudine can be improved by crystal engineering strategies. Solubility profiles were understood on the basis of structural and solvent-solute interaction approaches. At last, correlations between solubility and crystal structures allowed for a rational approach to understand how this physicochemical feature could be enhanced by engineering new salts of the drug.

The successes and failures of HIV drug discovery

Introduction: To date, several anti-human immunodeficiency virus (HIV) drugs, including reverse transcriptase inhibitors and protease inhibitors, have been developed and used clinically for the treatment of patients infected with HIV. Recently, novel drugs have been discovered which have different mechanisms of action from those of the above inhibitors, including entry inhibitors and integrase (IN) inhibitors; the clinical use of three of these inhibitors has been approved. Other inhibitors are still in development.Areas covered: This review article summarizes the history of the development of anti-HIV drugs and also focuses on successes in the development of these entry and IN inhibitors, along with looking at exploratory approaches for the development of other inhibitors.Expert opinion: Currently used highly active antiretroviral therapy can be subject to a loss of efficacy, due to the emergence of multi-drug resistant (MDR) strains; a change of regimens of the drug combination is required to combat this, along with careful monitoring of the virus and CD4 in the blood, by methods such as cellular tropism testing. In such a situation, entry inhibitors such as CCR5/CXCR4 antagonists, CD4 mimics, fusion inhibitors and IN inhibitors might be optional agents for an expansion of the drug repertoire available to patients at all stages of HIV infection.

Fc-receptor-mediated targeting of antibody-bearing liposomes containing dideoxycytidine triphosphate to human monocyte/macrophages.

Liposomes bearing surface-attached antibody (L-Ab) molecules can be used for various purposes including the immunospecific delivery of drugs or other materials to antigenic target cells. In this study, L-Ab were prepared to deliver an anti-human immunodeficiency virus (HIV) drug, dideoxycytidine triphosphate (ddCTP) to human monocyte/macrophages. Cells of the monocyte/macrophage lineage are an important reservoir of HIV-1. A mouse monoclonal antibody IgG2a was labelled with 125I and modified using N succinimidyl-3-(2-pyridyldithio)propionate (SPDP) as a heterobifunctional reagent in order to conjugate with liposomes to produce a covalent bond (thioether). SPDP-modified antibody was incubated with liposomes containing 5 mol% of maleimido phenyl butyrate phosphatidylethanolamine (MPB-PE) at room temperature (21 degrees C) for 24 h. L-Ab were separated from free and aggregated antibodies by centrifugation. L-Ab were characterized by measuring particle size and binding to anti-mouse IgG-sepharose. Ninety five per cent of the liposomal (L-Ab) lipid label was bound to anti-mouse IgG-sepharose, whereas only 7% of plain liposomes were bound, indicating non-specific binding. Uptake of L-Ab was measured in human monocyte/macrophages as a function of time and compared with that of plain liposomes. The uptake increased with time and it was 4-6 times greater than that of plain liposomes although part of that effect may have been due to unreacted MPB groups.

Differential Sensitivities of Retroviruses to Integrase Strand Transfer Inhibitors

Integrase inhibitors are emerging anti-human immunodeficiency virus (HIV) drugs, and multiple retroviruses and transposable elements were evaluated here for susceptibilities to raltegravir (RAL) and elvitegravir (EVG). All viruses, including primate and nonprimate lentiviruses, a Betaretrovirus, a Gammaretrovirus, and the Alpharetrovirus Rous sarcoma virus (RSV), were susceptible to inhibition by RAL. EVG potently inhibited all lentiviruses and intermediately inhibited Betaretrovirus and Gammaretrovirus infections yet was basically ineffective against RSV. Substitutions based on HIV type 1 (HIV-1) resistance changes revealed that integrase residue Ser150 contributed significantly to the resistance of RSV. The drugs intermediately inhibited intracisternal A-particle retrotransposition but were inactive against Sleeping Beauty transposition and long interspersed nucleotide element 1 (LINE-1) retrotransposition.

Dendrimers as Potential Inhibitors of the Dimerization of the Capsid Protein of HIV-1

Assembly of the mature human immunodeficiency virus type 1 capsid involves the oligomerization of the capsid protein, CA. The C-terminal domain of CA, CTD, participates both in the formation of CA hexamers and in the joining of hexamers through homodimerization. Intact CA and the isolated CTD are able to homodimerize in solution with similar affinity (dissociation constant in the order of 10 microM); CTD homodimerization involves mainly an alpha-helical region. In this work, we show that first-generation gallic acid-triethylene glycol (GATG) dendrimers bind to CTD. The binding region is mainly formed by residues involved in the homodimerization interface of CTD. The dissociation constant of the dendrimer-CTD complexes is in the range of micromolar, as shown by ITC. Further, the affinity for CTD of some of the dendrimers is similar to that of synthetic peptides capable of binding to the dimerization region, and it is also similar to the homodimerization affinity of both CTD and CA. Moreover, one of the dendrimers, with a relatively large hydrophobic moiety at the dendritic branching (a benzoate), was able to hamper the assembly in vitro of the human immunodeficiency virus capsid. These results open the possibility of considering dendrimers as lead compounds for the development of antihuman immunodeficiency virus drugs targeting capsid assembly.

HIV aspartyl protease inhibitors as promising compounds againstCandida albicansAndré Luis Souza dos Santos

Cells of Candida albicans (C. albicans) can invade humans and may lead to mucosal and skin infections or to deep-seated mycoses of almost all inner organs, especially in immunocompromised patients. In this context, both the host immune status and the ability of C. albicans to modulate the expression of its virulence factors are relevant aspects that drive the candidal susceptibility or resistance; in this last case, culminating in the establishment of successful infection known as candidiasis. C. albicans possesses a potent armamentarium consisting of several virulence molecules that help the fungal cells to escape of the host immune responses. There is no doubt that the secretion of aspartyl-type proteases, designated as Saps, are one of the major virulence attributes produced by C. albicans cells, since these hydrolytic enzymes participate in a wide range of fungal physiological processes as well as in different facets of the fungal-host interactions. For these reasons, Saps clearly hold promise as new potential drug targets. Corroborating this hypothesis, the introduction of new anti-human immunodeficiency virus drugs of the aspartyl protease inhibitor-type (HIV PIs) have emerged as new agents for the inhibition of Saps. The introduction of HIV PIs has revolutionized the treatment of HIV disease, reducing opportunistic infections, especially candidiasis. The attenuation of candidal infections in HIV-infected individuals might not solely have resulted from improved immunological status, but also as a result of direct inhibition of C. albicans Saps. In this article, we review updates on the beneficial effects of HIV PIs against the human fungal pathogen C. albicans, focusing on the effects of these compounds on Sap activity, growth behavior, morphological architecture, cellular differentiation, fungal adhesion to animal cells and abiotic materials, modulation of virulence factors, experimental candidiasis infection, and their synergistic actions with classical antifungal agents.

Antiretroviral tissue kinetics: in vivo imaging using positron emission tomography.

Our current knowledge on the antiviral efficacy, dosing, and toxicity of available highly active antiretroviral therapy regimens is mostly derived from plasma or blood kinetics of anti-human immunodeficiency virus (anti-HIV) drugs. However, the blood comprises only 2% of the total target cells in the body. Tissue drug levels may differ substantially from corresponding plasma levels, and drug distribution processes may be characterized by high intertissue variability, leading to suboptimal target site concentrations and the potential risk for therapeutic failures. Positron emission tomography has greatly expanded the scope of the pharmacokinetic measurements that can be performed noninvasively in animal models or humans. We have prepared [18F]FPMPA, a fluorine-18-radiolabeled analogue of tenofovir, to study antiretroviral tissue kinetics in vivo noninvasively and tested the imaging probe in rats. The biodistribution of the fluorine-18 analogue closely follows that of nonfluorinated tenofovir. Compared to that in the blood, the levels of penetration of the antiretroviral drug were found to be significantly reduced in the spleen and submandibular lymph nodes (approximately 2-fold), in the mesenteric lymph nodes and the testes (approximately 4-fold), and in the brain compartment (approximately 25-fold). Intersubject variability of the trough drug concentration (measured at 120 min) in certain tissues, like the colon (coefficient of variation, >100%), is not reflected by the intersubject variability in the blood compartment (coefficient of variation, 24%). Positron emission tomography imaging of the fluorine-18 analogue revealed the accumulation of the antiretroviral drug in the cortex of the kidneys, a potential correlate of tenofovir-induced nephrotoxicity observed in HIV-1-infected treated patients. Thus, [18F]FPMPA is a promising radiotracer for evaluation of tenofovir biodistribution under carefully controlled drug administration protocols.

Roles and Mechanisms of Human Immunodeficiency Virus Protease Inhibitor Ritonavir and Other Anti-Human Immunodeficiency Virus Drugs in Endothelial Dysfunction of Porcine Pulmonary Arteries and Human Pulmonary Artery Endothelial Cells

The objective of this study was to determine the effects of highly active antiretroviral therapy (HAART) drugs on pulmonary endothelial function. Porcine pulmonary arteries or human pulmonary arterial endothelial cells (HPAECs) were incubated with eight HAART drugs [ritonavir, indinavir, lopinavir, zidovudine (AZT), abacavir, stavudine, didanosine (ddI), and lamivudine] individually or in combination [three HAART drugs (3-plex; indinavir, stavudine, and ddI)] at their clinical plasma concentrations for 24 hours. Endothelium-dependent vasorelaxation in response to bradykinin was reduced significantly by the ritonavir in a concentration-dependent manner. Five other HAART drugs (indinavir, lamivudine, abacavir, AZT, and ddI) and the 3-plex significantly also impaired endothelium-dependent vasorelaxation in response to bradykinin. Five HAART drugs (ritonavir, indinavir, lamivudine, abacavir, and AZT) significantly decreased endothelial nitric oxide synthase (eNOS) expression and increased superoxide anion levels in both vessels and HPAECs. Furthermore, both ritonavir and AZT substantially activated ERK2 in HPAECs. Additionally, the antioxidants ginsenoside Rb1 and ginkgolide A effectively reversed HAART drug-induced vasomotor dysfunction and eNOS down-regulation. Inhibition of ERK1/2 also partially blocked ritonavir- and AZT-induced down-regulation of eNOS and vasomotor dysfunction. Thus, HAART drugs significantly impair endothelial functions of porcine pulmonary arteries and HPAECs, which may be mediated by eNOS down-regulation, oxidative stress, and ERK1/2 activation. These findings suggest that HAART drugs may contribute to the high incidence of pulmonary artery hypertension in human immunodeficiency virus-infected patients.

Expression and Functionality of Anti-Human Immunodeficiency Virus and Anticancer Drug Uptake Transporters in Immune Cells

Almost all drugs used in anti-human immunodeficiency virus (HIV)-1 and anticancer therapies require membrane proteins to get into the cell to develop their proper activity. Nevertheless, little is known regarding the expression and activity of specific carriers involved in the uptake of these drugs in immune cells. Here, we assessed the mRNA levels, protein expression profile, and activity of the gene families SLC28 (coding for concentrative nucleoside transporters, hCNT1-3), SLC29 (equilibrative nucleoside transporters, hENT1-2), and SLC22 (organic cation transporters, hOCT1-3 and hOCTN1-2). Both hENTs and hCNT2 were abundant in primary lymphocytes, with a preferential activity of hENT1. A significant up-regulation in hENTs expression (100-fold) and activity (30-fold) was seen under stimulation of primary T lymphocytes. In contrast, monocytes, monocyte-derived macrophages (MDMs), and immature monocyte-derived dendritic cells predominantly expressed hCNT3, a functional transporter in MDMs. Finally, in immune cells, hOCTs showed a more heterogeneous expression profile and a lower activity than human nucleoside transporters (hNTs), although up-regulation of hOCTs also occurred upon lymphocyte activation. Overall, the expression and activity of most of the studied transporters emphasize their relevance in relation to anti-HIV and anticancer therapies. The identification of the transporter involved in each specific drug uptake in immune cells could help to optimize pharmacological therapeutic responses.

Effect of Phosphonated Carbocyclic 2′-Oxa-3′-Aza-Nucleoside on Human T-Cell Leukemia Virus Type 1 Infection In Vitro

There is currently little research and development of new compounds with specific anti-human T-cell leukemia virus type 1 (HTLV-1) activity. The few antiretrovirals that have been tested against HTLV-1 in vitro have already been developed into anti-human immunodeficiency virus (HIV) drugs. Here, we show the effects of a newly synthesized family of phosphonated nucleoside compounds, phosphonated carbocyclic 2'-oxa-3'-aza-nucleosides (PCOANs), on HTLV-1 infection in vitro. To ascertain the anti-HTLV-1 activity of PCOANs, peripheral blood mononuclear cells from healthy donors were infected in vitro by coculture with an HTLV-1 donor cell line in the presence of three prototype PCOAN compounds. PCOANs were able to completely inhibit HTLV-1 infection in vitro at a concentration of 1 microM, similar to what has been observed for tenofovir and azidothymidine. Treatment with PCOANs was associated with inhibited growth of HTLV-1-infected cells, and their effects were 100 to 200 times more potent than that of tenofovir. The mechanisms involved in the anti-HTLV-1 effects of PCOANs can mainly be ascribed to their capacity to inhibit HTLV-1 reverse transcriptase activity, as ascertained by means of a cell-free assay. PCOANs caused little reduction in proliferation or induction of apoptotic cell death of uninfected cells, showing toxicity levels similar to tenofovir and lower than azidothymidine. Overall, these results indicate that the family of PCOANs includes potential candidate compounds for long-lasting control of HTLV-1 infection.

Phase I and II Study of the Safety, Virologic Effect, and Pharmacokinetics/Pharmacodynamics of Single-Dose 3-O-(3′,3′-Dimethylsuccinyl)Betulinic Acid (Bevirimat) against Human Immunodeficiency Virus Infection

Bevirimat [3-O-(3',3'-dimethylsuccinyl)betulinic acid] is the first in a new class of anti-human immunodeficiency virus (HIV) drugs that inhibit viral maturation by specifically blocking cleavage of the Gag capsid (CA) precursor, CA-SP1, to mature CA protein, resulting in defective core condensation and release of immature noninfectious virions. Four cohorts of six HIV-infected adults, with CD4 counts of >200 and plasma viral loads of 5,000 to 250,000 transcripts/ml and not currently receiving antiretroviral therapy, were randomized to receive a single oral dose of placebo, 75, 150, or 250 mg of bevirimat. Thirty blood samples for drug concentrations and 20 HIV RNA measures were collected from each subject over a 20-day period. Candidate pharmacokinetic/pharmacodynamic models were fit to individual subjects by maximum likelihood followed by Bayesian estimation; model discrimination was by corrected Akaike's Information Criterion. The bevirimat pharmacokinetics was well described by an oral two-compartment linear model (r(2), 0.98), with a mean (percent coefficient of variation) half-life of 60.3 (13.6) h and apparent oral clearance of bevirimat from the plasma compartment of 0.17 (18) liters/h. HIV RNA was modeled as being produced in infected CD4 cells, with bevirimat inhibiting infection of new CD4 cells thru a Hill-type function (r(2), 0.87). Single oral doses of bevirimat were well tolerated and demonstrated a dose-dependent reduction in viral load. The average maximum reduction from baseline following the 150- and 250-mg doses was greater than 0.45 log(10), with individual patients having reductions of greater than 0.7 log(10). No bevirimat resistance mutations were detected during the course of the study.

HIV gp41 C-terminal Heptad Repeat Contains Multifunctional Domains: RELATION TO MECHANISMS OF ACTION OF ANTI-HIV PEPTIDES

T20 (Fuzeon), a novel anti-human immunodeficiency virus (HIV) drug, is a peptide derived from HIV-1 gp41 C-terminal heptad repeat (CHR). Its mechanism of action has not yet been defined. We applied Pepscan strategy to determine the relationship between functional domains and mechanisms of action of five 36-mer overlapping peptides with a shift of five amino acids (aa): CHR-1 (aa 623-658), C36 (aa 628-663), CHR-3 (aa 633-668), T20 (aa 638-673), and CHR-5 (aa 643-678). C36 is a peptide with addition of two aa to the N terminus of C34. Peptides CHR-1 and C36 contain N-terminal heptad repeat (NHR)- and pocket-binding domains. They inhibited HIV-1 fusion by interacting with gp41 NHR, forming stable six-helix bundles and blocking gp41 core formation. Peptide T20 containing partial NHR- and lipid-binding domains, but lacking pocket-binding domain, blocked viral fusion by binding its N- and C-terminal sequences with gp41 NHR and cell membrane, respectively. Peptide CHR-3, which is located in the middle between C36 and T20, overlaps >86% of the sequences of these two peptides, and lacks pocket- and lipid-binding domains, exhibited marginal anti-HIV-1 activity. These results suggest that T20 and C36 contain different functional domains, through which they inhibit HIV-1 entry with distinct mechanisms of action. The multiple functional domains in gp41 CHR and their binding partners may serve as targets for rational design of new anti-HIV-1 drugs and vaccines.

Glucuronidation of Anti-HIV Drug Candidate Bevirimat: Identification of Human UDP-glucuronosyltransferases and Species Differences

Bevirimat [BVM, PA-457, 3-O-(3',3'-dimethylsuccinyl)-betulinic acid], a new anti-human immunodeficiency virus drug candidate, is metabolized to two monoglucuronides [mono-BVMG (I) and mono-BVMG (II)] and one diglucuronide (di-BVMG) both in vivo and in vitro. UDP-glucuronosyltransferase (UGT) reaction screening, enzyme kinetics, and species differences for the glucuronidation of BVM in vitro were investigated with pooled human liver microsomes (HLMs) and human intestinal microsomes (HIMs), animal liver microsomes, and 12 recombinant human UGT isoforms. Glucuronidation of BVM with HLMs predominantly involved the formation of mono-BVMG (I) (V(max) = 61 pmol/min/mg protein, K(m) = 27 microM) and mono-BVMG (II) (V(max) = 48 pmol/min/mg protein, K(m) = 16 microM). Di-BVMG was also observed but was a minor metabolite. HIMs mainly revealed glucuronidation to form mono-BVMG (II) (V(max) = 90 pmol/min/mg of protein, K(m) = 8.3 microM). UGT1A3 predominantly formed mono-BVMG (I) (V(max) = 65 pmol/min/mg of protein, K(m) = 13 microM), whereas UGT1A4 is a less active isoform (V(max) = 1.8 pmol/min/mg of protein, K(m) = 5.6 microM). UGT2B7 was involved in the formation of both mono-BVMG (I) (V(max) = 6.1 pmol/min/mg of protein, K(m) = 6.0 microM) and mono-BVMG (II) (V(max) = 6.5 pmol/min/mg of protein, K(m) = 7.8 microM). Among the animal liver microsomes examined, all species (rat, mouse, dog, and marmoset) demonstrated conjugation to form both mono-BVMG (I) and mono-BVMG (II), with dog liver microsomes exhibiting a higher formation rate for mono-BVMG (I), whereas marmoset liver microsomes showed a higher formation rate for mono-BVMG (II). The data suggest a primary role of UGT1A3 for the glucuronidation of BVM.

Patented HIV-1 Integrase Inhibitors (1998-2005)

Combination therapy, comprising at least three anti-human immunodeficiency virus (anti-HIV) drugs, has become the standard treatment of AIDS. Since 1996, highly active antiretroviral therapy (HAART) was designed to rapidly control HIV replication. It has had a significant impact on patient health and progression of AIDS in developed countries but its success has not been complete. HAART strategy still suffers from issues of patient compliance, cost, deleterious side effects and emerging drug resistance. Therefore it is logical to look for agents that inhibit different viral targets. In addition to the fusion, reverse transcription and protein formation processes, the HIV replicative cycle offers various other events that can be considered as potential targets for chemotherapeutic intervention. Amongst them integration is a key step and integrase (IN), one of the three viral enzymes, has been rapidly identified as a rational target for many years. To date, four molecules have entered in clinical trials. The present article reviews the increasing number of patents on small molecule HIV-1 integrase inhibitors in the 1998-2005 period, from the pioneer ones (discovery of selective strand transfer inhibitors) to the last patents including the actual molecules under clinical trials.

Oxadiazols: a New Class of Rationally Designed Anti-Human Immunodeficiency Virus Compounds Targeting the Nuclear Localization Signal of the Viral Matrix Protein

Despite recent progress in anti-human immunodeficiency virus (HIV) therapy, drug toxicity and emergence of drug-resistant isolates during long-term treatment of HIV-infected patients necessitate the search for new targets that can be used to develop novel antiviral agents. One such target is the process of nuclear translocation of the HIV preintegration complex. Previously we described a class of arylene bis(methylketone) compounds that inhibit HIV-1 nuclear import by targeting the nuclear localization signal (NLS) in the matrix protein (MA). Here we report a different class of MA NLS-targeting compounds that was selected using computer-assisted drug design. The leading compound from this group, ITI-367, showed potent anti-HIV activity in cultures of T lymphocytes and macrophages and also inhibited HIV-1 replication in ex vivo cultured lymphoid tissue. The virus carrying inactivating mutations in MA NLS was resistant to ITI-367. Analysis by real-time PCR demonstrated that the compound specifically inhibited nuclear import of viral DNA, measured by two-long terminal repeat circle formation. Evidence of the existence of this mechanism was provided by immunofluorescent microscopy, using fluorescently labeled HIV-1, which demonstrated retention of the viral DNA in the cytoplasm of drug-treated macrophages. Compounds inhibiting HIV-1 nuclear import may be attractive candidates for further development.

Viral dynamics during tenofovir therapy in patients infected with lamivudine‐resistant hepatitis B virus mutants

Tenofovir, an antihuman immunodeficiency virus (HIV) drug, has activity against lamivudine-resistant hepatitis B virus (HBV) mutants. To describe the efficacy of tenofovir in patients with lamivudine-resistant hepatitis B we applied two investigative approaches based on mathematical models of viral dynamics: the individual nonlinear fitting and the mixed-effect group fitting approaches. Eleven chronic HBV patients on lamivudine for a median of 176 weeks (range: 72-382) with YMDD mutation-related HBV-DNA breakthrough received 'add-on' tenofovir 300 mg once-daily, while maintaining their existing therapy. Sequential sera were taken at day 1 (t = 0 and t = 8 h), days 2, 4, 7, 10, 14, 21, 28 and every 4 weeks thereafter, and HBV-DNA levels were assessed using a validated quantitative polymerase chain reaction (PCR) assay. Median baseline log HBV-DNA was 8.62 (range: 6.48-9.76 log HBV-DNA). Tenofovir treatment resulted in a mean (+/-SD) log HBV-DNA decline of 1.37 +/- 0.51 in the first phase, 2.54 +/- 0.91 after 4 weeks, and 4.95 +/- 0.90 log HBV-DNA after 24 weeks. The median effectiveness of blocking viral replication in the individual fit model was 93% (range: 73-99) for eta = 0 and 93% (range: 59-99) for eta = 1. There was only a small difference between the efficacy parameter 'epsilon' of the individual nonlinear fitting and mixed-effect group fitting on the biphasic exponential model. These data show that tenofovir has good efficacy in blocking viral replication in HBV patients with lamivudine-induced drug-resistant HBV mutants, but effectiveness varies greatly among individuals. Both models can be used to describe viral decay during tenofovir therapy.

The effect of AZT and chloroquine on the activities of ricin and a saporin–transferrin chimeric toxin

This study deals with the combination of chloroquine (CQ, an anti-malaric drug) and 3′-azido-3′-deoxythymidine (AZT, anti-human immuno-deficiency virus (HIV) drug) with a chimeric toxin (TS) obtained by chemical linking of saporin (a ribosome inactivating protein from the plant Saponaria officinalis) and human transferrin, in the intoxication of the human chronic myeloid leukaemia cells (K562). Our data demonstrate that AZT, at concentrations comparable to those reached in the blood of HIV-infected patients under pharmacological treatment with this drug, can increase the toxicity of TS in cooperation with CQ inducing an increased effect on protein synthesis in K562 cells (∼50% inhibition of protein synthesis for TS alone, and TS with AZT and ∼70% with both AZT and CQ). Furthermore, pre-treatment of cells with AZT alone can induce an increase of apoptosis in K562 cells intoxicated with TS. By comparing data obtained with the model toxin ricin, we get indications that the two toxins partially differ in their intracellular routes, also suggesting that chimeric constructs containing ricin-like toxins (i.e. immunotoxins) could be coupled with the use of common and cheap drugs for the treatment of cancer in HIV-infected patients.

CD8+-cell-mediated suppression of virulent simian immunodeficiency virus during tenofovir treatment.

The ability of tenofovir to suppress viremia in simian immunodeficiency virus (SIV)-infected macaques for years despite the presence of virulent viral mutants with reduced in vitro susceptibility is unprecedented in this animal model. In vivo cell depletion experiments demonstrate that tenofovir's ability to suppress viremia during acute and chronic infection is significantly dependent on the presence of CD8+ lymphocytes. Continuous tenofovir treatment was required to maintain low viremia. Although it is unclear whether this immune-mediated suppression of viremia is linked to tenofovir's direct antiviral efficacy or is due to independent immunomodulatory effects, these studies prove the concept that antiviral immune responses can play a crucial role in suppressing viremia during anti-human immunodeficiency virus drug therapy.

Differential Expression Levels of MRP1, MRP4, and MRP5 in Response to Human Immunodeficiency Virus Infection in Human Macrophages

Multidrug resistance proteins (MRPs) have been reported to be involved in the efflux of some anti-human immunodeficiency virus (HIV) drugs. We show here that MRP1, MRP4, and MRP5 are expressed at the mRNA level in human monocyte-derived macrophages. HIV infection caused increased transcription of these MRPs; however, temporal differences in stimulation are reported.