Pharmacokinetic study of two acetylcholinesterase reactivators, trimedoxime and newly synthesized oxime K027, in rat plasma

K027 [1‐(4‐hydroxyiminomethylpyridinium)‐3‐(4‐carbamoylpyridinium)–propane dibromide] is a promising new reactivator of organophosphate‐ or organophosphonate‐inhibited acetylcholinesterase (AChE) with low acute toxicity and broad spectrum efficacy. The aim of the present study was to compare the pharmacokinetics of both compounds. Male Wistar rats (body weight = 320 ± 10 g) were administered a single intramuscular dose of K027 (22.07 mg kg^?1) and an equimolar dose of trimedoxime. Blood was collected at various time intervals until 180 min. Plasma samples were analyzed by reversed‐phase HPLC with ultraviolet (UV) detection. The recovery of both oximes from the plasma was approximately 90% and a linear relationship (R² > 0.998) was observed between the peak areas and concentrations of calibrated standards in the range 1–100 µg ml^?1. Near‐identical plasma profiles were obtained for both compounds. No differences were found in the mean ± SD values of C_max (18.6 ± 2.5 vs 20.0 ± 6.3 µg ml^?1, P = 0.72) and AUC_0–180min (2290 ± 304 vs 2269 ± 197 min µg ml^?1, P = 0.84). However, the percentage coefficient of variation of the first‐order rate constant of absorption (k_a) was 3‐fold higher (P < 0.01) providing evidence for more erratic absorption of intramuscular trimedoxime as compared with K027. In conclusion, oxime K027 might have superior pK properties that may be translated in its faster absorption and subsequent tissue distribution. Copyright © 2011 John Wiley & Sons, Ltd.     

In vitro oxime reactivation of red blood cell acetylcholinesterase inhibited by methyl-paraoxon

Oximes are cholinesterase reactivators of use in poisoning with organophosphorus ester enzyme inhibitors. Pralidoxime (PRX) is the oxime used in the United States. Clinical experience with pralidoxime (and other oximes) is disappointing and the routine use has been questioned. Furthermore oximes are not equally effective against all existent enzyme inhibitors. There is a clear demand for 'broad spectrum' cholinesterase reactivators with a higher efficacy than those clinically available. To meet this need over the years new reactivators of cholinesterase of potential clinical utility have been developed.The purpose of the study was to quantify 'in vitro' the extent of protection conferred by available (pralidoxime and methoxime) and experimental (K-27, K-33 and K-48) oximes, using methyl-paraoxon (methyl-POX) as an esterase inhibitor and to compare the results with those previously obtained using paraoxon (POX) as an inhibitor.Red blood cell (RBC) acetylcholinesterase (AChE) activities in whole blood were measured photometrically in the presence of different methyl-POX concentrations and IC(50) values calculated. Determinations were repeated in the presence of increasing oxime concentrations. The IC(50) of methyl-POX (59 nm) increased with the oxime concentration in a linear manner. The calculated IC(50) values were plotted against the oxime concentrations to obtain an IC(50) shift curve. The slope of the shift curve (tg alpha) was used to quantify the magnitude of the protective effect (nm IC(50) increase per microm reactivator).Based on our determinations the new K-series of reactivators is superior to pralidoxime (tg alpha = 1.9) and methoxime (tg alpha = 0.7), K-27 and K-48 being the outstanding compounds with a tg alpha value of 10 (nm IC(50) increase per microm reactivator), which is approximately five times the reactivator ability of PRX. The tg alpha value determined for K-33 was 6.3.The ranking of reactivator potencies of the examined oximes determined with methyl-POX as an inhibitor (K-27 = K-48 > K-33 > pralidoxime > methoxime) is similar to the ranking previously reported by us using POX as an inhibitor (K-27 > or = K-48 > K-33 > methoxime = pralidoxime). There is an (expected) inverse relationship between the binding constant K and the slope of the IC(50) shift curve (tg alpha) for all oximes examined. K-27 and K-48 (the most protective substances judging by the tg alpha) having the lowest K value (highest affinity).In vivo testing of the new oximes as methyl-paraoxon protective agents is necessary.     

In vitro oxime protection of human red blood cell acetylcholinesterase inhibited by diisopropyl-fluorophosphate

Oximes are enzyme reactivators used in treating poisoning with organophosphorus cholinesterase (AChE) inhibitors. The oxime dose which can be safely administered is limited by the intrinsic toxicity of the substances such as their own AChE-inhibiting tendency. Clinical experience with the available oximes is disappointing. To meet this need, new AChE reactivators of potential clinical utility have been developed. The purpose of the study was to estimate in vitro both the intrinsic toxicity and the extent of possible protection conferred by established (pralidoxime, obidoxime, HI-6, methoxime, trimedoxime) and experimental (K-type) oximes, using diisopropyl-fluoro-phosphate (DFP) as an AChE inhibitor. The IC50 of DFP against human red blood cell AChE was determined ( approximately 120 nm). Measurements were then repeated in the presence of increasing oxime concentrations, leading to an apparent increase in DFP IC50. Calculated IC50 values were plotted against oxime concentrations to obtain an IC50 shift curve. The slope of this shift curve (tan alpha) was used to quantify the magnitude of the protective effect (nm IC50 increase per microm oxime). We show that, in the case of a linear relationship between oxime concentration and IC50, the binding constant K, determined using the Schild equation, equals IC50/DFP/tan alpha. Based on the values of tan alpha and of the binding constant K, some of the new K-oxime reactivators are far superior to pralidoxime (tan alpha = 0.8), obidoxime (1.5), HI-6 (0.8), trimedoxime (2.9) and methoxime (5.9), with K-107 (17), K-108 (20), and K-113 (16) being the outstanding compounds.     

In vitro toxicology studies of extracellular vesicles

Extracellular vesicles (EVs) are membrane‐bound vesicles released from cells into the extracellular environment. There is emerging interest in the use of EVs as potential therapeutic interventions. We sought to evaluate the safety of EVs that may be therapeutically used by performing in vitro toxicological assessments. EVs were obtained from mesenchymal stem cells (MSC‐EV) or from bovine milk (BM‐EV) by differential ultracentrifugation, and quantitated using nanoparticle tracking analysis. Genotoxic effects, hematological effects, immunological effects and endotoxin production were evaluated at two dose levels. Neither MSC‐EVs nor BM‐EVs elicited detectable genotoxic effects using either the alkaline comet assay or micronucleus assay. Hemolysis was observed with BM‐EVs but not with MSC‐EVs. MSC‐EVs did not have any significant effect on either spontaneous or collagen‐induced platelet aggregation. In contrast, BM‐EVs were noted to increase collagen‐induced platelet aggregation, even though no spontaneous increase in platelet aggregation was noted. Both types of EVs induced leukocyte proliferation, which was greater with BM‐EV. Neither MSC‐EVs nor BM‐EVs induced HL‐60 phagocytosis, although BM‐EVs decreased zymosan‐induced phagocytosis. Furthermore, neither MSC‐EVs nor BM‐EVs induced nitric oxide production. Unlike MSC‐EVs, BM‐EVs tested positive for endotoxin and induced complement activation. There are significant differences in toxicological profiles between MSC‐EVs and BM‐EVs that may reflect variations in techniques for EV isolation, EV content or cross‐species differences. The safety of MSC‐EV supports their use for disease therapeutics, whereas detailed safety and toxicological assessment will be necessary before the use of BM‐EVs. Copyright © 2016 John Wiley & Sons, Ltd.     

红霉素A肟的新合成方法

目的 用一种全新的方法合成红霉素A肟(1)。方法 在酸碱缓冲溶液中由红霉素A(3)与盐酸羟胺缩合生成1。系统研究了反应体系的pH值、反应温度以及盐酸羟胺的加入量对反应结果的影响。同时对红霉素A肟化反应机理进行了推导。结果 红霉素A9—肟的收率达95％以上，产品纯度达92％。化合物1经红外、氢谱、碳谱、质谱鉴定，与文献报道相一致。结论 该方法有效地抑制了红霉素A的酸性分解，与文献方法相比，大幅度提高了反应的收率和产品的纯度。     

Application of a molecular networking approach for clinical and forensic toxicology exemplified in three cases involving 3‐MeO‐PCP,doxylamine, and chlormequat

Untargeted toxicological screening is an analytical challenge, given the high number of molecules and metabolites to be detected and the constant appearance of new psychoactive substances (NPS). The combination of liquid chromatography with high‐resolution tandem mass spectrometry (HRMS/MS) in a data‐dependent acquisition mode generates a large volume of high quality spectral data. Commercial software for processing MS data acquired during untargeted screening experiments usually compare measured features (mass, retention time, and fragmentation spectra) against a predefined list of analytes. However, there is a lack of tools for visualizing and organizing MS data of unknown compounds. Here, we applied molecular networking to untargeted toxicological screening. This bioinformatic tool allows the exploration and organization of MS/MS data without prior knowledge of the sample's chemical composition. The organization of spectral data is based on spectral similarity. Hence, important information can be obtained even before the annotation step. The link established between molecules enables the propagation of structural information. We applied this approach to three clinical and forensic cases with various matrices: (a) blood and a syringe content in a forensic case of death by self‐injection, (b) hair segments in a case of drug‐facilitated assault, and (c) urine and blood samples in a case of 3‐methoxyphencyclidine intoxication. Data preprocessing with MZmine allows sample‐to‐sample comparison and generation of multisample molecular networks. Our present study shows that molecular networking can be a useful complement to conventional approaches for untargeted screening interpretation, for example for xenobiotics identification or NPS metabolism elucidation.     

In vitro P-glycoprotein activity does not completely explain in vivo efficacy of novel centrally effective oxime acetylcholinesterase reactivators

Novel-substituted phenoxyalkyl pyridinium oxime acetylcholinesterase (AChE) reactivators (US patent 9,227,937) that showed convincing evidence of penetration into the brains of intact rats were developed by our laboratories. The oximes separated into three groups based on their levels of brain AChE reactivation following exposure of rats to the sarin surrogate nitrophenyl isopropyl methylphosphonate (NIMP). P-glycoprotein (P-gp) is a major blood–brain barrier (BBB) transporter and requires ATP for efflux. To determine if P-gp affinity screening could be used to reduce animal use, we measured in vitro oxime-stimulated ATPase activity to see if the in vivo reactivation efficacies related to the oximes’ functions as P-gp substrates. High efficacy oximes were expected to be poor P-gp substrates, thus remaining in the brain longer. The high efficacy oximes (24–35% brain AChE reactivation) were worse P-gp substrates than the low efficacy oximes (0–7% brain AChE reactivation). However, the oxime group with medium in vivo reactivation of 10–17% were even worse P-gp substrates than the high efficacy group so their reactivation ability was not reflected by P-gp export. The results suggest that in vitro P-gp ATPase activity can remove the low efficacy oximes from in vivo testing, but is not sufficient to differentiate between the top two tiers.     

高内涵分析在新药发现毒理学中的应用进展

在新药发现早期开展发现毒理学研究是提高新药研发效率的重要策略之一。高内涵分析(HCA)是基于高效新药筛选需求发展起来的一项新技术,其主要特点是基于活细胞、多参数、实时、高通量,能够实现化合物多种生物活性、毒性的早期、快速地检测,为发现毒理学研究提供了高效的技术手段。目前,HCA已用于多种靶器官细胞毒性、遗传毒性、神经毒性、血管毒性、生殖毒性等检测以及毒理学分子机制的研究,本文就HCA在新药发现毒理学方面的应用进展进行综述。     

Caco-2细胞模型在药物体外研究中的应用

Caco-2细胞模型是一种筛选药物离体口服特性的模型,已广泛用于评价药物在小肠的吸收特性和各种转运机制的研究.现综述Caco-2细胞单层模型的基本特点及其在药物的小肠吸收、代谢以及高通量筛选等方面的应用.     

Quantitative in vitro to in vivo extrapolation of cell-based toxicity assay results

The field of toxicology is currently undergoing a global paradigm shift to use of in vitro approaches for assessing the risks of chemicals and drugs in a more mechanistic and high throughput manner than current approaches relying primarily on in vivo testing. However, reliance on in vitro data entails a number of new challenges associated with translating the in vitro results to corresponding in vivo exposures. Physiologically based pharmacokinetic (PBPK) modeling provides an effective framework for conducting quantitative in vitro to in vivo extrapolation (QIVIVE). Their physiological structure facilitates the incorporation of in silico- and in vitro-derived chemical-specific parameters in order to predict in vivo absorption, distribution, metabolism and excretion. In particular, the combination of in silico- and in vitro parameter estimation with PBPK modeling can be used to predict the in vivo exposure conditions that would produce chemical concentrations in the target tissue equivalent to the concentrations at which effects were observed with in vitro assays of tissue/organ toxicity. This review describes the various elements of QIVIVE and highlights key aspects of the process, with an emphasis on extrapolation of in vitro metabolism data to predict in vivo clearance as the key element. Other important elements include characterization of free concentration in the toxicity assay and potential complications associated with intestinal absorption and renal clearance. Examples of successful QIVIVE approaches are described ranging from a simple steady-state approach that is suitable for a high throughput environment to more complicated approaches requiring full PBPK models.     

Maternal exposure causes mitochondrial dysfunction in brain,liver, and heart of mouse fetus: An explanation for perfluorooctanoic acid induced abortion and developmental toxicity

Perfluorooctanoic acid (PFOA) is an octanoic acid and is found in wildlife and humans. We have investigated mitochondrial toxicity in isolated mitochondria from, placenta, brain, liver, and heart after oral exposure with PFOA in mice during gestational days (7‐15). Histopathological examination and mitochondrial toxicity parameters were assayed. Results indicated that PFOA decreased the weight of the fetus and placenta, the length of the fetus and the diameter of the placenta, dead fetuses and dead macerated fetuses in treated mice with 25 mg/kg. Histopathological examination showed that PFOA induced pathological abnormalities in liver, brain, heart, and placenta. Also, PFOA induced mitochondria toxicity in brain, liver, heart of mouse fetus. Our results indicate that PFOA up to 20 mg/kg exposure adversely affect embryofetal/developmental because for mitochondria dysfunction. These results suggested that mitochondrial dysfunction induced by PFOA in liver, heart, and brain lead to developmental toxicity and abnormality in tissues.     

A high throughput screening approach to identify isocitrate lyase inhibitors from traditional Chinese medicine sources

Isocitrate lyase (ICL) catalyses the first step of the glyoxylate bypass pathway, which reversibly cleaves isocitrate into succinate and glyoxylate. This pathway occurs in a wide range of pathogens and plays a key role in the pathogenesis of Mycobacterium tuberculosis (MTB) suggesting that it may represent a drug target for the treatment of tuberculosis. ICL was cloned, expressed, and purified, and a high‐throughput screen (HTS) developed to screen active extracts derived from traditional Chinese medicines (TCMs) for inhibition of ICL. A colorimetric assay based on the formation of glyoxylate‐phenylhydrazone was used to measure ICL activity. The assay had signal to noise (S/N) of 12.74 and Z′ factor of 0.72, indicating that the assay was suitable for HTS. Screening of a collection of 465 extracts derived from TCMs resulted in the identification of two extracts from Illicium verum Hook.f (Illiciaceae, XHD‐1) and Zingiber officinale Rosc (Zingiberaceae, XHD‐2), which inhibited ICL with IC₅₀ values of 47.7 ± 16.9 and 18.2 ± 0.9 µg/ml, respectively. Drug Dev. Res. 67:818–823, 2006. © 2007 Wiley‐Liss, Inc.     

A framework for in vitro systems toxicology assessment of e-liquids

Various electronic nicotine delivery systems (ENDS), of which electronic cigarettes (e-cigs) are the most recognized prototype, have been quickly gaining ground on conventional cigarettes because they are perceived as less harmful. Research assessing the potential effects of ENDS exposure in humans is currently limited and inconclusive. New products are emerging with numerous variations in designs and performance parameters within and across brands. Acknowledging these challenges, we present here a proposed framework for an in vitro systems toxicology assessment of e-liquids and their aerosols, intended to complement the battery of assays for standard toxicity assessments. The proposed framework utilizes high-throughput toxicity assessments of e-liquids and their aerosols, in which the device-to-device variability is minimized, and a systems-level investigation of the cellular mechanisms of toxicity is an integral part. An analytical chemistry investigation is also included as a part of the framework to provide accurate and reliable chemistry data solidifying the toxicological assessment. In its simplest form, the framework comprises of three main layers: (1) high-throughput toxicity screening of e-liquids using primary human cell culture systems; (2) toxicity-related mechanistic assessment of selected e-liquids, and (3) toxicity-related mechanistic assessment of their aerosols using organotypic air–liquid interface airway culture systems. A systems toxicology assessment approach is leveraged to enable in-depth analyses of the toxicity-related cellular mechanisms of e-liquids and their aerosols. We present example use cases to demonstrate the suitability of the framework for a robust in vitro assessment of e-liquids and their aerosols.     

A metabonomic approach for mechanistic exploration of pre-clinical toxicology

Metabonomics involves the application of advanced analytical tools to profile the diverse metabolic complement of a given biofluid or tissue. Subsequent statistical modelling of the complex multivariate spectral profiles enables discrimination between phenotypes of interest and identifies panels of discriminatory metabolites that represent candidate biomarkers. This review article presents an overview of recent developments in the field of metabonomics with a focus on application to pre-clinical toxicology studies. Recent research investigations carried out as part of the international COMET 2 consortium project on the hepatotoxic action of the aminosugar, galactosamine (galN) are presented. The application of advanced, high-field NMR spectroscopy is demonstrated, together with complementary application of a targeted mass spectrometry platform coupled with ultra-performance liquid chromatography. Much novel mechanistic information has been gleaned on both the mechanism of galN hepatotoxicity in multiple biofluids and tissues, and on the protection afforded by co-administration of glycine and uridine. The simultaneous identification of both the metabolic fate of galN and its associated endogenous consequences in spectral profiles is demonstrated. Furthermore, metabonomic assessment of inter-animal variability in response to galN presents enhanced mechanistic insight on variable response phentoypes and is relevant to understanding wider aspects of individual variability in drug response. This exemplar highlights the analytical and statistical tools commonly applied in metabonomic studies and notably, the approach is applicable to the study of any toxin/drug or intervention of interest. The metabonomic approach holds considerable promise and potential to significantly advance our understanding of the mechanistic bases for adverse drug reactions.     

Autophagic and apoptotic mechanisms of curcumin-induced death in K562 cells

Curcumin (1), a natural polyphenolic compound, has shown strong antioxidant and anticancer activities. Several molecular mechanisms have been attributed to its inhibitory effects on a wide range of tumor cells. In this study, the response of the chronic myeloid leukemia cell line K562 cells to 1 is investigated. Curcumin inhibited the viability of K562 cells in a dose- and time-dependent manner. Furthermore, curcumin-induced cell death was associated with the formation of the apoptosome complex, the collapse of the mitochondrial membrane potential, and caspase-3 activation. Curcumin treatment also induced Bid cleavage and downregulated the expression of Bcl-2 protein. Surprisingly, even with these molecular features of apoptosis, we showed that 1 stimulated autophagy, which was evidenced by microtubule-associated protein light chain 3 (LC3) immunoreactivty. Curcumin also increased the protein levels of beclin 1 and membrane form LC3 (LC3-II). Autophagy inhibitor bafilomycin A1 and the pan-caspase inhibitor Z-VAD-fmk suppressed curcumin-induced K562 cell death. Overall, these results suggest that curcumin induces autophagic and apoptotic death of K562 cells. These findings suggest that both apoptotic and autophagic mechanisms contribute to the curcumin-induced K562 cell death.     

Cytoprotective effects of adenosine and inosine in an in vitro model of acute tubular necrosis

Background and purpose:

We have established an in vitro model of acute tubular necrosis in rat kidney tubular cells, using combined oxygen-glucose deprivation (COGD) and screened a library of 1280 pharmacologically active compounds for cytoprotective effects.

Experimental approach:

We used in vitro cell-based, high throughput, screening, with cells subjected to COGD using hypoxia chambers, followed by re-oxygenation. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the Alamar Blue assay measured mitochondrial respiration and the lactate dehydrogenase assay was used to indicate cell death. ATP levels were measured using a luminometric assay.

Key results:

Adenosine markedly reduced cellular injury, with maximal cytoprotective effect at 100 µM and an EC₅₀ value of 14 µM. Inosine was also found to be cytoprotective. The selective A₃ adenosine receptor antagonist MRS 1523 attenuated the protective effects of adenosine and inosine, while an A₃ adenosine receptor agonist provided a partial protective effect. Adenosine deaminase inhibition attenuated the cytoprotective effect of adenosine but not of inosine during COGD. Inhibition of adenosine kinase reduced the protective effects of both adenosine and inosine during COGD. Pretreatment of the cells with adenosine or inosine markedly protected against the fall in cellular ATP content in the cells subjected to COGD.

Conclusions and implications:

The cytoprotection elicited by adenosine and inosine in a model of renal ischaemia involved both interactions with cell surface adenosine receptors on renal tubular epithelial cells and intracellular metabolism and conversion of adenosine to ATP.     

新药研发过程中毒理学研究方法的进展--毒性的原因和早期毒性筛选

毒性已成为新药开发过程中淘汰的主要原因。近年来制药和生物技术工业的研究人员开发了多种新技术以便在药物发现和开发过程中尽早确定化合物的安全特性。笔者首先对药物毒性产生的原因如药靶的特异性、药物的分子结构、药物代谢和代谢动力学等方面进行了分析，再介绍了近几年早期毒性筛选的新技术，包括预测模型、体外高通量毒性筛选、活性代谢产物的检测、高内涵筛选技术、动物实验。