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.

Cultured CD4T cells and primary human lymphocytes express hOATPs: intracellular accumulation of saquinavir and lopinavir.

BACKGROUND AND PURPOSE: Drug efflux tranporters (P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP)) limit the cellular uptake of human immunodeficiency virus protease inhibitors but the contribution of influx transporters in cells that (over)express P-gp or MRP is less clear. Here, we studied the expression of one influx transporter system, human organic anion-transporting polypeptide (hOATP), in some T-cell lines (CEM, CEM(VBL), CEM(E1000)) and in peripheral blood mononuclear cells (PBMCs) and examined the effects of manipulation of influx/efflux transporters on the uptake of saquinavir and lopinavir. EXPERIMENTAL APPROACH: The expression of hOATPs was studied by PCR. We used hOATP substrate or inhibitor (estrone-3-sulphate (E-3-S) or montelukast, respectively) and inhibitors of P-gp (XR9576) and MRP (MK571 and frusemide) to study functional interactions between influx and efflux transporters in the uptake of saquinavir and lopinavir. Lipophilicity of the drugs was measured by octanol/saline partition coefficient. KEY RESULTS: CEM cells, their variants and PBMCs express various hOATP isoforms, with OATP3A1 detected in all of the cells. MK571, XR9576 and frusemide increased the uptake of saquinavir and lopinavir. E-3-S and montelukast reduced the uptake of saquinavir and lopinavir in some, but not all, of the cells. Pretreatment of the cells with MK571, XR9576 or frusemide, followed by E-3-S co-incubation reduced the cellular accumulation of saquinavir and lopinavir. Lopinavir is much more lipophilic than saquinavir. CONCLUSIONS AND IMPLICATIONS: Human OATPs, MRP, P-gp and lipophilicity determine the cellular uptake and retention of saquinavir and lopinavir. These data may have important implications for drug-drug interactions, drug safety and efficacy.

Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with pfcrt polymorphism in Papua New Guinea and South America.

The mechanistic basis for chloroquine resistance (CQR) in Plasmodium falciparum recently has been linked to the polymorphic gene pfcrt. Alleles associated with CQR in natural parasite isolates harbor threonine (T), as opposed to lysine (K) at amino acid 76. P. falciparum CQR strains of African and Southeast Asian origin carry pfcrt alleles encoding an amino acid haplotype of CVIET (residues 72-76), whereas most South American CQR strains studied carry an allele encoding an SVMNT haplotype; chloroquine-sensitive strains from malarious regions around the world carry a CVMNK haplotype. Upon investigating the origin of pfcrt alleles in Papua New Guinean (PNG) P. falciparum we found either the chloroquine-sensitive-associated CVMNK or CQR-associated SVMNT haplotypes previously seen in Brazilian isolates. Remarkably we did not find the CVIET haplotype observed in CQR strains from Southeast Asian regions more proximal to PNG. Further we found a previously undescribed CQR phenotype to be associated with the SVMNT haplotype from PNG and South America. This CQR phenotype is significantly less responsive to verapamil chemosensitization compared with the effect associated with the CVIET haplotype. Consistent with this, we observed that verapamil treatment of P. falciparum isolates carrying pfcrt SVMNT is associated with an attenuated increase in digestive vacuole pH relative to CVIET pfcrt-carrying isolates. These data suggest a key role for pH-dependent changes in hematin receptor concentration in the P. falciparum CQR mechanism. Our findings also suggest that P. falciparum CQR has arisen through multiple evolutionary pathways associated with pfcrt K76T.

Cellular uptake of chloroquine is dependent on binding to ferriprotoporphyrin IX and is independent of NHE activity in Plasmodium falciparum.

Here we provide definitive evidence that chloroquine (CQ) uptake in Plasmodium falciparum is determined by binding to ferriprotoporphyrin IX (FPIX). Specific proteinase inhibitors that block the degradation of hemoglobin and stop the generation of FPIX also inhibit CQ uptake. Food vacuole enzymes can generate cell-free binding, using human hemoglobin as a substrate. This binding accounts for CQ uptake into intact cells and is subject to identical inhibitor specificity. Inhibition of CQ uptake by amiloride derivatives occurs because of inhibition of CQ-FPIX binding rather than inhibition of the Na+/H+ exchanger (NHE). Inhibition of parasite NHE using a sodium-free medium does not inhibit CQ uptake nor does it alter the ability of amilorides to inhibit uptake. CQ resistance is characterized by a reduced affinity of CQ-FPIX binding that is reversible by verapamil. Diverse compounds that are known to disrupt lysosomal pH can mimic the verapamil effect. These effects are seen in sodium-free medium and are not due to stimulation of the NHE. We propose that these compounds increase CQ accumulation and overcome CQ resistance by increasing the pH of lysosomes and endosomes, thereby causing an increased affinity of binding of CQ to FPIX.

Chloroquine uptake and activity is determined by binding to ferriprotoporphyrin IX in Plasmodium falciparum.

The selective antimalarial activity of chloroquine and related compounds stems from the extensive saturable uptake of these drugs into malaria parasites. Chloroquine resistant strains of Plasmodium falciparum have evolved a mechanism to reduce the saturable uptake. The molecular mechanism of saturable chloroquine uptake is controversial and attention is currently focused on mutually exclusive models of active chloroquine uptake and intracellular chloroquine binding. We sum up recent evidence which conclusively proves that the saturable accumulation of chloroquine is due to intracellular binding to ferriprotoporphyrin IX rather than active transport into the parasite via the sodium/hydrogen exchanger. We discuss recent findings that the affinity of chloroquine binding to ferriprotoporphyrin IX is reduced in resistant parasites. The mechanism responsible for reduced binding affinity can be overcome by verapamil and various lysosomotropic agents, and is thought to be the basis of chloroquine resistance.

The effect of benomyl on neurite outgrowth in mouse NB2A and human SH-SY5Y neuroblastoma cells in vitro.

The commercial fungicide methyl 1-[(butylamino) carbonyl]-1H-benzimidazol-2-ylcarbamate (benomyl) is teratogenic in rats. Its mode of action is believed to be related to its ability to inhibit the polymerization of brain tubulin. In this study its effects were studied in cultured neuronal cells during differentiation and neurite outgrowth. Mouse NB2a and human SH-SY5Y neuroblastoma cells were induced to differentiate by addition of dibutyryl cyclic AMP and at the same time were exposed to various concentrations of benomyl. Benomyl significantly inhibited neurite outgrowth in both cell lines at concentrations of 10(-8) M and above with IC50 values of 5.9 x 10(-7) M and 1.0 x 10(-6) M in the NB2a and SH-SY5Y cells respectively. The results show that benomyl inhibits neuronal cell differentiation at concentrations likely to be achieved during the development of fetal abnormalities in rats in vivo.