Variation in Phenolic Profile, ß-Carotene and Flavonoid Contents, Biological Activities of Two Tagetes Species from Pakistani Flora.

The objective of present study was to evaluate the variation in phenolic profile, ß-carotene, flavonoid contents, antioxidant and antimicrobial properties of Tagetes erecta and Tagetes patula (T. erecta and T. patula) through different in vitro assays. Antioxidant activity was determined through 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and inhibition of linoleic acid peroxidation assays and antibacterial and antifungal activities studied using the disc diffusion and resazurin microtiter-plate assays against bacterial and fungal strains. Moreover, total phenolics (TP), total carotenoids (TC) and total flavonoids (TF) were also determined. Highest (TP 35.8 mg GAE/g) and TF (16.9 mg CE/g) contents were found in MeOH extract of T. patula. T. erecta extract showed higher TC contents (6.45 mg/g) than T. patula extract (6.32 mg/g). T. erecta exhibited the highest DPPH radical-scavenging activity (IC50 ) (5.73 µg/mL) and inhibition of linoleic acid peroxidation (80.1%). RP-HPLC revealed the presence of caffeic acid, sinapic acid and ferulic acid in Tagetes extracts, m-coumaric acid in T. erecta whereas chlorogenic acid in T. patula extract only. Both extracts possessed promising antimicrobial activity compared to the ciprofloxacin and flumequine (+ve controls) against Bacillus subtilis and Alternaria alternate. Both extract were rich source of polyphenols exhibiting excellent biological activities.

Investigation into the cardiotoxic effects of doxorubicin on contractile function and the protection afforded by cyclosporin A using the work-loop assay.

Doxorubicin is known to cause cardiotoxicity through multiple routes including the build-up of reactive oxygen species and disruption of the calcium homeostasis in cardiac myocytes, but the effect of drug treatment on the associated biomechanics of cardiac injury remains unclear. Detecting and understanding the adverse effects of drugs on cardiac contractility is becoming a priority in non-clinical safety pharmacology assessment. The work-loop technique enables the assessment of force-length work-loop contractions, which mimic those of the pressure-volume work-loops experienced by the heart in vivo. During this study we evaluated whether the work-loop technique could potentially provide improved insight into the biomechanics associated with drug-induced cardiac dysfunction. In order to do this we investigated the cardiotoxic effects of doxorubicin and characterised the protection afforded by the co-administration of cyclosporin A (CsA). This study provides detailed biomechanical in vitro insight into the cardiac dysfunction associated with Doxorubicin treatment, including reduction in peak force, force during shortening and power output. These effects were significantly abrogated in doxorubicin-CsA co-treatment studies. Closely mimicking the in vivo pressure-volume muscle mechanics, this assay provides a quick and easy technique to gain a better understanding of the detailed biomechanics of drug-induced cardiac dysfunction.

Synthesis and evaluation of the antimalarial, anticancer, and caspase 3 activities of tetraoxane dimers.

The synthesis of a range of mono spiro and dispiro 1,2,4,5-tetraoxane dimers is described. Selected molecules were examined in in vitro assays to determine their antimalarial and anticancer potential. Our studies reveal that several molecules possess potent nanomolar antimalarial and single digit micromolar antiproliferative IC(50)s versus colon (HT29-AK and leukemia (HL60) cell lines.

Attenuation of doxorubicin-induced cardiotoxicity by mdivi-1: a mitochondrial division/mitophagy inhibitor.

Doxorubicin is one of the most effective anti-cancer agents. However, its use is associated with adverse cardiac effects, including cardiomyopathy and progressive heart failure. Given the multiple beneficial effects of the mitochondrial division inhibitor (mdivi-1) in a variety of pathological conditions including heart failure and ischaemia and reperfusion injury, we investigated the effects of mdivi-1 on doxorubicin-induced cardiac dysfunction in naïve and stressed conditions using Langendorff perfused heart models and a model of oxidative stress was used to assess the effects of drug treatments on the mitochondrial depolarisation and hypercontracture of cardiac myocytes. Western blot analysis was used to measure the levels of p-Akt and p-Erk 1/2 and flow cytometry analysis was used to measure the levels p-Drp1 and p-p53 upon drug treatment. The HL60 leukaemia cell line was used to evaluate the effects of pharmacological inhibition of mitochondrial division on the cytotoxicity of doxorubicin in a cancer cell line. Doxorubicin caused a significant impairment of cardiac function and increased the infarct size to risk ratio in both naïve conditions and during ischaemia/reperfusion injury. Interestingly, co-treatment of doxorubicin with mdivi-1 attenuated these detrimental effects of doxorubicin. Doxorubicin also caused a reduction in the time taken to depolarisation and hypercontracture of cardiac myocytes, which were reversed with mdivi-1. Finally, doxorubicin caused a significant elevation in the levels of signalling proteins p-Akt, p-Erk 1/2, p-Drp1 and p-p53. Co-incubation of mdivi-1 with doxorubicin did not reduce the cytotoxicity of doxorubicin against HL-60 cells. These data suggest that the inhibition of mitochondrial fission protects the heart against doxorubicin-induced cardiac injury and identify mitochondrial fission as a new therapeutic target in ameliorating doxorubicin-induced cardiotoxicity without affecting its anti-cancer properties.

Artemisinin-polypyrrole conjugates: synthesis, DNA binding studies and preliminary antiproliferative evaluation.

Greater than the sum of its parts: Artemisinins are currently in phase I-II clinical trials against breast, colorectal and non-small-cell lung cancers. In an attempt to offer increased specificity, a series of hybrid artemisinin-polypyrrole minor groove binder conjugates are described. DNA binding/modelling studies and preliminary biological evaluation give insights into their mechanism of action and the potential of this strategy.

Evaluation of antimalarial, free-radical-scavenging and insecticidal activities of Artemisia scoparia and A. Spicigera, Asteraceae

Artemisia species (Asteraceae), widespread throughout the world, are a group of important medicinal plants. The extracts of two medicinal plants of this genus, Artemisia scoparia Waldst. & Kit. and A. spicigera C. Koch, were evaluated for potential antimalarial, free-radical-scavenging and insecticidal properties, using the heme biocrystallisation and inhibition assay, the DPPH assay and the contact toxicity bioassay using the pest Tribolium castaneum, respectively. The methanol extracts of both species showed strong free-radical-scavenging activity and the RC50 values were 0.0317 and 0.0458 mg/mL, respectively, for A. scoparia and A. spicigera. The dichloromethane extracts of both species displayed a moderate level of potential antimalarial activity providing IC50 at 0.778 and 0.999 mg/mL for A. scoparia and A. spicigera, respectively. Both species of Artemisia showed insecticidal properties. However, A. spicigera was more effective than A. scoparia.

Composition, antioxidant and chemotherapeutic properties of the essential oils from two Origanum species growing in Pakistan

The GC-MS analyses of Origanum majorana L. (OME) and Origanum vulgare L. (OVE), Lamiaceae, essential oils helped identification of 39 (96.4 percent of the total oils) and 43 (92.9 percent of the total oils) components, respectively. The major constituents of OME were terpinene-4-ol (20.9 percent), linalool (15.7 percent), linalyl-acetate (13.9 percent), limonene (13.4 percent) and α-terpineol (8.57 percent), whereas, thymol (21.6 percent), carvacrol (18.8 percent), o-cymene (13.5 percent) and α-terpineol (8.57 percent) were the main components of OVE. In the disc diffusion and the resazurin microtitre assays, OME showed better antibacterial activity than OVE with larger zones of inhibition (16.5-27.0 mm) and smaller MIC (40.9-1250.3 μg/mL) against the tested bacterial strains. Only OVE displayed anti-heme biocrystallization activity with an IC50 at 0.04 mg/mL. In the DPPH assay, OVE showed better radical-scavenging activity than OME (IC50=65.5 versus 89.2 μg/mL) and both OME and OVE inhibited lionleic acid oxidation. However, in the bleaching β-carotene assay, OVE exhibited better antioxidant activity than OME. In the MTT assay, OME was more cytotoxic than OVE against different cancer cell types, such as MCF-7, LNCaP and NIH-3T3, with IC50s of 70.0, 85.3 and 300.5 μg/mL, respectively. Overall, some components of OME and OVE may have antiparasitic and chemotherapeutic activity.

Interactions of tenofovir, Lamivudine, abacavir and Didanosine in primary human cells.

Certain triple nucleoside/tide reverse transcriptase inhibitor (NRTI) regimens containing tenofovir (TDF) have been associated with rapid early treatment failure. The mechanism is unknown, but may be at the level of drug transport. We measured the lipophilicity of the drugs [3H]-lamivudine (3TC), -didanosine (ddI), -TDF and -ABC. Peripheral blood mononuclear cells (PBMCs) were used to evaluate drug-drug interactions at the level of drug transport. PBMCs were measured for the expression of P-glycoprotein (P-gp), multidrug resistance-associated protein-1 (MRP-1) and breast cancer resistance protein (BCRP) by flow cytometry. The rank order of the lipophilicity of the drugs were ABC>>>3TC³ddI>TDF. The accumulation of [3H]-3TC, -ddI and -TDF were temperature sensitive (suggesting facilitated transport), in contrast to [3H]-ABC. ABC reduced the accumulation of [3H]-3TC, and cell fractionation experiments suggested this was mainly in membrane-bound [3H]-3TC. ABC/TDF and ABC/ddI increased the accumulation of [3H]-3TC and 3TC/TDF also increased the accumulation of [3H]-TDF. In contrast, none of the NRTI/NtRTI incubations (alone or in combination) altered the accumulation of [3H]-ABC and -ddI. PBMC expression of P-gp, MRP1 and BCRP were detected, but none correlated with the accumulation of the drugs. The high failure rates seen with TDF, ABC and 3TC are not fully explained by an interaction at transporter level.

Comparative efflux of saquinavir, ritonavir and lopinavir from primary human cells.

Therapeutic drug monitoring is an important element in the management of drug treatment in HIV-1 infected patients. We have examined the effect of temperature on the egress of HIV-1 protease inhibitors from primary T lymphocytes to determine optimum conditions to be adopted in the processing of blood samples in order to accurately estimate intracellular or plasma drug concentrations. Peripheral blood mononuclear cells or U937 cells were incubated with radiolabelled saquinavir, ritonavir or lopinavir at a concentration of 1 µM. The cells were washed and resuspended in RPMI medium (without radiolabelled drug) and further incubated at 37°C, room temperature (21°C) or at 4°C. We observed that release of drug ensued upon the removal of cells from bathing media containing drug, with the rate of efflux being slower at 4°C and fastest at 37°C for all the protease inhibitors. There was a more rapid efflux of saquinavir and ritonavir than lopinavir from both cultured monocytic and primary human cells. The rank order of the partition coefficient of the drugs were lopinavir > saquinavir > ritonavir. All factors that may limit optimal estimation of cell-associated drug concentrations must be considered so that intracellular concentrations of drug can be accurately estimated.

Concentration-dependent effects and intracellular accumulation of HIV protease inhibitors in cultured CD4 T cells and primary human lymphocytes.

BACKGROUND: The intracellular and plasma concentrations of HIV protease inhibitors (HPIs) vary widely in vivo. It is unclear whether there is a concentration-dependent effect of HPIs such that at increasing concentration they may either block their own efflux (leading to 'autoboosting') or influx (leading to saturability/decreased intracellular accumulation). METHOD: The effects of various concentrations (0-30 microM) of lopinavir, saquinavir, ritonavir and atazanavir on the accumulation of [(14)C]lopinavir, [(3)H]saquinavir, [(3)H]ritonavir and [(3)H]atazanavir, respectively, were investigated in CEM(parental), CEM(VBL) [P-glycoprotein (ABCB1) overexpressing], CEM(E1000) (MRP1 overexpressing) and in peripheral blood mononuclear cells (PBMCs). We also investigated the effects of inhibitors of ABCB1/ABCG2 (tariquidar), ABCC (MK571) and ABCC1/2 (frusemide), singly and in combination with HPIs, on cellular accumulation. RESULTS: In all the cell lines, with increasing concentration of lopinavir, saquinavir and ritonavir, there was a significant increase in the cellular accumulation of [(14)C]lopinavir, [(3)H]saquinavir and [(3)H]ritonavir. Tariquidar, MK571 and frusemide (alone and in combination with lopinavir, saquinavir and ritonavir) significantly increased the accumulation of [(14)C]lopinavir, [(3)H]saquinavir and [(3)H]ritonavir. Ritonavir (alone or in combination with tariquidar) decreased the intracellular accumulation of [(3)H]ritonavir in PBMCs. Atazanavir decreased the accumulation of [(3)H]atazanavir in a concentration-dependent manner in all of the cells tested. CONCLUSIONS: There are complex and variable drug-specific rather than class-specific effects of the HPIs on their own accumulation.

The accumulation and metabolism of zidovudine in 3T3-F442A pre-adipocytes.

BACKGROUND AND PURPOSE: Cultured pre-adipocytes accumulate and metabolize zidovudine (ZDV), but its mode of accumulation into these cells is unclear. We investigated the mode of accumulation of [(3)H]-ZDV, and the impact of changes in external pH and modulators of drug transporters on its accumulation and metabolism. EXPERIMENTAL APPROACH: The initial rate and steady-state accumulation of [(3)H]-ZDV were measured in 3T3-F442A cells. P-glycoprotein (P-gp) expression was detected by Western blotting. External pH was varied, and modulators of intracellular pH and drug transporters were used to study the mode of accumulation of ZDV. Phosphorylated ZDV metabolites were detected by high-performance liquid chromatography. KEY RESULTS: Intracellular accumulation of ZDV was rapid, reaching equilibrium within 20 min; nigericin increased accumulation by 1.9-fold, but this did not alter the generation of ZDV mono-, di- and triphosphate. The accumulation and metabolism were pH dependent, being maximal at pH 7.4 and least at pH 5.1. Monensin, carbonyl cyanide p-trifluoromethoxy) phenyl hydrazone, brefeldin A, bafilomycin A1 and concanamycin A increased accumulation; 2-deoxyglucose, dipyridamole, thymidine and tetraphenylphosphonium inhibited accumulation. The accumulation was saturable; the derived K(d) and capacity of binding were 250 nmol per 10(6) cells and 265 nM respectively. 3T3-F442A cells express P-gp; inhibitors of P-gp (XR9576 and verapamil), P-gp/BCRP (GF120918), multidrug resistance protein (MRP) (MK571) and MRP/OATP (probenecid) increased the accumulation of ZDV. Saquinavir, ritonavir, amprenavir and lopinavir increased accumulation. CONCLUSIONS AND IMPLICATIONS: The accumulation of ZDV in 3T3-F442A cells was rapid, energy dependent, saturable and pH sensitive. Western blot analysis showed that 3T3-F442A cells express P-gp, and direct inhibition assays suggest that ZDV is a substrate of P-gp and MRP.

P-glycoprotein, multidrug resistance-associated proteins and human organic anion transporting polypeptide influence the intracellular accumulation of atazanavir.

BACKGROUND: Drug efflux (for example, P-glycoprotein [P-gp], multidrug resistance-associated proteins [MRPs] and breast cancer resistance protein [BCRP]) and influx (for example, human organic anion transporting polypeptide [hOCTP] or human organic anion transporting polypeptide [hOATP]) transporters alter the cellular concentrations of some HIV protease inhibitors (HPIs). Here, we studied the lipophilicity and uptake of [(3)H]-atazanavir (ATV) in CEM (parental), CEM(VBL) (P-gp-overexpressing), CEM(E1000) (MRP1-overexpressing) and peripheral blood mononuclear cells (PBMCs), and evaluate the effects of modulators of drug transporters on uptake. METHODS: Lipophilicity was measured by octanol/saline partition method. The influence of influx/efflux transporters on uptake was evaluated in the absence and presence of inhibitors of P-gp (GPV031), P-gp/BCRP (tariquidar and GF120918), P-gp/MRP1 (dipyridamole and daidzein), MRP1/2 (frusemide and genistein), hOATP/hOCTP (estrone-3-sulfate [E-3-S]) and hOATP/hOCTP/MRP (probenecid). The effects of a number of HPIs on uptake were also evaluated. Data from digitonin permeabilized cells allowed the evaluation of the contribution of cellular binding to total drug uptake, whereas the inhibitory effect of ATV on P-gp was assessed by daunomycin efflux/uptake assays. RESULTS: [(3)H]-ATV is lipophilic and accumulates in the cultured cells as follows: CEM>CEM(E1000)>CEM(VBL). Tariquidar, GF120918 and daidzein significantly increased the uptake of [(3)H]-ATV in the cultured cells. By contrast, only daidzein and tipranavir significantly increased uptake in PBMCs, with tariquidar and frusemide devoid of effects, whereas dipyridamole, E-3-S, GPV031 and genistein significantly decreased accumulation. ATV inhibits P-gp activity; manipulation of uptake with digitonin suggests binding of [(3)H]-ATV to P-gp. CONCLUSIONS: [(3)H]-ATV is lipophilic, a P-gp, MRP and hOATP substrate and an inhibitor of P-gp. Concomitant administration of ATV with drugs and dietary components (for example, daidzein and genistein) that interact with these transporters could alter its pharmacokinetics.

Intracellular 'boosting' of darunavir using known transport inhibitors in primary PBMC.

AIMS: ABCB1, some ABCCs and SLCOs have been reported to affect the intracellular accumulation of various protease inhibitors in vitro and ex vivo. Darunavir is the most recently licensed protease inhibitor and we sought to investigate the ability of transport inhibitors to influence its intracellular accumulation in lymphocytes from healthy volunteers. METHODS: The intracellular accumulation of radiolabelled darunavir was assessed using CEM cells and ABCB1-overexpressing CEM(VBL) cells. Apical and basolateral transport of radiolabelled darunavir through MDCKII monolayers was also studied. Finally the ability of known inhibitors to influence intracellular accumulation of darunavir in peripheral blood mononuclear cells (PBMC) was investigated. RESULTS: CEM(VBL) cells (1.4 +/- 0.6, P < 0.001, 95% CI for the difference = 0.46, 0.80, n= 7) had significantly lower accumulation of darunavir compared with CEM cells (5.6 +/- 0.7, n= 7) and this was reversed by addition of tariquidar (30 nm, 4.6 +/- 0.8, P < 0.001, 95% CI =-0.64, -0.41, n= 4). In MDCKII-ABCBI cells, transport from the basal to the apical compartment was observed and this was also reversible with the addition of tariquidar. In PBMCs, dipyridamole (6.9 +/- 1.3, P < 0.01, 95% CI for the difference =-1.16, -0.30, (n= 8) significantly increased whilst montelukast (5.7 +/- 1.0, P < 0.01, 95% CI for the difference = 0.16, 0.79, n= 8) significantly decreased the intracellular accumulation of darunavir when compared with control (6.2 +/- 1.1, n= 8). CONCLUSIONS: Darunavir is a substrate for efflux and influx transporters in PBMC and intracellular concentrations can be manipulated using known inhibitors.

Intracellular accumulation of efavirenz and nevirapine is independent of P-glycoprotein activity in cultured CD4 T cells and primary human lymphocytes.

BACKGROUND: Interaction of antiretrovirals with drug transporters such as P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), breast cancer resistance protein (BCRP) and solute carrier organic anion transporter (SLCO) may influence the emergence of viral mutants by altering intracellular drug concentrations. Here we characterize the effect of transporter expression in a variety of cell types such as control CEM, CEM(VBL) (P-gp-overexpressing), CEM(E1000) (MRP1-overexpressing), MT4, control MDCKII, MDCKII(MDR1) (P-gp-overexpressing) and peripheral blood mononuclear cells (PBMCs) on the uptake of [(14)C]efavirenz and [(3)H]nevirapine. We also investigated the lipophilicity of [(14)C]efavirenz and [(3)H]nevirapine. METHODS: The expression of P-gp, MRP1, MRP2, SLCO1A2, 1B1, 1B3, 2B1, 3A1 and 4A1 was assessed by PCR. Inhibitors of P-gp (XR9576, GF120918, dipyridamole) and MRP (MK571, frusemide, dipyridamole), and SLCO substrate or inhibitor (estrone-3-sulphate or montelukast, respectively) were used to study the role of drug transporters in the accumulation of [(14)C]efavirenz and [(3)H]nevirapine. Lipophilicity was measured by the octanol/saline partition coefficient. RESULTS: CEM cells, MT4 cells and PBMCs express various SLCO isoforms, with SLCO3A1 detected in all of the cells. XR9576, dipyridamole and GF120918 had no effects on the accumulation of [(14)C]efavirenz, while MK571 and frusemide produced variable effects in the cells. The accumulation of [(14)C]efavirenz was significantly decreased in all the cells by montelukast and estrone-3-sulphate. CONCLUSIONS: P-gp expression had no effect on the accumulation of [(14)C]efavirenz and [(3)H]nevirapine. MRP1/2 expression, lipophilicity and SLCO-like transporters (possibly SLCO3A1) may have greater influence on the accumulation of [(14)C]efavirenz than [(3)H]nevirapine.

Antitumour and antimalarial activity of artemisinin-acridine hybrids.

Artemisinin-acridine hybrids were prepared and evaluated for their in vitro activity against tumour cell lines and a chloroquine sensitive strain of Plasmodium falciparum. They showed a 2-4-fold increase in activity against HL60, MDA-MB-231 and MCF-7 cells in comparison with dihydroartemisinin (DHA) and moderate antimalarial activity. Strong evidence that the compounds induce apoptosis in HL60 cells was obtained by flow cytometry, which indicated accumulation of cells in the G1 phase of the cell cycle.

Inhibition of P-glycoprotein and multidrug resistance-associated proteins modulates the intracellular concentration of lopinavir in cultured CD4 T cells and primary human lymphocytes.

BACKGROUND: HIV protease inhibitors (HPIs) are an important component of highly active antiretroviral therapy. However, the activity of drug efflux transporters, such as P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRP1/MRP2), may limit intracellular drug accumulation. Drugs that inhibit the activity of drug efflux proteins may, in combination with HPIs, enhance the clinical efficacy of the drugs. METHODS: The transport of [(14)C]lopinavir was evaluated in peripheral blood mononuclear cells (PBMCs) in the absence or presence of known inhibitors: tariquidar (P-gp), MK571 (MRP), frusemide (MRP1/2), dipyridamole (MRP1/P-gp) and probenecid (MRP2/OATP). The effects of ritonavir, amprenavir and atazanavir on the accumulation of lopinavir were also evaluated in cultured CD4(+) T-lymphoblastoid cells [CEM (parental), CEM(VBL) (P-gp-overexpressing) and CEM(E1000) (MRP1-overexpressing)] and PBMCs. The relative expression of the drug efflux proteins on the PBMCs was assessed by flow cytometric and real-time PCR methods. RESULTS: Tariquidar, MK571, frusemide and dipyridamole all significantly increased the intracellular accumulation of lopinavir in the PBMCs, whereas probenecid decreased it. The cellular accumulation ratio (CAR) of lopinavir was also increased by ritonavir, amprenavir and atazanavir in a concentration-dependent manner in both cell types. The expression of P-gp, MRP1 and MRP2 mRNA were variable and individually did not correlate with CARs of lopinavir. CONCLUSIONS: We provide evidence that lopinavir is a substrate of P-gp, MRP1 and MRP2. The intracellular concentration of lopinavir is increased when co-incubated with ritonavir, amprenavir and atazanavir in PBMCs. Manipulation of drug efflux transporters may be a useful strategy for increasing the intracellular concentration and thereby enhancing the clinical efficacy of lopinavir.

Modulation of the intracellular accumulation of saquinavir in peripheral blood mononuclear cells by inhibitors of MRP1, MRP2, P-gp and BCRP.

BACKGROUND: The efflux transporters P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRP) and breast cancer resistance protein (BCRP) limit the accumulation of antiretrovirals in cell lines but it is more important to know whether the expression of these transporters in peripheral blood mononuclear cells (PBMC) impacts cellular drug concentrations. OBJECTIVES: To study the transport and accumulation of saquinavir (SQV) in PBMC and the effects of specific inhibitors of MRP1, MRP2, P-gp and BCRP. METHODS: Transport and accumulation of [H]-SQV was measured in PBMC in the absence or presence of specific and non-specific inhibitors of MRP1, MRP2, P-gp and BCRP. Flow cytometric, western blot and real-time PCR assays were used to examine the relative expression of the drug efflux transporters in the same batches of PBMC. RESULTS: MRP2 is present in PBMC. The expression of P-gp, MRP1, MRP2 (mRNA) and BCRP all displayed batch-to-batch variability. Specific and non-specific inhibitors of MRP1, P-gp and MRP2 significantly increased the baseline accumulation of SQV. Accumulation of SQV was not correlated with the expression of any single transporter. CONCLUSIONS: Multiple drug efflux transporters are important in the intracellular accumulation of SQV in PBMC. If drug efflux contributes towards virological failure, then all contributing transporters will need to be inhibited.

In vitro synergy and enhanced murine brain penetration of saquinavir coadministered with mefloquine.

Highly active antiretroviral therapy has substantially improved prognosis in human immunodeficiency virus (HIV). However, the integration of proviral DNA, development of viral resistance, and lack of permeability of drugs into sanctuary sites (e.g., brain and lymphocyte) are major limitations to current regimens. Previous studies have indicated that the antimalarial drug chloroquine (CQ) has antiviral efficacy and a synergism with HIV protease inhibitors. We have screened a panel of antimalarial compounds for activity against HIV-1 in vitro. A limited efficacy was observed for CQ, mefloquine (MQ), and mepacrine (MC). However, marked synergy was observed between MQ and saquinavir (SQV), but not CQ in U937 cells. Furthermore, enhancement of the antiviral activity of SQV and four other protease inhibitors (PIs) by MQ was observed in MT4 cells, indicating a class specific rather than a drug-specific phenomenon. We demonstrate that these observations are a result of inhibition of multiple drug efflux proteins by MQ and that MQ also displaces SQV from orosomucoid in vitro. Finally, coadministration of MQ and SQV in CD-1 mice dramatically altered the tissue distribution of SQV, resulting in a >3-fold and >2-fold increase in the tissue/blood ratio for brain and testis, respectively. This pharmacological enhancement of in vitro antiviral activity of PIs by MQ now warrants further examination in vivo.

Altered adipokine response in murine 3T3-F442A adipocytes treated with protease inhibitors and nucleoside reverse transcriptase inhibitors.

BACKGROUND: Abnormal levels of tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and adiponectin have been observed in HIV patients with lipodystrophy. However, because these patients have long drug and disease histories, it is unclear which drugs are responsible for these abnormalities. We have therefore investigated the effects of individual antiretrovirals on adipokine expression and adipogenesis using a murine cell line in vitro. METHODS: Differentiating murine 3T3-F442A adipocytes were incubated with 20 microM nucleoside reverse transcriptase inhibitors (NRTIs) zidovudine or stavudine, or protease inhibitors (PIs) indinavir, ritonavir, saquinavir or nelfinavir, in the presence and absence of rosiglitazone (10 microM). Adipogenesis was assessed using glycerol-3-phosphate dehydrogenase activity, while expression of TNF-alpha, IL-6 and adiponectin at protein and mRNA levels was assessed by ELISA and quantitative real-time PCR, respectively. RESULTS: Nelfinavir, ritonavir and saquinavir inhibited adipogenesis and up-regulated the expression of TNF-alpha and IL-6, but this effect was not seen with indinavir, zidovudine and stavudine. Adiponectin expression was significantly reduced in both NRTI- and PI-treated cells, although the most profound reductions were found with ritonavir and saquinavir. Co-incubation with rosiglitazone led to a partial attenuation of the change in TNF-alpha, IL-6 and adiponectin secretion. CONCLUSIONS: Our data suggest that the PIs nelfinavir, ritonavir and saquinavir have potent effects in inhibiting adipocyte differentiation whilst up-regulating TNF-alpha and IL-6 mRNA levels and decreasing adiponectin levels. These changes were partially attenuated by rosiglitazone. Taken together, the data show that antiretrovirals have complex effects on adipocyte function, which may be mediated by an altered adipokine response.

Intracellular disposition and metabolic effects of zidovudine, stavudine and four protease inhibitors in cultured adipocytes.

OBJECTIVE: The pathogenesis of lipodystrophy caused by the HIV protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs) is unclear. We have investigated the disposition of these drugs in adipocytes and the consequent effect on adipocyte metabolism and viability. DESIGN: Laboratory study utilizing two murine cell lines, 3T3-L1 and 3T3-F442A. METHODS: Intracellular NRTI phosphate and PI concentrations were determined by HPLC and HPLC-MS/MS, respectively. The cytotoxicity of the drugs was examined on the different adipogenic stages together with their effects on glucose uptake plus or minus insulin, and on glycerol and triglyceride levels. RESULTS: There was rapid intracellular accumulation and phosphorylation of [3H]-zidovudine and -stavudine to their phosphate metabolites in adipocytes. The NRTIs were not cytotoxic, did not affect preadipocyte protein synthesis and did not inhibit adipogenesis or induce lipolysis. PIs accumulated in adipocytes (nelfinavir>saquinavir>ritonavir>indinavir). All PIs, except indinavir, were cytotoxic and inhibited adipogenesis, increased lipolysis and impaired preadipocyte protein synthesis. PIs inhibited glucose uptake in the rank order: indinavir>saquinavir>ritonavir>nelfinavir. CONCLUSION: These data demonstrate that PIs may play a role in the insulin resistance observed in lipodystrophy by affecting glucose uptake, adipogenesis and lipolysis. NRTIs alone do not seem to have any effect on adipocyte metabolism despite undergoing phosphorylation to their triphosphorylated anabolites, although their effects in combination with PIs in perturbing adipocyte metabolism warrants further investigation.