Omar Janneh

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 [3H]-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.

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.

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.

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% of the total oils) and 43 (92.9% of the total oils) components, respectively. The major constituents of OME were terpinene-4-ol (20.9%), linalool (15.7%), linalyl-acetate (13.9%), limonene (13.4%) and α-terpineol (8.57%), whereas, thymol (21.6%), carvacrol (18.8%), o-cymene (13.5%) and α-terpineol (8.57%) 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.

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.

Doxorubicin induced myocardial injury is exacerbated following ischaemic stress via opening of the mitochondrial permeability transition pore.

Chemotherapeutic agents such as doxorubicin are known to cause or exacerbate cardiovascular cell death when an underlying heart condition is present. However, the mechanism of doxorubicin-induced cardiotoxicity is unclear. Here we assess the cardiotoxic effects of doxorubicin in conditions of myocardial ischaemia reperfusion and the mechanistic basis of protection, in particular the role of the mitochondrial permeability transition pore (mPTP) in such protection. The effects of doxorubicin (1μM)±cyclosporine A (CsA, 0.2μM; inhibits mPTP) were investigated in isolated male Sprague-Dawley rats using Langendorff heart and papillary muscle contraction models subjected to simulated ischaemia and reperfusion injury. Isolated rat cardiac myocytes were used in an oxidative stress model to study the effects of drug treatment on mPTP by confocal microscopy. Western blot analysis evaluated the effects of drug treatment on p-Akt and p-Erk 1/2 levels. Langendorff and the isometric contraction models showed a detrimental effect of doxorubicin throughout reperfusion/reoxygenation as well as increased p-Akt and p-Erk levels. Interestingly, CsA not only reversed the detrimental effects of doxorubicin, but also reduced p-Akt and p-Erk levels. In the sustained oxidative stress assay to study mPTP opening, doxorubicin decreased the time taken to depolarization and hypercontracture, but these effects were delayed in the presence of CsA. Collectively, our data suggest for the first that doxorubicin exacerbates myocardial injury in an ischaemia reperfusion model. If the inhibition of mPTP ameliorates the cardiotoxic effects of doxorubicin, then more selective inhibitors of mPTP should be further investigated for their utility in patients receiving doxorubicin.

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.

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.

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.

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 [3H]‐ZDV, and the impact of changes in external pH and modulators of drug transporters on its accumulation and metabolism.