A robust control system for targeting melanoma by a supermolecular DDMC/paclitaxel complex.

A DEAE-dextran-MMA copolymer (DDMC)-paclitaxel (PTX) conjugate was prepared using PTX as the guest and DDMC as the host. The resistance of B16F10 melanoma cells to PTX was confirmed, while the DDMC-PTX conjugate showed excellent anticancer activity that followed the Hill equation. The robustness in the tumor microenvironment of the allosteric system was confirmed via BIBO stability. This feedback control system, explained via a transfer function, was very stable and showed the sustainability of the system via a loop, and it showed superior anti-cancer activity without drug resistance from cancer cells. The block diagram of this signal system in the tumor microenvironment used its loop transfer function G(s) and the dN(s) of the external force. This indicial response is an ideal one without a time lag for the outlet response. The cell death rate of DDMC-PTX is more dependent on the Hill coefficient n than on the Michaelis constant Km. This means that this supermolecular reaction with tubulin follows an "induced fit model".

In vivo blockade of the programmed cell death-1 pathway using soluble recombinant PD-1-Fc enhances CD4+ and CD8+ T cell responses but has limited clinical benefit.

The programmed cell death-1 (PD-1)/programmed cell death ligand-1 pathway has been shown to limit cell-mediated effector functions during chronic viral infections impeding clearance of pathogens. As a strategy to reverse this exhaustion and increase T cell polyfunctionality, PD-1 ligands were blocked in vivo using a recombinant macaque PD-1 fused to a macaque Ig-Fc (rPD-1-Fc) in SIVmac239-infected rhesus macaques during the early chronic phase of infection, either alone or in combination with antiretroviral therapy. In vitro blockade showed improvement of Ag-specific CD4(+) and CD8(+) T cells from monkeys chronically infected with SIV. Of note, a prolonged 5-d blockade in culture was beneficial for both gag-specific CD4(+) and CD8(+) T cells based on proliferation and dual cytokine production. Although the in vivo administration of rPD-1-Fc induced enhanced SIV-specific CD4 and CD8 T cell proliferation both in the blood and gut, it failed to alter plasma viremia. However, rPD-1-Fc administration in the context of antiretroviral therapy interruption induced a significant delay of viral load rebound. In addition, rPD-1-Fc administration in MamuA*001(+) monkeys led to both an increase in the frequencies and Ki67 expression of GagCM9(+) CD8(+) T cells in the blood and rectal mucosa and polyfunctionality of GagCM9(+) CD8(+) T cells in blood. In conclusion, however, our data suggest that PD-1/programmed cell death ligand-1 blockade using soluble rPD-1-Fc instead of anti-PD-1 mAb, although effective in rescuing the effector function of SIV-specific CD4(+) and CD8(+) T cells during the early chronic phase of infection, has limited clinical benefit.

Complete conformational space of the potential HIV-1 reverse transcriptase inhibitors d4U and d4C. A quantum chemical study.

A comprehensive quantum-chemical conformational analysis of two nucleoside analogues, 2',3'-didehydro-2',3'-dideoxyuridine (d4U) and 2',3'-didehydro-2',3'-dideoxycytidine (d4C), is reported. The electronic structure calculations were performed at the MP2/6-311++G(d,p)//B3LYP/6-31++G(d,p) level of theory. It was found that d4U and d4C adopt 20 conformers and 19 conformers, respectively, which correspond to local minima on the respective potential energy landscapes. QTAIM and NBO analyses show that the d4U and d4C conformers are stabilised by a complicated network of specific intramolecular interactions, which includes conventional (OHO) and non-conventional (CHO, CHHC) H-bonds as well as closed-shell van der Waals (CO) contacts. A satisfactory linear correlation was found between Grunenberg's compliance constants for closed-shell intramolecular interactions and their energy. It is shown that there are no conformational obstacles for incorporation of d4U and d4C into the double helical A and B forms of DNA. The less pronounced biological activity of d4U as compared to 2',3'-didehydro-2',3'-dideoxythymidine (d4T) is most likely due to the presence of the bulky methyl group at the 5-position of d4T, which can be recognised by target enzymes.

Strong antiviral activity of the new l-hydroxycytidine derivative, l-Hyd4FC, in HBV-infected human chimeric uPA/SCID mice.

BACKGROUND: Suppression of viral replication with nucleoside/nucleotide inhibitors has been shown to greatly improve the outcome of chronic HBV infection. ß-l-nucleoside analogues, especially ß-l-deoxycytidine derivatives represent one of the most efficient groups of antiretroviral compounds. We recently described that hydroxylation of the amino group of these ß-l-deoxycytidine derivatives preserved their strong HBV inhibitory activity in vitro, but strongly reduced their cytotoxicity. From this new group of compounds we selected ß-l-2',3'-didehydro-2',3'-dideoxy-N(4)-hydroxy-5-fluorocytidine (l-Hyd4FC) for a first in vivo investigation. The aim of this study was to determine the antiviral activity of l-Hyd4FC in HBV-infected human liver chimeric urokinase plasminogen activator (uPA)/SCID mice. METHODS: Stably infected animals (median 6×10(7) HBV DNA/ml) were injected daily with either l-Hyd4FC (50 mg/kg) or saline as controls. Mice treated with lamivudine served to compare the in vivo antiviral potency of l-Hyd4FC. Virological changes were determined by quantitative PCR. RESULTS: Treatment with l-Hyd4FC for 4 weeks induced a 2-log reduction of viraemia, while a median 1.5-log decline was achieved with lamivudine. Intrahepatically, l-Hyd4FC induced a median eightfold decline of viral activity (relaxed circular DNA/covalently closed circular DNA), and threefold reduction of pregenomic RNA/GAPDH levels. No significant decline of subgenomic HBV transcripts, as well as of circulating hepatitis B e antigen and hepatitis B surface antigen was detected. Maintenance of human serum albumin concentrations throughout the study, negative TUNEL staining and occurrence of viral rebound after drug withdrawal indicated that l-Hyd4FC was not toxic in human hepatocytes. CONCLUSIONS: Administration of l-Hyd4FC in uPA/SCID mice harbouring HBV-infected human hepatocytes demonstrated the high antiviral potency of this drug in vivo. Such characteristics make l-Hyd4FC a good candidate for further investigations a as potential HBV therapeutic agent.

The base component of 3'-azido-2',3'-dideoxynucleosides influences resistance mutations selected in HIV-1 reverse transcriptase.

We recently reported that HIV-1 resistant to 3'-azido-3'-deoxythymidine (AZT) is not cross-resistant to 3'-azido-2',3'-dideoxypurines. This finding suggested that the nucleoside base is a major determinant of HIV-1 resistance to nucleoside analogs. To further explore this hypothesis, we conducted in vitro selection experiments by serial passage of HIV-1(LAI) in MT-2 cells in increasing concentrations of 3'-azido-2',3'-dideoxyguanosine (3'-azido-ddG), 3'-azido-2',3'-dideoxycytidine (3'-azido-ddC), or 3'-azido-2',3'-dideoxyadenosine (3'-azido-ddA). 3'-Azido-ddG selected for virus that was 5.3-fold resistant to 3'-azido-ddG compared to wild-type HIV-1(LAI) passaged in the absence of drug. Population sequencing of the entire reverse transcriptase (RT) gene identified L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain. However, when introduced into HIV-1 by site-directed mutagenesis, these 5 mutations only conferred ∼2.0-fold resistance. Single-genome sequencing analyses of the selected virus revealed a complex population of mutants that all contained L74V and L214F linked to other mutations, including ones not identified during population sequencing. Recombinant HIV-1 clones containing RT derived from single sequences exhibited 3.2- to 4.0-fold 3'-azido-ddG resistance. In contrast to 3'-azido-ddG, 3'-azido-ddC selected for the V75I mutation in HIV-1 RT that conferred 5.9-fold resistance, compared to the wild-type virus. Interestingly, we were unable to select HIV-1 that was resistant to 3'-azido-ddA, even at concentrations of 3'-azido-ddA that yielded high intracellular levels of 3'-azido-ddA-5'-triphosphate. Taken together, these findings show that the nucleoside base is a major determinant of HIV-1 resistance mechanisms that can be exploited in the design of novel nucleoside RT inhibitors.

Transgenic cardiac-targeted overexpression of human thymidylate kinase.

Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes phosphorylation of thymidine monophosphate to thymidine diphosphate. TMPK also mediates phosphorylation of monophosphates of thymidine nucleoside analog (NA) prodrugs on the pathway to their active triphosphate antiviral or antitumor moieties. Novel transgenic mice (TG) expressing human (h) TMPK were genetically engineered using the alpha-myosin heavy chain promoter to drive its cardiac-targeted overexpression. In '2 by 2' protocols, TMPK TGs and wild-type (WT) littermates were treated with the NA zidovudine (a deoxythymidine analog, 3'-azido-3'deoxythymidine (AZT)) or vehicle for 35 days. Alternatively, TGs and WTs were treated with a deoxycytidine NA (racivir, RCV) or vehicle. Changes in mitochondrial DNA (mtDNA) abundance and mitochondrial ultrastructure were defined quantitatively by real-time PCR and transmission electron microscopy, respectively. Cardiac performance was determined echocardiographically. Results showed TMPK TGs treated with either AZT or RCV exhibited decreased cardiac mtDNA abundance. Cardiac ultrastructural changes were seen only with AZT. AZT-treated TGs exhibited increased left ventricle (LV) mass. In contrast, LV mass in RCV-treated TGs and WTs remained unchanged. In all cohorts, LV end-diastolic dimension remained unchanged. This novel cardiac-targeted overexpression of hTMPK helps define the role of TMPK in mitochondrial toxicity of antiretrovirals.

Cellular pharmacology of the anti-hepatitis B virus agent beta-L-2',3'-didehydro-2',3'-dideoxy-N4-hydroxycytidine: relevance for activation in HepG2 cells.

Beta-l-2',3'-didehydro-2',3'-dideoxy-N(4)-hydroxycytidine (l-Hyd4C) was demonstrated to be an effective and highly selective inhibitor of hepatitis B virus (HBV) replication in HepG2.2.15 cells (50% effective dose [ED(50)] = 0.03 microM; 50% cytotoxic dose [CD(50)] = 2,500 microM). In the present study, we investigated the intracellular pharmacology of tritiated l-Hyd4C in HepG2 cells. l-[(3)H]Hyd4C was shown to be phosphorylated extensively and rapidly to the 5'-mono-, 5'-di-, and 5'-triphosphate derivatives. Other metabolites deriving from a reduction or removal of the NHOH group of l-Hyd4C could not be detected, although both reactions were described as the primary catabolic pathways of the stereoisomer ss-d-N(4)-hydroxycytidine in HepG2 cells. Also, the formation of liponucleotide metabolites, such as the 5'-diphosphocholine derivative of l-Hyd4C, as described for some l-deoxycytidine analogues, seems to be unlikely. After incubation of HepG2 cells with 10 microM l-[(3)H]Hyd4C for 24 h, the 5'-triphosphate accumulated to 19.4 +/- 2.7 pmol/10(6) cells. The predominant peak belonged to 5-diphosphate, with 43.5 +/- 4.3 pmol/10(6) cells. The intracellular half-life of the 5'-triphosphate was estimated to be 29.7 h. This extended half-life probably reflects a generally low affinity of 5'-phosphorylated l-deoxycytidine derivatives for phosphate-degrading enzymes but may additionally be caused by an efficient rephosphorylation of the 5'-diphosphate during a drug-free incubation. The high 5'-triphosphate level and its extended half-life in HepG2 cells are consistent with the potent antiviral activity of l-Hyd4C.

Reacciones cutáneas adversas a fármacos en los pacientes con infección por el VIH en la era TARGA / Cutaneous Drug Reactions in HIV-Infected Patients in the HAART Era

La introducción del tratamiento antirretroviral de gran actividad (TARGA) en 1996 supuso un cambio radical en la historia natural de la infección por el virus de la inmunodeficiencia humana (VIH) al lograr reducir drásticamente la mortalidad en estos pacientes. No obstante, estos tratamientos no están exentos de limitaciones que incluyen efectos adversos, fracaso del tratamiento, interacciones farmacocinéticas, aparición de resistencias y respuestas inmunes anómalas. En este artículo se revisa la situación actual de las reacciones cutáneas adversas a fármacos en los pacientes con infección por el VIH (AU)

The introduction of highly active antiretroviral treatment (HAART) in 1996 radically changed the clinical course of human immunodeficiency virus (HIV) infection as it led to a dramatic reduction in mortality in these patients. However, these treatments have their limitations, including adverse effects, therapeutic failure, pharmacokinetic interactions, the development of resistance, and abnormal immune responses. In this article we review the current situation of cutaneous drug reactions in HIV-infected patients (AU)

Multiple-dose pharmacokinetic behavior of elvucitabine, a nucleoside reverse transcriptase inhibitor, administered over 21 days with lopinavir-ritonavir in human immunodeficiency virus type 1-infected subjects.

The purpose of this study was to describe the plasma pharmacokinetics (PK) of elvucitabine at different doses when administered daily or every other day for 21 days with lopinavir-ritonavir (Kaletra) in human immunodeficiency virus (HIV)-infected subjects. Three different dosing regimens of elvucitabine were administered with lopinavir-ritonavir to 24 subjects with moderate levels of HIV. Plasma samples were collected over 35 days. Elvucitabine concentrations were analyzed using a validated liquid chromatography-tandem mass spectrometry assay. The PK of elvucitabine was determined using both noncompartmental and compartmental analyses. Models were developed and tested using ADAPT II, while a population analysis was performed using IT2S. The PK behavior of elvucitabine was best described by a two-compartment linear model using two absorption rates and an increase in the bioavailability after day 1. The augmentation in the bioavailability after day 1 was variable, with some subjects demonstrating a major increase while others had little or no increase. Elvucitabine has a long half-life of approximately 100 h. The increase in elvucitabine bioavailability may be due to ritonavir inhibiting an efflux gut transporter with activity present in various levels between subjects. The proposed PK model may be utilized and improved further by linking the PK behavior of elvucitabine to various markers of efficacy.

Effect of a single dose of ritonavir on the pharmacokinetic behavior of elvucitabine, a nucleoside reverse transcriptase inhibitor, administered in healthy volunteers.

The purpose of this study was to determine the effect of a single dose of 300 mg of ritonavir on the plasma pharmacokinetics (PK) of a single dose of 20 mg of elvucitabine when the two drugs were coadministered in healthy subjects. In a three-way crossover design, 30 subjects received 20 mg of elvucitabine, 300 mg of ritonavir, or 20 mg of elvucitabine coadministered with 300 mg of ritonavir. Elvucitabine concentrations were analyzed using a validated liquid chromatography-tandem mass spectrometry assay. The PK of elvucitabine was determined using both noncompartmental and compartmental analyses. Models were developed and tested using ADAPT-II, while a population analysis was performed using IT2S. Comparisons of PK parameters between groups were done with SAS. The pharmacokinetic behavior of elvucitabine was best described by a two-compartment linear model using two absorption rates and a first-order elimination rate. Ritonavir significantly impacted the PK of elvucitabine by reducing elvucitabine's bioavailability, with the most plausible explanation being an inhibition on influx transporters by ritonavir. The decrease in elvucitabine bioavailability when elvucitabine was coadministered with ritonavir may be due to ritonavir's inhibiting influx gut transporters. Continued development of elvucitabine is warranted to better characterize its PK and to determine its in vivo efficacy against human immunodeficiency virus.

Simian immunodeficiency virus infection induces severe loss of intestinal central memory T cells which impairs CD4+ T-cell restoration during antiretroviral therapy.

BACKGROUND: Simian immunodeficiency virus (SIV) infection leads to severe loss of intestinal CD4(+) T cells and, as compared to peripheral blood, restoration of these cells is slow during antiretroviral therapy (ART). Mechanisms for this delay have not been examined in context of which specific CD4(+) memory subsets or lost and fail to regenerate during ART. METHODS: Fifteen rhesus macaques were infected with SIV, five of which received ART (FTC/PMPA) for 30 weeks. Viral loads were measured by real-time PCR. Flow cytometric analysis determined changes in T-cell subsets and their proliferative state. RESULTS: Changes in proliferative CD4(+) memory subsets during infection accelerated their depletion. This reduced the central memory CD4(+) T-cell pool and contributed to slow CD4(+) T-cell restoration during ART. CONCLUSION: There was a lack of restoration of the CD4(+) central memory and effector memory T-cell subsets in gut-associated lymphoid tissue during ART, which may contribute to the altered intestinal T-cell homeostasis in SIV infection.

Antiviral and cellular metabolism interactions between Dexelvucitabine and lamivudine.

Studies on cellular drug interactions with antiretroviral agents prior to clinical trials are critical to detect possible drug interactions. Herein, we demonstrated that two 2'-deoxycytidine antiretroviral agents, dexelvucitabine (known as beta-d-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine, DFC, d-d4FC, or RVT) and lamivudine (3TC), combined in primary human peripheral blood mononuclear (PBM) cells infected with human immunodeficiency virus 1 strain LAI (HIV-1(LAI)), resulted in additive-to-synergistic effects. The cellular metabolism of DFC and 3TC was studied in human T-cell lymphoma (CEM) and in primary human PBM cells to determine whether this combination caused any reduction in active nucleoside triphosphate (NTP) levels, which could decrease with their antiviral potency. Competition studies were conducted by coincubation of either radiolabeled DFC with different concentrations of 3TC or radiolabeled 3TC with different concentrations of DFC. Coincubation of radiolabeled 3TC with DFC at concentrations up to 33.3 microM did not cause any marked reduction in 3TC-triphosphate (TP) or any 3TC metabolites. However, a reduction in the level of DFC metabolites was noted at high concentrations of 3TC with radiolabeled DFC. DFC-TP levels in CEM and primary human PBM cells decreased by 88% and 94%, respectively, when high concentrations of 3TC (33.3 and 100 microM) were added, which may influence the effectiveness of DFC-5'-TP on the HIV-1 polymerase. The NTP levels remained well above the median (50%) inhibitory concentration for HIV-1 reverse transcriptase. These results suggest that both beta-d- and beta-l-2'-deoxycytidine analogs, DFC and 3TC, respectively, substrates of 2'-deoxycytidine kinase, could be used in a combined therapeutic modality. However, it may be necessary to decrease the dose of 3TC for this combination to prove effective.

The triphosphate of beta-D-4'-C-ethynyl-2',3'-dideoxycytidine is the preferred enantiomer substrate for HIV reverse transcriptase.

The enantioselective synthesis of the beta-d (1) enantiomer of 4'-C-ethynyl-2',3'-dideoxycytidine confirms an earlier stereochemical assignment that was strictly based on the ability of HIV reverse transcriptase and its M184V mutant to discriminate between the d- and l-configuration of nucleoside 5'-triphosphates.

Meeting notes from the 3rd IAS Conference. New drugs.

CCR5 antagonists created quite a buzz at IAS: several are entering phase III trials and could be available in the next 2 to 3 years.

In vitro selection and analysis of human immunodeficiency virus type 1 resistant to derivatives of beta-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine.

Serial passage of human immunodeficiency virus type 1 in MT-2 cells in increasing concentrations of the d- and l-enantiomers of beta-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (d4FC) resulted in the selection of viral variants with reverse transcriptase substitutions M184I or M184V for l-d4FC and I63L, K65R, K70N, K70E, or R172K for d-d4FC. Phenotypic analysis of site-directed mutants defined the role of these mutations in reducing susceptibility to l- or d-d4FC.