Validation of membrane vesicle-based breast cancer resistance protein and multidrug resistance protein 2 assays to assess drug transport and the potential for drug–drug interaction to support regulatory submissions

1. Breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) can play a role in the absorption, distribution, metabolism, and excretion of drugs, impacting on the potential for drug–drug interactions. This study has characterized insect cell– and mammalian cell–derived ABC-transporter–expressing membrane vesicle test systems and validated methodologies for evaluation of candidate drugs as substrates or inhibitors of BCRP or MRP2.

2. Concentration-dependent uptake of BCRP ([³H]oestrone 3-sulfate, [³H]methotrexate, [³H]rosuvastatin) and MRP2 ([³H]oestradiol 17β-glucuronide, [³H]pravastatin, carboxydichlorofluorescein) substrates, and inhibitory potencies (IC₅₀) of BCRP (sulfasalazine, novobiocin, fumitremorgin C) and MRP2 (benzbromarone, MK-571, terfenadine) inhibitors were determined.

3. The apparent K_m for probes [³H]oestrone 3-sulfate and [³H]oestradiol 17β-glucuronide was determined in insect cell vesicles to be 7.4?±?1.7 and 105?±?8.3?µM, respectively. All other substrates exhibited significant uptake ratios. Positive control inhibitors sulfasalazine and benzbromarone gave IC₅₀ values of 0.74?±?0.18 and 36?±?6.1 µM, respectively. All other inhibitors exhibited concentration-dependent inhibition. There was no significant difference in parameters generated between test systems.

4. On the basis of the validation results, acceptance criteria to identify substrates/inhibitors of BCRP and MRP2 were determined for insect cell vesicles. The approach builds on earlier validations to support drug registration and extends from those cell-based systems to encompass assay formats using membrane vesicles.

     

(±)-3,4-methylenedioxymethamphetamine and metabolite disposition in plasma and striatum of wild-type and multidrug resistance protein 1a knock-out mice

Mice lacking multidrug resistance protein 1a (mdr1a) are protected from methylenedioxymethamphetamine (MDMA)-induced neurotoxicity, suggesting mdr1a might play an important role in this phenomenon. We characterized MDMA pharmacokinetics in murine plasma and brain to determine if mdr1a alters MDMA distribution. Wild-type (mdr1a?/?) and mdr1a knock-out (mdr1a?/?) mice received i.p. 10, 20 or 40 mg/kg MDMA. Plasma and brain specimens were collected 0.3-4 h after MDMA, and striatum were dissected. MDMA and metabolites were quantified in plasma and striatum by gas chromatography-mass spectrometry. MDMA maximum plasma concentrations (C(max)) for both strains were 916- 1363, 1833-3546, and 5979-7948 μg/L, whereas brain C(max) were 6673-14,869, 23,428-29,433, and 52,735-66,525 μg/kg after 10, 20, or 40 mg/kg MDMA, respectively. MDMA and metabolite striatum/plasma AUC ratios were similar in both strains, inconsistent with observed MDMA neuroprotective effects in mdr1a?/? mice. Ratios of methylenedioxyamphetamine (MDA) and 4-hydroxy-3-methoxymethamphetamine (HMMA) AUCs exceeded 18% of MDMA's in plasma, suggesting substantial MDMA hepatic metabolism in mice. MDMA, MDA, HMMA, and 4-hydroxy-3-methoxyamphetamine maximum concentrations and AUCs exhibited nonlinear relationships during dose-escalation studies, consistent with impaired enzymatic demethylenation. Nonlinear increases in MDMA plasma and brain concentrations with increased MDMA dose may potentiate MDMA effects and toxicity.     

Mutation and evolution of antibiotic resistance: antibiotics as promoters of antibiotic resistance?

Antibiotic resistance appearance and spread have been classically considered the result of a process of natural selection, directed by the use of antibiotics. Bacteria, that have to face the antibiotic challenge, evolve to acquire resistance and, under this strong selective pressure, only the fittest survive, leading to the spread of resistance mechanisms and resistant clones. Horizontal transference of resistance mechanisms seems to be the main way of antibiotic resistance acquisition. Nevertheless, recent findings on hypermutability and antibiotic-induced hypermutation in bacteria have modified the landscape. Here, we present a review of the last data on molecular mechanisms of hypermutability in bacteria and their relationship with the acquisition of antibiotic resistance. Finally, we discuss the possibility that antibiotics may act not only as selectors for antibiotic resistant bacteria but also as resistance promoters.     

Anti-platelet therapy and aspirin resistance - clinically and chemically relevant?

Platelets play a central role in the pathogenesis of the atherothrombosis which ultimately causes myocardial infarction, stroke and peripheral vascular disease. Commonly used oral anti-platelet drugs include aspirin (an irreversible inhibitor of cyclo-oxygenase), clopidogrel (an ADP receptor antagonist), other thienopyridines such as ticlopidine and prasgruel, and dipyridamole (an inhibitor of adenosine reuptake and platelet phosphodiesterase). Newer agents are in development and one, ticagrelor, a reversible ADP receptor antagonist has shown promise. Despite their proven benefit, recurrent vascular events still occur in those taking anti-platelet drugs. This has led to the concept of anti-platelet resistance, most commonly aspirin resistance as this drug is the cornerstone of most regimens. The causes of aspirin resistance are numerous but potential mechanisms include lack of patient adherence, non COX-1 mediated thromboxane A2 synthesis, increased activity of alternate platelet activation pathways, interference of aspirin action by other drugs and probably pharmacogenetic factors. Measurement of platelet response to aspirin is made possible using a number of in-vitro laboratory assays of platelet function which include measurement of thromboxane A2 metabolites as well as newer point-of-care assays of platelet aggregation. The phenomenon of aspirin resistance is important as it raises the possibility of developing strategies to identify those who respond best to a particular anti-platelet regimen, or to development of newer anti-platelet therapies to which more patients respond. This review discusses important aspects of aspirin resistance both in terms of clinical medicine, alternative anti-platelet strategies, and the potential to overcome its various causes.     

β-Adrenoceptors and resistance to stress: old problems and new possibilities

The commentators suggest that our approach may be too limited to account for many of the factors which influence stress-related behaviour, yet too general to help us interpret the neurochemical coding of behavioural responses to stress. In our reply we argue that the strategy described in our Critique both confronts the issues discussed in the Commentaries and is more objective and rigorous than approaches used currently.     

What is the precise role of human MDR 1 protein in chemotherapeutic drug resistance?

Elucidating the molecular function of hu MDR 1 protein (also called P-glycoprotein or P-gp 1) and the precise role this protein plays in clinically relevant tumor drug resistance remains a perplexing problem. Hundreds of reports over the past decades summarize a dizzying array of observations relevant to hu MDR 1 protein function. A dominant model in the MDR literature that is used to explain many observations is the well known "drug pump" model first suggested by Keld Dano in 1973 [1]. Although this model has proved useful in conceptualizing additional experiments, it violates fundamental laws of biology and chemistry and in well over a decade of intense effort, active outward drug pumping via hu MDR 1 protein has still never been unequivocally measured. Also, in recent years it has become clear that the drug pump model cannot explain several important phenomena that are highly relevant to the cancer clinic. Thus, other models have also proved increasingly popular. One is the altered partitioning model, which does not violate fundamental laws, is consistent with the vast majority of available data, and has important predictive ability. This newer model has several novel facets that are relevant for cancer pharmacology, and that help explain phenomena not explained by the drug pump model. The basic principle of this model is that MDR proteins do not directly transport drugs, but that their altered expression leads to altered regulation of ion transport or signal transduction that is critical for setting key biophysical parameters of the cell (e.g. compartmental pH and membrane potentials) that dictate relative passive diffusion of drugs as well as important signal transduction linked to the cytotoxic actions of these drugs. Along with debate over the molecular details of hu MDR 1 function, additional controversy surrounds the precise role of hu MDR 1 in the clinic. Many investigators now debate the significance of its function (regardless of precise mechanism) with regard to "real" drug resistance phenotypes exhibited in the clinic. I believe that thorough debate on the pros and cons of various molecular models for hu MDR 1 function will help to address confusion over the clinical relevance of hu MDR1. In the current atmosphere of disappointment over the relative success of clinical trials based in large part on the logic of the drug pump model, it is important that we not lose sight of critical points. Namely, hu MDR 1 protein remains an extremely important window in on the complex pathways that lead to induced chemotherapeutic drug resistance. Exploring the rationale behind newer models for hu MDR 1 function leads to key predictions that can be tested.     

Cryptotanshinone switches target from estrogen receptor α to breast cancer resistance protein(BCRP/ABCG2) to reverse multidrug resistance in breast cancer

OBJECTIVE Targeting individual molecule is not only difficult to cure multigenic cancers, but also prone to inducing resistance. The so-cal ed multidrug resistance(MDR) in breast cancer is highly associated with estrogen receptor α induction and the overexpression of breast cancer resistance protein(BCRP) transporter.Thus, one of the strategies to overcome MDR is to inhibit the expression of the transporter by small molecule inhibitors. METHODS Western blotting and RT-PCR were used respectively for quantification of protein expression and m RNA, non-reducing gradientgel electrophoresis and fluorescence resonance energy transfer(FRET) microscopy imaging for determination of BCRP dimer. RESULTS Cryptotanshinone(CPT) a natural anti-cancer compound,was found to bind BCRP and inhibit its membrane dimerization to attenuate its transport function. And this process is dependent on estrogen receptor α(ERα) in breast cancer. Furthermore, the resistant breast cancer cells with high BCRP expression are also sensitive to CPT followed with the inhibition of membrane dimer of BCRP although they are ERα-negative, suggesting that BCRP expression is essential to CPT reversing the resistance.Meanwhile, the combination of CPT and chemotherapy drugs could obviously enhance the chemotherapeutic effect in vitro. CONCLUSION Totally, we thought that CPT is a novel natural BCRP inhibitor via blocking the formation of BCRP membrane dimer. At the presence of ERα, it functions via ERα regulation, but directly propels BCRP at the absence of ER in resistant cells. CPT can target multi molecules and switch the target from ERα to BCRP in ERα-negative breast cancer, contributing to decrease the occurrence of resistance and reverse multidrug resistance in breast cancer.     

Inhibition of protein–protein interaction

Tracts of repeated glutamine regions can cause aggregation of the proteins that contain them, causing misfolding, loss of function and ultimately a disease state, both from the loss of function and from the plaques and tangle that result from the protein precipitates. The authors propose the use of oligopeptides to prevent the formation of such aggregates.     

Effect of quercetin on activities of protein kinase C and tyrosine protein kinase from HL-60 cells

目的：研究槲皮素（Ｑｕｅ）对ＨＬ６０细胞中细胞溶质和胞膜蛋白激酶Ｃ（ＰＫＣ）、酪氨酸蛋白激酶（ＴＰＫ）活性的影响．方法：活细胞数的计数用苔盼兰拒染法；用组蛋白ⅢＳ、［γ３２Ｐ］ＡＴＰ与ＰＫＣ酶液一起保温测定ＰＫＣ活性；用聚谷氨酸·酪氨酸（４∶１）多肽）、［γ３２Ｐ］ＡＴＰ与ＴＰＫ酶液一起保温测定ＴＰＫ活性．结果：Ｑｕｅ对ＨＬ６０细胞的增殖有抑制作用，呈剂量依赖关系，处理４８小时后，其ＩＣ５０为２９（２２－３７）μｍｏｌ·Ｌ－１；在体外，Ｑｕｅ能强烈抑制细胞溶质ＰＫＣ和胞膜ＴＰＫ活性，其ＩＣ５０分别为：３１（２０－４８）μｍｏｌ·Ｌ－１，２４（１３－４５）μｍｏｌ·Ｌ－１，但不影响胞膜ＰＫＣ和细胞溶质ＴＰＫ活性．结论：这为Ｑｕｅ对癌细胞的生长有抑制作用与其抑制ＰＫＣ和／或ＴＰＫ有关提供了直接证据．     

Is there a relationship between insulin resistance and frailty syndrome?

Due to the fact that the percentage of aged subjects in the populations of industrialized countries is dramatically increasing, the scientific community has been obligated to focus their attention on age related disease states and peculiar consequences of aging such as, frailty. Frailty is defined as a syndrome of decreased reserve and resistance to stressors and is clinically expressed as muscle weakness, poor exercise tolerance, factors related to body composition, sarcopenia, and lower extremity mobility. Some biochemical markers of frailty in older persons, including pro-inflammatory markers, hormones and free radicals have been suggested. However, there is growing evidence that a rise in insulin resistance [IR] occurs as individuals age and IR is not only considered a simple metabolic finding, but has been identified as a major risk factor for many age-related diseases due to altered lipid metabolism, increased inflammatory state, impaired endothelial functioning, pro-thrombotic status and atherosclerosis. Considering that IR is related to many of the clinical features of frailty such as, skeletal muscle weakness, lower extremity mobility disability, cognitive decline and body composition changes, we will analyze the relationships among IR and such individual components while highlighting potential pathophysiologic mechanisms of IR on the activation of the downward spiral of the frailty syndrome in older persons. In particular, we will address the issue that IR may also be considered a pivotal biological component of some clinical aspects of the frailty syndrome in aging individuals.     

Immune suppressive protein of stress (ISPS) and psoriasis

To find out the relationship between ISPS and psoriasis, radioimmunoassay (RIA) was used to determine the effect of sera from 49 psoriatic patients and 34 healthy controls on lymphocyte proliferation of normal mouse lymphocytes.The lymphocyte proliferation of normal mouse lymphocytes incubated with the sera of psoriatic patients was 41.55 %, and that of controls was 68.09 %.The suppressive effect of sere in patients during the progressive stage (more extensive lesions) was stronger than that     

Disruption of nNOS-PSD95 protein–protein interaction inhibits acute thermal hyperalgesia and chronic mechanical allodynia in rodents

Background and purpose:

Post-synaptic density protein 95 (PSD95) contains three PSD95/Dosophilia disc large/ZO-1 homology domains and links neuronal nitric oxide synthase (nNOS) with the N-methyl-D-aspartic acid (NMDA) receptor. This report assesses the effects of disruption of the protein–protein interaction between nNOS and PSD95 on pain sensitivity in rodent models of hyperalgesia and neuropathic pain.

Experimental approach:

We generated two molecules that interfered with the nNOS–PSD95 interaction: IC87201, a small molecule inhibitor; and tat-nNOS (residues 1–299), a cell permeable fusion protein containing the PSD95 binding domain of nNOS. We then characterized these inhibitors using in vitro and in vivo models of acute hyperalgesia and chronic allodynia, both of which are thought to require nNOS activation.

Key results:

IC87201 and tat-nNOS (1–299) inhibited the in vitro binding of nNOS with PSD95, without inhibiting nNOS catalytic activity. Both inhibitors also blocked NMDA-induced 3′,5′-cyclic guanosine monophosphate (cGMP) production in primary hippocampal cultures. Intrathecal administration of either inhibitor potently reversed NMDA-induced thermal hyperalgesia in mice. At anti-hyperalgesic doses, there was no effect on acute pain thresholds or motor coordination. Intrathecal administration of IC87201 and tat-nNOS also reversed mechanical allodynia induced by chronic constriction of the sciatic nerve.

Conclusions and implications:

nNOS–PSD95 interaction is important in maintaining hypersensitivity in acute and chronic pain. Disruption of the nNOS–PSD95 interaction provides a novel approach to obtain selective anti-hyperalgesic compounds.