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D J Back

Publications and source records attributed to D J Back.

At least 19 recordsLinked to original sources
PubMed2004

The intracellular pharmacology of antiretroviral protease inhibitors.

Therapeutic drug monitoring (TDM) of antiretroviral protease inhibitors (PIs) has been suggested to have the potential to both reduce toxicity and optimize individual therapy. However, the major target of PIs is within cells infected with HIV. Therefore clinical outcome ultimately must be related to intracellular drug concentrations since antiviral activity of PIs is highly correlated with intracellular concentrations in vitro. Intracellular pharmacokinetics provides information regarding drug disposition in a compartment where HIV replication occurs and combined with plasma data may be useful in understanding therapeutic failure in relation to cellular resistance. In order to improve therapeutic efficacy, it is therefore important that the intracellular pharmacokinetics of drugs, such as PIs, is studied in addition to plasma pharmacokinetics. Multidrug resistance transporters may result in a lower cellular concentration of drug via an efflux mechanism, thus contributing to sanctuary site formation. However, conclusive proof that transporters contribute to clinical drug resistance is still lacking, although recent studies have attempted to address this issue. In relation to host and cellular factors, this review considers several issues involved in influencing intracellular drug concentrations and discusses the intracellular levels of PIs recently published from cellular studies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Effect of protease inhibitor-containing regimens on lymphocyte multidrug resistance transporter expression.

BACKGROUND: Increased expression of multidrug resistance transporters, such as P-glycoprotein (P-gp), has been suggested as a potential mechanism for decreased protease inhibitor (PI) availability at certain intracellular sites and tissue compartments. OBJECTIVES: To investigate the effect of PIs on the surface lymphocyte expression of P-gp in vitro and in vivo. PATIENTS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy subjects (n = 15) and incubated (72 h) with 10 microM of each PI studied (saquinavir, ritonavir, nelfinavir, indinavir, amprenavir and lopinavir), or dimethyl sulphoxide (DMSO) control. PBMCs were also isolated from HIV-infected subjects (n = 50; viral load <50 copies/mL) on a PI- or a non-PI-containing combination antiretroviral regimen. P-gp expression was analysed by flow cytometry. RESULTS: No differences in surface P-gp expression on lymphocytes, CD4+ or CD8+ lymphocyte subsets were observed following incubation with 10 microM saquinavir, ritonavir, indinavir, amprenavir or lopinavir in vitro. Nelfinavir, however, increased P-gp expression. In vivo, no difference in P-gp expression on total lymphocytes was observed between patients receiving a PI-containing regimen [saquinavir n = 9, ritonavir n = 6, nelfinavir n = 7, indinavir n = 7 and lopinavir/ritonavir n = 13, and two nucleoside reverse transcriptase inhibitors (NRTIs)] and patients receiving a control regimen of three NRTIs alone (n = 8). CONCLUSION: This study suggests that, of the PIs, only nelfinavir increases P-gp expression in vitro, and in vivo the PI class of antiretrovirals do not increase P-gp expression on lymphocytes. It is clear that factors other than PI induction are important in the inter-individual variability in the lymphocyte expression of P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

A simplified approach to determining P-glycoprotein expression in peripheral blood mononuclear cell subsets.

P-glycoprotein (P-gp), encoded by the MDR-1 (multidrug resistance) gene mediates the cellular efflux of several therapeutic agents with the potential of treatment failure. The differential expression of P-gp in many localised tissues and cells of the hematopoietic system implies diverse physiological and pharmacological roles. The exact function of P-gp involved in multidrug resistance remains unclear owing to the numerous discrepancies between different laboratories. The ability to characterise accurately P-gp expression has important clinical implications. However, a complete consensus recommendation regarding methods of P-gp detection has been difficult to reach. With the advancement in immune technology and new commercially available antibodies, we describe a simplified direct immunofluorescent assay capable of detecting surface P-gp expression in peripheral blood mononuclear cells (PBMCs) and subpopulations of lymphocytes in vivo by dual colour flow cytometry. Results were expressed as mean increase in fluorescence (MI) compared to isotypically matched controls. Using this assay, differential basal P-gp expression was found to exist in the following significant hierarchy CD56+ (MI=0.684+/-0.273; n=15)>CD8+ (MI=0.312+/-0.117; n=15)>CD4+ (MI=0.194+/-0.086; n=15). This method is rapid and reproducible and has potential use for in vitro and in vivo application.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

A longitudinal study of maternal dose response to low molecular weight heparin in pregnancy.

OBJECTIVE: To assess the maternal response to low molecular weight heparin during pregnancy, by estimation of plasma anti-Xa activity, at three specified gestation points and in the nonpregnant state. METHODS: A longitudinal, prospective, observational study was set in a tertiary referral recurrent miscarriage clinic. Twenty-four women, attending consecutively, were invited to participate and gave informed consent. Each woman had a history of recurring pregnancy loss and positive preconception screening for antiphospholipid syndrome. After confirmation of a viable pregnancy all subjects began taking 5000 IU of dalteparin once daily subcutaneously. Serial measurement of plasma anti-Xa activity after administration of dalteparin was performed at three standard gestation points (12, 24, and 36 weeks) and in the nonpregnant state (6 weeks postpartum). RESULTS: Peak anti-Xa levels occurred at 4 hours postbolus in pregnancy, as compared with 2 hours in the nonpregnant state. The mean anti-Xa levels at 12, 24, and 36 weeks' gestation were significantly reduced, at 2 hours postinjection, as compared with the nonpregnant state (P <.001, P <.01, P <.001, respectively). The lowest dose-response curve was at 36 weeks' gestation. A repeated-measures analysis of variance found a significant difference (P <.05) between the 36-week group and the postterm group but not between any of the other groups. CONCLUSION: During pregnancy, differences in the pharmacokinetics of low molecular weight heparin were observed, with an overall reduction in anti-Xa activity. On the basis of this study it is questionable to extrapolate dosing and lack of dose monitoring, in pregnant women, using data derived from a nonpregnant population.

Abortion, Habitual↗
PubMed2002

Determination of P-gp and MRP1 expression and function in peripheral blood mononuclear cells in vivo.

P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) mediate the efflux of many therapeutic agents and have been implicated in the treatment failure of many infectious diseases and cancers. The ability to characterise the expression and function of these transporters in vivo is important when assessing the pharmacological activity of drugs. We investigated some of the problems involved in screening the multidrug resistance status of individuals using flow cytometry. Expression of P-gp and MRP1 on the surface of lymphocytes isolated from blood samples (30 ml) was determined by indirect immunofluorescence. Functional ability was assessed by measuring the efflux of specific fluorescent dyes. Results were expressed as a mean fold increase in fluorescence from the isotype control (expression) and a change in fluorescence compared to the load (function). Using these assays, we determined the expression of P-gp to be 2.01+/-0.40, n=30 and MRP1 to be 1.46+/-0.23, n=25. Functional ability was 6.98+/-4.97, n=25 for P-gp and 1.55+/-0.25, n=25 for MRP1. The dye efflux studies were associated with a lack of specificity and a number of methodological difficulties. There was no correlation between the expression and function of P-gp (r=0.338; p=0.10) or MRP1 (r=0.283; p=0.17). Therefore, we considered determination of P-gp and MRP1 expression to be a more reproducible and accurate approach to clinical investigation into the role of multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

CYP3A4-mediated hepatic metabolism of the HIV-1 protease inhibitor saquinavir in vitro.

1. The aim was to identify the major metabolites of saquinavir (SQV) from human hepatic microsomal incubations and the CYP isoform(s) responsible. 2. Ten fractions containing various metabolites were separated by isocratic reversed-phase HPLC and characterized by HPLC, mass spectrometry and NMR. 3. Metabolites were either mono- or di-hydroxylated derivatives of SQV. Fast-atom bombardment and electrospray MS showed that hydroxylation was predominantly situated on the decahydroisoquinoline ring. A major metabolite (M4) was rigorously identified as 6-equatorial-hydroxy SQV. 4. Metabolism of saquinavir to all metabolites was inhibited by the CYP3A4-selective inhibitor ketoconazole (IC50 = 0.55 +/- 0.12 microM). Other isoform-selective inhibitors were non-inhibitory. The protease inhibitors ritonavir, indinavir and nelfinavir potently inhibited SQV metabolism in hepatic microsomes with IC50 = 0.025 +/- 0.004, 0.82 +/- 0.26 and 0. 58 +/- 0.14 microM, respectively. 5. Saquinavir metabolism correlated with immunochemically determined CYP3A4 levels and testosterone 6beta-hydroxylation, but it failed to correlate with either immunochemically determined CYPIA2 levels or marker activities for CYP1A2, 2C9 or 2E1. 6. Heterologously expressed CYP3A4 metabolized saquinavir with a similar metabolic profile to that of human liver microsomes. 7. Km, and Vmax for total SQV metabolism were 0.61 +/- 0.19 microM and 1.82 +/- 1.13 nmol mg(-1) min(-1), respectively. 8. The extensive involvement of hepatic CYP3A4 in the metabolism of saquinavir predicts high intrinsic clearance of saquinavir. Inhibitors of CYP3A4 such as other protease inhibitors will substantially increase the bioavailability of saquinavir.

Adult↗
PubMed2002

P-glycoprotein and MRP1 expression and reduced ritonavir and saquinavir accumulation in HIV-infected individuals.

OBJECTIVES: Efflux transporters may play a role in lowering intracellular drug concentrations. As the HIV protease inhibitors are substrates for the efflux transporters P-glycoprotein and MRP, we wished to investigate whether differences in expression of these transporters on human lymphocytes correlated with intracellular concentrations of ritonavir and saquinavir. PATIENTS AND METHODS: Drug efflux transporter expression (P-glycoprotein and MRP1) on peripheral blood mononuclear cells isolated from HIV-positive patients was investigated using flow cytometry. In addition, plasma and intracellular ritonavir and saquinavir concentrations were measured by HPLC/mass spectrometry. The ratio of intracellular:plasma drug concentration was used to quantify intracellular drug accumulation. RESULTS: Patients with lower MRP1 expression (<median) had a significantly higher accumulation of both ritonavir and saquinavir than those with higher MRP1 expression (P = 0.035, CI = -1.70 to -0.06 and P = 0.043, CI = -12.79 to -0.11, respectively). Ritonavir accumulation was significantly greater in patients with lower P-glycoprotein expression (<median) than in patients with higher expression (P = 0.014, CI = -1.56 to -0.14). There was no relationship between saquinavir accumulation in patients and P-glycoprotein expression (P = 0.219, CI = -5.02 to 2.40). Combining expression of P-glycoprotein and MRP1 (expression index, EI = [(P-glycoprotein - 1) + (MRP1 - 1) x 100]) resulted in a statistically significant relationship between transporter expression and intracellular accumulation of both saquinavir (r(2) = 0.195, P = 0.035) and ritonavir (r(2) = 0.220, P = 0.049). CONCLUSION: Increased expression of P-glycoprotein and MRP1 on lymphocytes is associated with lower intracellular accumulation of saquinavir and ritonavir. These two transporters may play a role in the efflux of ritonavir and saquinavir from lymphocytes in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Zidovudine phosphorylation and mitochondrial toxicity in vitro.

Zidovudine (ZDV) is a thymidine analogue activated to its triphosphate (ZDVTP) by the host's intracellular enzymes. The initial phosphorylation step is conversion to ZDV monophosphate (ZDVMP). The poor affinity of ZDVMP for thymidylate kinase results in intracellular accumulation of ZDVMP. Clinical use of ZDV is associated with cytotoxicity, thought to be mediated through mitochondrial damage. It has been suggested that ZDV cytotoxicity correlates with intracellular ZDVMP. Here we have further studied the role of ZDVMP in cytotoxicity and some of the mechanisms involved. Intracellular metabolism of ZDV in five lymphocyte/monocyte cell lines, U937, BSM, MOLT 4, JJAHN, and RAJI (4 x 10(6) cells), was investigated following 24 h incubation with [(3)H]ZDV (1.2 microCi; 0.1 microM) and cytotoxicity was determined by the MTT assay. Cytotoxicity was closely related to intracellular concentrations of the major metabolite (ZDVMP) but not with the active metabolite ZDVTP. ZDVMP was the only metabolite detected following incubation of viable mitochondria isolated from U937 cells with ZDV (1.2 microCi; 0.1 microM; 24 h) with mitochondrial levels of 0.27 +/- 0.11 pmol/microg protein (mean +/- SD; n = 3). No MTT toxicity was seen in isolated mitochondria. Following phytohemagglutinin (PHA) stimulation of peripheral blood mononuclear cells there was an increase in ZDV cytotoxicity compared to unstimulated cells. The results suggest that the mitochondrial isozyme of thymidine kinase (TK2) plays only a minor part in ZDVMP formation. Following PHA stimulation, activation of the cytosolic thymidine kinase isozyme (TK1) is associated with increased toxicity of ZDV. We conclude that ZDVMP responsible for mitochondrial toxicity is formed in the cytosol.

Dose-Response Relationship, Drug↗
PubMed2001

Penetration of efavirenz into the male genital tract: drug concentrations and antiviral activity in semen and blood of HIV-1-infected men.

Efavirenz is a potent non-nucleoside reverse transcriptase inhibitor, licensed for the treatment of HIV-1. Data on sanctuary site penetration are limited. Therefore, we measured efavirenz concentrations in the blood and semen of 19 HIV-1-positive men and found concentrations in seminal plasma averaged 10% of those in blood plasma. Furthermore, seminal plasma viral loads were suppressed by 24 weeks of therapy in all patients. These data suggest that efavirenz-containing regimens have antiviral activity within the male genital tract.

Alkynes↗
PubMed2001

Expression of P-glycoprotein and multidrug resistance-associated protein in healthy volunteers and HIV-infected patients.

Increased expression of the multidrug efflux transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) has been suggested as a potential mechanism for decreased drug availability at certain intracellular sites that provide sanctuary for HIV. Here we investigate the expression of these transporters in peripheral blood mononuclear cells (PBMCs) of HIV-infected patients and healthy volunteers. Venous blood (30 ml) was taken from healthy volunteers (n = 21) and HIV-infected patients (n = 21; 4 antiretroviral drug naive, 17 antiretroviral drug experienced). PBMCs were isolated and fixed. To assess P-gp expression, PBMCs were incubated with an isotype control antibody or an antibody directed to an external epitope of P-gp (UIC2). To assess MRP expression, cells were permeabilized before incubation with either a control antibody or an antibody directed to an internal epitope of MRP (MRPm5). After washing, a secondary phycoerythrin-bound antibody was incubated. After additional wash steps, samples were fixed and analyzed by flow cytometry. The median fluorescence intensity of 5000 events was recorded. Results are expressed as fold increase between isotype control and UIC2/MRPm5 samples. Expression of P-gp in HIV-infected patients (1.42 +/- 0.36) was significantly lower (p = 0.0021; 95% CI, -0.633 to -0.164) than in healthy volunteers (1.82 +/- 0.55). However, MRP expression was similar in HIV-infected patients (1.37 +/- 0.34) and healthy volunteers (1.37 +/- 0.21; p = 0.91; 95% CI, -0.148941 to 0.165191). We conclude that in HIV infection, P-gp expression in total PBMCs is reduced whereas MRP expression appears to be unaltered.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

P-Glycoprotein and transporter MRP1 reduce HIV protease inhibitor uptake in CD4 cells: potential for accelerated viral drug resistance?

BACKGROUND: The multidrug transporters P-glycoprotein (P-gp) and MRP1 are functionally expressed in several subclasses of lymphocytes. HIV-1 protease inhibitors interact with both; consequently the transporters could reduce the local concentration of HIV-1 protease inhibitors and, thus, influence the selection of viral mutants. OBJECTIVES: To study the effect of the expression of P-gp and MRP1 on the transport and accumulation of HIV-1 protease inhibitors in human lymphocytes and to study the effects of specific P-gp and MRP1 inhibitors. METHODS: The initial rate and the steady-state intracellular accumulation of radiolabelled ritonavir, indinavir, saquinavir and nelfinavir was measured in three human lymphocyte cell lines: control CEM cells, CEM-MDR cells, which express 30-fold more P-gp than CEM cells, and CEM-MRP cells, which express fivefold more MRP1 protein than CEM cells. The effect of specific inhibitors of P-gp (GF 120918) and MRP1 (MK 571) was also examined. RESULTS: Compared with CEM cells, the initial rates of uptake and the steady-state intracellular concentrations of all protease inhibitors are significantly reduced in CEM-MDR cells. The intracellular concentrations of the protease inhibitors are increased upon co-administration with GF 120918, in some cases to levels approaching those in CEM cells. The intracellular concentrations of the protease inhibitors are also significantly reduced in CEM-MRP cells. Co-administration with MK -571 can partially overcome these effects. CONCLUSIONS: The overexpression of multidrug transporters significantly reduces the accumulation of protease inhibitors at this major site of virus replication, which, potentially, could accelerate the acquisition of viral resistance. Targeted inhibition of P-gp may represent an important strategy by which this problem can be overcome.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Differences in the intracellular accumulation of HIV protease inhibitors in vitro and the effect of active transport.

OBJECTIVES: To investigate the intracellular accumulation of HIV protease inhibitors (PI) and to assess the effect of active transport on this accumulation. METHODS: CEM cells were incubated with a PI for 18 h and the intracellular concentration determined using cell number and radioactivity. The effect of active transport was investigated using cells expressing P-glycoprotein (CEM(VBL)) and cells expressing multidrug resistance-associated protein 1 (MRP1; CEM(E1000)). Incubations were also carried out at 4 degrees C and in the presence of 2-deoxyglucose plus rotenone to examine the effect of inhibiting active transport. RESULTS: Nelfinavir (NFV) accumulated to the greatest extent (> 80-fold) followed by saquinavir (SQV; approximately 30-fold), ritonavir (RTV; 3-7-fold) and finally indinavir (IDV; extracellular equivalent to intracellular). In CEM(VBL) cells there was a significant reduction in the intracellular accumulation of NFV, SQV and RTV and in CEM(E1000) cells there was reduced accumulation of SQV and RTV. Inhibition of active transport processes caused a reduction in SQV and RTV accumulation but had no effect on IDV accumulation in all cell types. NFV accumulation was increased in CEM(VBL) cells as a result of inhibition of active transport. CONCLUSIONS: Marked differences can be detected in the intracellular accumulation of HIV PI drugs in vitro. Both P-glycoprotein and MRP1 may play a role in limiting the intracellular concentration of the PI and active influx mechanisms may contribute to drug accumulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

The pharmacokinetics of methadone in HIV-positive patients receiving the non-nucleoside reverse transcriptase inhibitor efavirenz.

AIMS: Methadone is predominantly metabolized by cytochrome P450 3A4 and the non nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz is a recognized inducer of this enzyme. We evaluated the pharmacokinetics of methadone in the presence and absence of efavirenz when administered to HIV infected patients with a history of injection drug use (IDU). METHODS: Eleven patients on stable methadone maintenance therapy, due to commence antiretroviral therapy (ART), participated in this study. Steady state methadone kinetic profiles were obtained on two occasions 0, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h post dosing. Following centrifugation, separated plasma was heated at 58 degrees C for 30 min to inactivate HIV and stored at -80 degrees C until methadone analysis by high performance liquid chromatography. RESULTS: When combined with efavirenz there was a marked decrease in the maximum plasma concentration (Cmax) of methadone from 689 (range 212-1568) to 358 (range 205-706) ng ml(-1), P = 0.007 : 95% confidence interval (CI) 112-549. The mean area under the methadone concentration curve 0-24 h (AUC(0,24 h)) was also significantly reduced from 12341 (range 3682-34147) to 5309 (range 2430-10349) ng ml(-1) h in the presence of efavirenz, P = 0.012 : 95% CI 1921-12143. Nine patients described symptoms of methadone withdrawal and received a dose increase. Although methadone AUC(0,24 h) was reduced by over 50% following efavirenz the mean increase in methadone dose required was 22% (range 15-30 mg). CONCLUSION: The inclusion of the NNRTI efavirenz in once daily ART for HIV patients with a history of IDU receiving methadone maintenance results in a significant reduction in methadone plasma concentrations mediated by enzyme induction. It is important to distinguish efavirenz neurological effects which were observed between days 1-5 of therapy from symptoms of methadone withdrawal which occurred from day 8 onwards. The dose of methadone was adjusted by increments of 10 mg to counteract the efavirenz inducing effect.

Adult↗
PubMed2001

Effect of alpha1-acid glycoprotein on the intracellular accumulation of the HIV protease inhibitors saquinavir, ritonavir and indinavir in vitro.

AIMS: Since alpha1-acid glycoprotein (AGP) levels may be raised during HIV infection, we have examined in vitro the effect of increasing the concentration of AGP on the intracellular accumulation of the HIV protease inhibitors saquinavir (SQV), ritonavir (RTV) and indinavir (IDV). METHODS: U937 cells (5 x 10(6) cells in 5 ml RPMI growth medium) were incubated at 37 degrees C for 18 h with [14C]-SQV (0.1 microCi), [3H]-RTV and [3H]-IDV (0.135 microCi) to a final concentration of 1 microM in the presence of 0, 0.5 and 2.0 mg x ml(-1) AGP. Following extraction in 60% methanol the intracellular drug concentration was determined by liquid scintillation counting. RESULTS: Binding to AGP (2.0 mg x ml(-1)) reduced the mean intracellular concentration of SQV from 31.5 microM to 7.4 microM (P < 0.0001; 95% CI 19.4-28.8). RTV concentration was also reduced (8.8 microM to 1.6 microM; P < 0.0001; 95% CI 5.4-9.0) as was the concentration of IDV (3.0 microM to 1.5 microM; P < 0.0001; 95% CI 1.1-1.9). CONCLUSIONS: Reduced intracellular protease inhibitor concentrations in the presence of increasing concentrations of AGP will certainly impact on the antiviral activity in vitro. However, since protease inhibitors are high clearance drugs, free drug concentration will likely remain unaffected in the presence of elevated AGP during chronic oral dosing although there will be an increase in total plasma drug concentration.

Cell Line↗