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D J Back

Publications and source records attributed to D J Back.

At least 37 records · Page 2Linked to original sources
PubMed2001

Antiretroviral drug concentrations in semen of HIV-infected men: differential penetration of indinavir, ritonavir and saquinavir.

Variable drug penetration of antiretroviral drugs into the genital tract may contribute to the differential evolution of HIV and the emergence of drug resistance. This, in turn, may have an impact on the sexual transmission of resistant HIV in patients treated with antiretroviral drugs. We have measured concentrations of the HIV-1 protease inhibitors indinavir, ritonavir and saquinavir in the blood plasma (BP) and seminal plasma (SP) of 23 HIV-1-positive men. Forty-five time-matched blood and semen samples were obtained. SP concentrations of indinavir exceeded the EC95 of indinavir, corrected for protein binding, of 42 ng/mL at all time intervals. In contrast, the median ritonavir and saquinavir SP concentrations were below the relevant EC95 at all times post drug ingestion. The median SP:BP concentration ratios for indinavir were 0.6, 0.8 and 1.4, respectively, at 0-2, 2-6 and 6-8 h post-drug ingestion. In contrast, the median SP:BP concentration ratios at 0-3, 3-9 and 9-12 h post-drug ingestion were <0.02, <0.04 and <0.04, respectively, for both ritonavir and saquinavir. These differences justify further study of HIV-1 evolution and development of resistance in the genital tract of men taking these anti-HIV drugs.

Adult↗
PubMed2001

Zidovudine phosphorylation determined sequentially over 12 months in human immunodeficiency virus-infected patients with or without previous exposure to antiretroviral agents.

We sought to determine whether the intracellular activation of zidovudine (ZDV) varied over time and with previous antiretroviral exposure in human immunodeficiency virus-infected individuals and to examine whether there is an association between virological responses and intracellular phosphorylation. A total of 23 patients (12 treatment naïve, 11 previously treated with ZDV) who commenced ZDV as part of dual nucleoside therapy were prospectively monitored for 12 months or until withdrawal from the study. No association was observed between virological responses at 2 weeks and 3 months and ZDV phosphorylation. The mean intracellular concentrations of ZDV mono-, di-, and triphosphates did not change significantly over time or with previous ZDV exposure. The rate of formation of total ZDV phosphates was increased in patients with CD4 counts <100 cells/mm(3). Previous reports from in vitro cell culture experiments or cross-sectional cohort studies suggesting alterations of ZDV phosphorylation over time are not confirmed by this longitudinal study.

Adult↗
PubMed2001

Influence of prior exposure to zidovudine on stavudine phosphorylation in vivo and ex vivo.

Intracellular phosphorylation of stavudine (d4T) and zidovudine (ZDV) was investigated in peripheral blood mononuclear cells (PBMCs) isolated from ZDV-naive and ZDV-experienced human immunodeficiency virus (HIV)-positive patients. An in vivo study measured the amount of d4T triphosphate (d4TTP), while an ex vivo study assessed the capacity of cells to phosphorylate added d4T. Endogenous dTTP was also measured. d4TTP and dTTP were determined in vivo using a reverse transcriptase chain termination assay. In ex vivo studies, d4T (1 microM) was incubated in resting and phytohemagglutinin-stimulated (10 microg ml(-1); 72 h) PBMCs for 24 h. After washing and methanol extraction, radiolabeled anabolites were detected by high-performance liquid chromatography. d4TTP reached its highest level 2 to 4 h after dosing (0.21 +/- 0.14 pmol/10(6) cells; n = 27 [mean +/- standard deviation]). Comparison of ZDV-naive and ZDV-experienced individuals showed no significant difference in levels of d4TTP (ZDV naive, 0.23 +/- 0.17 pmol/10(6) cells [n = 7] versus ZDV experienced, 0.20 +/- 0.14 pmol/10(6) cells [n = 20]; P = 0.473) or the d4TTP/dTTP ratio (0.14 +/- 0.12 [n = 7] and 0.10 +/- 0.08 [n = 20], respectively; p = 0.391). Ex vivo data demonstrated no significant difference in the formation of d4TTP or total d4T phosphates in naive and experienced patients (0.086 +/- 0.055 pmol/10(6) cells in ZDV-naive patients [n = 17] versus 0.081 +/- 0.038 pmol/10(6) cells in ZDV-experienced patients [n = 22]; P = 0.767). The ability of HIV-infected patients to phosphorylate d4T in vivo and ex vivo was unchanged with increasing exposure to ZDV.

Anti-HIV Agents↗
PubMed2001

Elevated alpha-1-acid glycoprotein reduces the volume of distribution and systemic clearance of saquinavir.

The purpose of this study was to characterize the relationship between plasma protein binding and the pharmacokinetic disposition of saquinavir during a normal and elevated alpha-1-acid glycoprotein condition. The extent of plasma binding of [14C]saquinavir to human plasma, human albumin, and human alpha-1-acid glycoprotein was also assessed. Transgenic mice, which overexpress plasma alpha-1-acid glycoprotein, and control mice were given a single intravenous injection of saquinavir (10 mg/kg) and plasma samples were harvested as a function of time. The extent of [14C]saquinavir (0.5-30 microg/ml) plasma protein binding in each group of mice was determined by ultrafiltration. Plasma saquinavir concentrations from in vivo administration were determined by high performance liquid chromatography with tandem mass spectrometry. Saquinavir binding in human plasma and control mouse plasma was similar (approximately 3% unbound). In contrast, the extent of binding was significantly increased in transgenic mice (1.5% unbound). Furthermore, saquinavir was more extensively bound to alpha-1-acid glycoprotein than to albumin (2.1 versus 11.5% unbound). The systemic clearance and volume of distribution of saquinavir were significantly reduced in transgenic mice compared with control mice. The results of this study show that alpha-1-acid glycoprotein is the predominant plasma protein to which saquinavir binds. In addition, elevations in plasma alpha-1-acid glycoprotein considerably alter the pharmacokinetic disposition of saquinavir. This is consistent with the observations that systemic exposure to saquinavir in human immunodeficiency virus patients is greater than that in healthy volunteers and that alpha-1-acid glycoprotein levels increase with the degree of HIV infection.

Animals↗
PubMed2000

The intracellular activation of lamivudine (3TC) and determination of 2'-deoxycytidine-5'-triphosphate (dCTP) pools in the presence and absence of various drugs in HepG2 cells.

AIMS: Lamivudine (3TC, 2'-deoxy-3'-thiacytidine) requires intracellular metabolism to its active 5'-triphosphate, 3TC-5'-triphosphate (3TCTP), to inhibit the replication of hepatitis B virus (HBV). We have investigated the activation of 3TC, in the presence and absence of a range of compounds, in HepG2 cells. The intracellular levels of the endogenous competitor of 3TCTP, 2'-deoxycytidine-5'-triphosphate (dCTP), were also determined and 3TCTP/dCTP ratios calculated. METHODS: The effects of a number of compounds on 3TC (3H; 1 microM) phosphorylation were investigated by radiometric h.p.l.c. dCTP levels were determined using a template primer extension assay. 3TCTP/dCTP ratios were calculated from these results. RESULTS: The phosphorylation of 3TC was significantly increased in the presence of either hydroxyurea (HU), methotrexate (MTX), or fludarabine (FLU). For example, at 100 microM HU, control 3TCTP levels were increased to 361% of control, whereas at 100 microM FLU, control 3TCTP levels were increased to 155%. dCTP pools were significantly reduced in the presence of HU and FLU, at 100 microM concentrations only. However, for all the above three compounds investigated, the ratio of 3TCTP/dCTP was favourably enhanced (e.g. at 1 microM MTX, 255% of control). Neither ganciclovir (GCV), lobucavir (LCV), penciclovir (PCV), adefovir dipivoxil (ADV), nor foscarnet (FOS) had any significant effects on 3TC phosphorylation or dCTP pools. CONCLUSIONS: These results suggest that the activity of 3TC may be potentiated when combined with one of the modulators studied. The lack of an interaction between 3TC and the other anti-HBV agents is reassuring. These in vitro studies can be used as an initial screen to examine potential interactions at the phosphorylation level.

Anti-HIV Agents↗
PubMed2000

Therapeutic drug monitoring of antiretrovirals in human immunodeficiency virus infection.

The era of antiviral therapy directed against HIV-1 has now entered its second decade. In the twelve years since the FDA approved the first antiretroviral drug zidovudine there have been a number of seminal developments that have revolutionized the approach to therapy. These advances converged to change the treatment paradigm from one of therapeutic nihilism to that of cautious optimism. First, several trials demonstrated that combination therapy of nucleoside reverse transcriptase inhibitors (NRTIs) is superior to monotherapy in extending survival and delaying disease progression. Second, the concept of virologic latency in asymptomatic HIV-infected patients was revised. Mathematic modelling demonstrated that there is an ongoing high level of virus production driving a rapid turnover of CD4 cells at all stages of infection. Hence it was concluded that the aim of antiretroviral therapy (ART) should be to "hit early and hit hard." Third, significant advances in molecular virology facilitated the development of quantitative methods to measure the circulating HIV plasma RNA. HIV viral load has been shown to be a sensitive predictor of disease progression and a valuable marker of response to therapy. However, none of these developments would have translated into improved patient care without the advent of two new classes of drugs-the protease inhibitors (PIs) and the nonnucleoside reverse transcriptase inhibitors (NNRTIs).

Anti-HIV Agents↗
PubMed2000

Pharmacokinetics of zidovudine phosphorylation in human immunodeficiency virus-positive thai patients and healthy volunteers.

We assessed the pharmacokinetics of zidovudine (ZDV) in plasma and intracellular ZDV phosphate anabolites in peripheral blood mononuclear cells in Thai human immunodeficiency virus (HIV) type 1-infected patients and healthy volunteers. The plasma ZDV area under the concentration-time curve from 0 to 6 h (AUC(0-6)) was similar in patients and healthy volunteers (32.77 and 22.77 micromol/liter. h, respectively; confidence interval, -3.37 to 19. 92). Although the concentration of ZDV triphosphate (ZDVTP) was similar in the two groups, the ZDV monophosphate (ZDVMP) AUC(0-6) was significantly greater in HIV patients (1.12 pmol/10(6) cells) than in healthy volunteers (0.15 pmol/10(6) cells). In agreement with previously published data obtained with Caucasians, the significant difference in intracellular phosphorylation in Thai volunteers and HIV patients is primarily due to ZDVMP. Comparing the data from this study with the data obtained with Caucasians suggests no marked ethnic differences in ZDV phosphorylation.

Adult↗
PubMed2000

Correlation between intracellular pharmacological activation of nucleoside analogues and HIV suppression in vitro.

Following intracellular activation of HIV nucleoside analogue reverse transcriptase inhibitors, their triphosphates (ddNTPs) compete with endogenous nucleoside triphosphates (dNTPs) for incorporation into proviral DNA. In this study we have examined the effect of combinations of two thymidine analogues, stavudine (d4T) and zidovudine (ZDV), and two cytidine analogues, lamivudine (3TC) and zalcitabine (ddC) on intracellular drug activation and on the relevant competing dNTP in uninfected and persistently HIV-infected cells. Endogenous triphosphates of deoxycytidine (dCTP) and deoxythymidine (dTTP) were measured using a template primer assay and the ratio of ddNTP:dNTP was calculated. Antiviral activity of two-drug combinations was also assayed by p24 ELISA. A significant reduction in d4T triphosphate (d4TTP) [0.11+/-0.09 pmol/10(6) cells to undetectable (<0.01); P=0.039] in the presence of equimolar concentrations of ZDV and d4T, resulted in a decrease in the d4TTP/dTTP ratio of 90%. ZDVTP/dTTP was not significantly altered in the presence of d4T. 3TC (10 microM) reduced total ddC phosphates by 57% and ddCTP/dCTP by 27%. 3TC phosphorylation was comparatively unaffected by ddC, up to a concentration of 10 microM ddC (>100 times the plasma concentration achieved following standard dosing). 3TC plus ddC resulted in greater p24 inhibition than 3TC or ddC alone (P<0.001). Combining one thymidine analogue (ZDV or d4T) with one cytidine analogue (3TC or ddC) resulted in greater inhibition of p24 inhibition than with any single agent. From a pharmacological viewpoint, the combination of ZDV plus d4T should be avoided, but in vitro the combination of 3TC plus ddC confers modest benefit over either drug alone. This in vitro study illustrates that decreases in ddNTP/dNTP are consistent with a reduction in antiviral effect.

Anti-HIV Agents↗
PubMed1999

Interaction of sildenafil and indinavir when co-administered to HIV-positive patients.

OBJECTIVES: The prevalence of erectile dysfunction in HIV-infected men is estimated to be 33%. Sildenafil citrate (Viagra; Pfizer Ltd, Sandwich, Kent, UK) is the first oral drug for this condition. Since sildenafil and the protease inhibitors are both metabolized by, and act as inhibitors of cytochrome P450 3A4, we evaluated the pharmacokinetics of the combination sildenafil plus indinavir in HIV-infected patients. DESIGN AND METHODS: Six patients at steady state in treatment with indinavir participated in the study. On the first day blood samples for indinavir assay were drawn at times 0, 1, 2, 3, 4, 6 and 8 h after dosing. On the second study day patients received a single dose of 25 mg of sildenafil in addition to their routine morning medication. Blood samples were taken as described. Separated plasma was stored at -80 degrees C until analysis by high performance liquid chromatography. In a parallel study, the effect of indinavir, ritonavir, saquinavir and nelfinavir on the in vitro hepatic metabolism of sildenafil was assessed. RESULTS: The geometric mean area under the concentration curve for 0-8 h (AUC0-8h) and maximum plasma concentration (Cmax) for indinavir were 19.69 microg/ml h (range, 9.19-31.99 microg/ml h) and 7.02 microg/ml (range, 2.33-16.17 microg/ml), respectively, on the first study day. In the presence of sildenafil, the mean AUC0-8h and Cmax of indinavir were 22.37 microg/ml h [range, 10.08-37.25 microg/ml h; 95% confidence interval (CI) for difference between means, -15 to 13.25) and 9.11 microg/ml (range, 3.41-22.78 microg/ml; 95% CI, -13 to 6.37), respectively. The geometric mean AUC0-8h and Cmax for sildenafil were 1631 ng/ml h (range, 643-2970 ng/ml h) and 384 ng/ml (range, 209-766 ng/ml) respectively. The AUC for sildenafil was 4.4 times higher than data from historical controls given either 50 mg or 100 mg of sildenafil and dose normalized to 25 mg. Indinavir was a potent inhibitor of sildenafil hepatic metabolism in vitro [concentration producing 50% inhibition of control enzyme activity (IC50) = 0.39 +/- 0.17 microM, mean +/- SD]. CONCLUSIONS: Co-administration of sildenafil 25 mg did not significantly alter the plasma indinavir levels. However, plasma sildenafil AUC was markedly increased in the presence of indinavir compared with historical controls. From the in vitro data, the mechanism of increase is indinavir inhibition of the hepatic metabolism of sildenafil. The magnitude of this interaction suggests a lower starting dose of sildenafil may be more appropriate in this clinical setting.

Adult↗
PubMed1999

Modulation of P-glycoprotein function in human lymphocytes and Caco-2 cell monolayers by HIV-1 protease inhibitors.

OBJECTIVES: To determine the effect of the protease inhibitors ritonavir, nelfinavir and indinavir on the P-glycoprotein (P-gp)-mediated transport of saquinavir in Caco-2 cell monolayers. To study the modulation of P-gp function in human lymphocytes by saquinavir, ritonavir, nelfinavir and indinavir. METHODS: We examined the effect of the protease inhibitors on P-gp function in human lymphocytes by using Rhodamine 123 (Rh 123; a fluorescent substrate of P-gp) by flow cytometry. Efflux of Rh 123 correlates with P-gp function and inhibition of P-gp results in dye retention. Verapamil, a P-gp modulator and inhibitor of active transport at 4 degrees C was used as a positive control. The transport of [14C]saquinavir (1 microM) across Caco-2 cell monolayers was investigated, alone and in the presence of verapamil and ketoconazole (500 microM) and the protease inhibitors at 100 microM. Caco-2 cells are an in vitro model of the intestinal epithelium that is widely used for the study of P-gp function. The transport of saquinavir was determined in both the apical to basolateral (AP-BL) and basolateral to apical (BL-AP) directions. RESULTS: Saquinavir and ritonavir (10 microM) markedly inhibited Rh 123 efflux with an increase in fluorescence intensity similar to that obtained with verapamil. A small but statistically significant increase in fluorescence intensity was observed with nelfinavir; however indinavir did not modulate Rh 123 efflux. In Caco-2 cells the apparent permeability coefficient for BL-AP efflux of saquinavir exceeded that for AP-BL efflux by a factor of 26: this is indicative of an active efflux pump. Known P-gp modulators caused a decrease in BL-AP efflux and an increase in AP-BL transport. The protease inhibitors displayed some P-gp modulation with ritonavir having the most potent effect. CONCLUSIONS: We have demonstrated that saquinavir is a substrate for P-gp and that ritonavir, nelfinavir and indinavir modulate P-gp function in both human lymphocytes and Caco-2 cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

The rabbit study: ritonavir and saquinavir in combination in saquinavir-experienced and previously untreated patients.

Thirteen protease inhibitor-naive patients with HIV-1 infection, and 12 patients with a median of 58 months prior treatment with saquinavir (SQV) monotherapy, were treated with SQV (400 mg twice daily) and ritonavir (RIT, 500 mg twice daily) in a study designed to assess the effect of prior treatment with SQV monotherapy on the antiretroviral activity of RIT-SQV combination therapy. Median baseline viral load and CD4+ cell counts were 155,000 and 262,000 copies/ml and 333 and 225 cells/mm3 in the naive and experienced groups, respectively. Mean viral load changes at 24 weeks were -1.63 and -0.27 log copies/ml in the naive and SQV-experienced groups, respectively (intent-to-treat analysis). Baseline genotype by point mutation assay and sequencing in the SQV-experienced group was highly predictive of virological response. Eight of 11 SQV-experienced patients had evidence of phenotypic resistance to RIT at baseline, despite previous treatment with SQV only. There was strong correlation between phenotypic resistance to RIT and the presence of the L90M mutation. We conclude that prolonged prior treatment with saquinavir monotherapy may produce cross-resistance to ritonavir and reduce the subsequent response to ritonavir-saquinavir in combination. In this study, both phenotypic resistance to ritonavir and presence of the L90M mutation predicted the viral load response to ritonavir-saquinavir.

Adult↗
PubMed1999

Intracellular activation of 2',3'-dideoxyinosine and drug interactions in vitro.

Didanosine (2',3'-dideoxyinosine; ddI) requires intracellular metabolism to its active triphosphate, 2',3'-dideoxyadenosine 5'-triphosphate (ddATP), to inhibit the replication of human immunodeficiency virus (HIV). We have investigated the metabolism of ddI to ddATP in the presence and absence of a range of compounds. In addition, we determined the levels of the endogenous competitor of ddATP, 2'-deoxyadenosine 5'-triphosphate (dATP), and calculated ddATP/dATP ratios. None of the nucleoside analogs studied had any effect on ddI phosphorylation at 1 and 10 microM concentrations. At 100 microM concentrations, ddC reduced total ddA phosphates (82% of control total ddA phosphates; p < 0.001). ZDV significantly decreased the levels of dATP, whereas ddC significantly increased dATP pools (e.g., at 100 microM ZDV, 82% of control dATP levels; p < 0.001). Hence, the ddATP/dATP ratio was increased in the presence of ZDV, but was decreased in the presence of ddC. Neither d4T nor 3TC affected the ddATP/dATP ratio. Deoxyinosine (dI) significantly reduced ddA phosphate production at 100 microM concentrations, with ddATP reduced to undetectable levels (p < 0.001). Hydroxyurea (HU) did not affect the activation of ddI, but significantly reduced dATP pools at 100 microM concentrations (67% of control dATP levels; p < 0.001), enhancing the ddATP/dATP ratio. ddA phosphate production was significantly reduced by pentoxyfylline (PXF) at 10 and 100 microM concentrations. dATP levels were unaffected, but the ddATP/dATP ratio was reduced. Finally, 8-aminoguanosine (8-AMG) had no effect on either ddI activation or dATP pools. These studies demonstrate the importance of determining both the active TP and the competing endogenous TP, as changes to the resulting ratio could alter the efficacy of the nucleoside analog in question.

Anti-HIV Agents↗
PubMed1999

Pharmacological issues relating to viral resistance.

Anti-HIV drug regimens can fail for a number of reasons including virological resistance, difficulties of adherence and poor tolerability. However, this brief review focuses on the development of "pharmacological" resistance as an area of great importance in drug failure. For nucleoside reverse transcriptase inhibitors (NRTIs) this is related to the possible down-regulation of the intracellular phosphorylation of an NRTI with time. For protease inhibitors the concern is cells expressing transmembrane energy-dependent transporters (such as p-glycoprotein, p-gp; or multi-drug resistance protein, MRP) which efflux drug (particularly protease inhibitors) out of the cell so that intracellular concentrations of drug are insufficient for antiviral effect.

Anti-HIV Agents↗
PubMed1999

Inhibition of the CYP3A4-mediated metabolism and P-glycoprotein-mediated transport of the HIV-1 protease inhibitor saquinavir by grapefruit juice components.

AIMS: Cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) are both expressed in the intestinal mucosa and present a barrier to oral drug delivery. CYP3A4 and P-gp share both overlapping tissue distribution and substrate specificity. Grapefruit juice interactions with CYP3A4 substrates are well documented and occur as a consequence of down regulation of intestinal CYP3A4. The aim of the present study was to screen grapefruit juice components against the CYP3A4-mediated metabolism and P-gp mediated transport of the HIV-1 protease inhibitor saquinavir. METHODS: Five grapefruit juice components: quercetin, naringin, naringenin, 6', 7'-dihydroxybergamottin and bergamottin were screened as potential inhibitors of the metabolism of saquinavir by human liver microsomes. The known CYP3A4 inhibitor ketoconazole was also screened for inhibitory potential. These compounds were also screened as modulators of P-gp activity by assessing the directional transport of saquinavir across Caco-2 cell monolayers which express P-gp. The effect of verapamil, a known modulator of P-gp function, was also determined in these cell lines. RESULTS: On preincubation, 6', 7'-dihydroxybergamottin and bergamottin inhibited the metabolism of saquinavir, with IC50 values of 0.33+/-0.23 muM and 0.74+/-0.13 muM, respectively (n=3). Ketoconazole achieved an IC50 of 0. 55+/-0.12 muM (n=4). The other compounds studied failed to reach IC50 at concentrations of up to 100 muM. The transport of saquinavir in the basolateral-->apical (BL-->AP) direction exceeded that in the apical -->basolateral direction (AP-->BL), with apparent permeability coefficients of 199.2+/-15.8x10-7 cm s-1 and 8.00+/-1. 13x10-7 cm s-1, respectively (n=3) which is indicative of a polarized efflux mechanism. The ratio of BL-->AP/AP-->BL for saquinavir was 25, but in the presence of verapamil and ketoconazole this ratio was reduced to 3.6 and 4.0, respectively (n=3), indicating extensive inhibition of P-gp mediated saquinavir efflux. Of the grapefruit juice components studied only naringin and 6', 7'-dihydroxybergamottin had any appreciable effect, reducing the ratio to 7.6 and 7.1, respectively (n=3); but this was due solely to increased AP-->BL transport. CONCLUSIONS: Grapefruit juice components inhibit CYP3A4-mediated saquinavir metabolism and also modulate, to a limited extent, P-gp mediated saquinavir transport in Caco-2 cell monolayers. The in vivo effects of grapefruit juice coadministration are most likely the result of effects on CYP3A4 (inhibition and down regulation) and only to a minor extent on modulation of P-gp function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗