Overcoming P-Glycoprotein-Mediated Doxorubicin Resistance

Intracellular concentration of doxorubicin in target cancer cells is a major determinant of therapeutic success of doxorubicin-based regimens. As known, doxorubicin is a substrate of P-glycoprotein (P-gp), the drug efflux transporter in the ABC superfamily. High expression level of P-gp in cancer cells can prevent intracellular accumulation of doxorubicin up to its effective level, leading to doxorubicin resistance and treatment failure. Moreover, these P-gp-overexpressed cells display multi-drug resistance (MDR) phenotype. Regarding this, application of P-gp modulators (suppressor of P-gp activity and expression) is likely to reverse MDR and restore cell sensitivity to doxorubicin treatment. In searching for potential chemo-sensitizer against resistant cancer, a number of phytochemicals or dietary compounds have been studied extensively for their P-gp modulating effects. Furthermore, combination between doxorubicin and P-gp modulators (e.g., plant-derived compounds, siRNA) given through specific target delivery platforms have been an effective strategic approach for MDR reversal and restore doxorubicin effectiveness for cancer treatment

Assessing the relative contribution of CYP3A-and P-gp-mediated pathways to the overall disposition and drug-drug interaction of dabigatran etexilate using a comprehensive mechanistic physiological-based pharmacokinetic model

Dabigatran etexilate (DABE) is a clinical probe substrate for studying drug-drug interaction (DDI) through an intestinal P-glycoprotein (P-gp). A recent in vitro study, however, has suggested a potentially significant involvement of CYP3A-mediated oxidative metabolism of DABE and its intermediate monoester BIBR0951 in DDI following microdose administration of DABE. In this study, the relative significance of CYP3A- and P-gp-mediated pathways to the overall disposition of DABE has been explored using mechanistic physiologically based pharmacokinetic (PBPK) modeling approach. The developed PBPK model linked DABE with its 2 intermediate (BIBR0951 and BIBR1087) and active (dabigatran, DAB) metabolites, and with all relevant drug-specific properties known to date included. The model was successfully qualified against several datasets of DABE single/multiple dose pharmacokinetics and DDIs with CYP3A/P-gp inhibitors. Simulations using the qualified model supported that the intestinal CYP3A-mediated oxidation of BIBR0951, and not the gut P-gp-mediated efflux of DABE, was a key contributing factor to an observed difference in the DDI magnitude following the micro-versus therapeutic doses of DABE with clarithromycin. Both the saturable CYP3A-mediated metabolism of BIBR0951 and the solubility-limited DABE absorption contributed to the relatively modest nonlinearity in DAB exposure observed with increasing doses of DABE. Furthermore, the results suggested a limited role of the gut P-gp, but an appreciable, albeit small, contribution of gut CYP3A in mediating the DDIs following the therapeutic dose of DABE with dual CYP3A/P-gp inhibitors. Thus, a possibility exists for a varying extent of CYP3A involvement when using DABE as a clinical probe in the DDI assessment, across DABE dose levels

Stereoselective influence of isomalathion on neurotoxicity: Inhibition of acetylcholinesterase and neurotoxic esterase.

Isomalathion contamination of malathion formulations increases cholinergic toxicity and may be involved in sporadic reports of delayed neurotoxicity from malathion. With two asymmetric centers at phosphorus and diethyl thiosuccinyl carbon, isomalathion has four stereoisomers: (1R,3R), (1R,3S), (1S,3R), and (1S,3S), which differ in their inhibitory potencies against acetylcholinesterase (AChE). Comparative studies of the in vitro kinetics of inhibition of AChE and neurotoxic esterase (NTE) were carried out to investigate the stereoselective contributions of either asymmetric center on the inhibitory potencies and mechanism of inhibition of each stereoisomer toward different sources of AChEs (hen brain; rat, human and bovine erythrocyte) and hen brain NTE. Regardless of enzyme source, interdependent steric effects from both asymmetric centers contributed to inhibitory strength. The order of potencies against these brain and erythrocyte AChEs was (1R,3R) $>$ (1R,3S) $>$ (1S,3R) $>$ (1S,3S). Moreover, the postinhibitory kinetic profiles of hen brain and bovine erythrocyte AChEs inhibited by (1R) isomalathions were similar to those of O,S-dimethyl phosphoryl conjugates generated from (S) isoparathion methyl. In contrast, the expected pattern of this similarity in the postinhibitory kinetic profiles between AChEs inhibited by (1S) isomalathions and by (R) isoparathion methyl was not observed, indicating that the (1S) isomalathion-AChE complexes were not O,S-dimethyl phosphoryl conjugates. The results suggest that the mechanism of inhibition of AChE inhibited by (1R) isomalathions involves loss of diethyl thiosuccinyl as the primary leaving group, whereas the mechanism of inhibition by (1S) stereoisomers likely involves loss of thiomethyl. Hence, stereoisomerism in isomalathion does not only affect inhibitory potency, but the mechanism of inhibition as well. This apparent mechanistic shift in AChE inhibition with (1S) isomalathion suggested the possibility that NTE inhibition might occur with these compounds. However, extrapolated I\sb{50} values of the isomalathion against hen brain NTE were 1.2 to 29 mM, indicating that none of the isomers were effective inhibitors of this enzyme. Because the inhibitory potencies against hen brain AChE were 1.5\times 10\sp3 to 1.5 \times 10\sp5 times greater than that for NTE, it is extremely unlikely that delayed neurotoxicity associated with malathion exposure could have arisen from any isomalathion contamination that may have been present.Ph.D.Agricultural chemistryHealth and Environmental SciencesOccupational safetyPure SciencesToxicologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/131237/2/9840566.pd

Alteration of the Heme Prosthetic Group of Neuronal Nitric- Oxide Synthase during Inactivation by N G -Amino-L-arginine in Vitro and in Vivo

ABSTRACT It is established that N G -amino-L-arginine (NAA) is a metabolism-based inactivator of all three major nitric-oxide synthase (NOS) isoforms. The mechanism by which this inactivation occurs, however, is not well understood. In the current study, we discovered that inactivation of the neuronal isoform of NOS (nNOS) by NAA in vitro results in covalent alteration of the heme prosthetic group, in part, to products that contain an intact porphyrin ring and are either dissociable from or irreversibly bound to the protein. The alteration of the heme is concomitant with the loss of nNOS activity. Studies with nNOS containing a 14 C-labeled prosthetic heme moiety indicate that the major dissociable product and the irreversibly bound heme adduct account for 21 and 28%, respectively, of the heme that is altered. Mass spectral analysis of the major dissociable product gave a molecular ion of m/z 775.3 that is consistent with the mass of an adduct of heme and NAA minus a hydrazine group. Peptide mapping of the irreversibly bound heme adduct indicates that the heme is bound to a residue in the oxygenase domain of nNOS. We show for the first time that metabolismbased inactivation of nNOS occurs in vivo as highly similar heme products are formed. Because inactivation and alteration may trigger ubiquitination and proteasomal degradation of nNOS, NAA may be a useful biochemical tool for the study of these basic regulatory processes