Quantitative assessment of cardiomyocyte mechanobiology through high-throughput cantilever-based functional well plate systems.

Proper regulation of the in vitro cell culture environment is essential for disease modelling and drug toxicity screening. The main limitation of well plates used for cell culture is that they cannot accurately maintain energy sources and compounds needed during cell growth. Herein, to understand the importance of perfusion in cardiomyocyte culture, changes in contractile force and heart rate during cardiomyocyte growth are systematically investigated, and the results are compared with those of a perfusion-free system. The proposed perfusion system consists of a Peltier refrigerator, a peristaltic pump, and a functional well plate. A functional well plate with 12 wells is made through injection moulding, with two tubes integrated in the cover for each well to continuously circulate the culture medium. The contractile force of cardiomyocytes growing on the cantilever surface is analysed through changes in cantilever displacement. The maturation of cardiomyocytes is evaluated through fluorescence staining and western blot; cardiomyocytes cultured in the perfusion system show greater maturity than those cultured in a manually replaced culture medium. The pH of the culture medium manually replaced at intervals of 3 days decreases to 6.8, resulting in an abnormal heartbeat, while cardiomyocytes cultured in the perfusion system maintained at pH 7.4 show improved contractility and a uniform heart rate. Two well-known ion channel blockers, verapamil and quinidine, are used to measure changes in the contractile force of cardiomyocytes from the two systems. Cardiomyocytes in the perfusion system show greater stability during drug toxicity screening, proving that the perfusion system provides a better environment for cell growth.

Hibiscus-cisplatin combination treatment decreases liver toxicity in rats while increasing toxicity in lung cancer cells via oxidative stress- apoptosis pathway.

Cisplatin (CIS) is a broad-spectrum anti-carcinogen that causes cytotoxic effects both in normal and cancer cells. The purpose of this study was to test whether Hibiscus sabdariffa (HS) extract can reduce CIS-induced hepatotoxicity in rodents and to assess its anticancer activity in vitro. Treatment with HS extract at daily doses of 500 mg/kg before and after a single dose of CIS (10 mg/kg) reduced hepatotoxicity in Wistar male albino rats. HS extract reduced activity of hepatic damage marker enzymes ( i.e. alanine and aspartate aminotransferases), necrosis, and apoptosis in liver tissues of CIS-treated rats. This hepatic protection was associated with reduced oxidative stress in liver tissues. The antioxidant effects of HS were manifested as a normalization of malondialdehyde levels and glutathione levels which were all raised after CIS-induction. In addition, HS treatment resulted in a decrease of catalase, and superoxide dismutase activity. The combined effects of CIS and HS were also studied in two human lung cancer cell lines (A549 and H460). Treatment with HS (20 µg /mL) enhanced the cytotoxic activity of CIS both in A549 and H460 cell lines. Interestingly, HS increased CIS-induced apoptosis and oxidative stress more clearly in A549 cells indicating that HS extract in combination with CIS could increase the efficacy of CIS in the treatment of cancer.

In Vitro/In Vivo Hepatoprotective and Antioxidant Effects of Defatted Extract and a Phenolic Fraction Obtained from Phlomis Tuberosa.

An in vitro/in vivo hepatotoxicity and hepatoprotection evaluation of a defatted extract and a phenolic fraction from Phlomis tuberosa, administered alone and in a carbon tetrachloride (CCl4)-induced metabolic bioactivation model, was performed. The extract and the phenolic fraction were analysed by high performance liquid chromatography (HPLC) to determine the total flavonoid content, to identify flavonoids and to quantify verbascoside. In addition, total polyphenolics in the samples were expressed as gallic acid equivalents. Applied alone, the extract and the fraction (5, 10 and 50 µg/mL) did not show a statistically significant hepatotoxic effect on isolated rat hepatocytes in vitro. In a CCl4-induced hepatotoxicity model, the samples exhibited a concentration-dependent, statistically significant hepatoprotective effect, which was most pronounced at 50 µg/mL for both. The phenolic fraction exhibited a more pronounced hepatoprotective effect compared to the extract. Data from the in vitro study on the effects of the extract were also confirmed in the in vivo experiment conducted in a CCl4-induced hepatotoxicity model in rats. A histopathological study showed that the animals treated with CCl4 and the extract had an unaltered histoarchitecture of the liver. The effects of the extract were the same as those of silymarin.

Ferulic acid prevents Diosbulbin B-induced liver injury by inhibiting covalent modifications on proteins.

Diosbulbin B (DIOB) has been reported to cause serious liver injury. However, in traditional medicine, DIOB-containing herbs are highly safe in combination with ferulic acid (FA)-containing herbs, suggesting potential neutralizing effect of FA on the toxicity of DIOB. DIOB can be metabolized to generate reactive metabolites (RMs), which can covalently bind to proteins and lead to hepatoxicity. In the present study, the quantitative method was firstly established for investigating the correlation between DIOB RM-protein adducts (DRPAs) and hepatotoxicity. Then, we estimated the detoxication effect of FA in combination with DIOB and revealed the underlying mechanism. Our data indicated that the content of DRPAs positively correlate with the severity of hepatotoxicity. Meanwhile, FA is able to reduce the metabolic rate of DIOB in vitro. Moreover, FA suppressed the production of DRPAs and decreased the serum alanine/aspartate aminotransferase (ALT/AST) levels elevated by DIOB in vivo. Thus, FA can ameliorate DIOB-induced liver injury through reducing the production of DRPAs.

ZnO-NPs alleviate aflatoxin B1-induced hepatoxicity in ducklings by promoting hepatic metallothionein expression.

Aflatoxin B1 (AFB1) is a mycotoxin widely present in animal feed and human food, posing a serious threat to animal and human health. This study was aim to illustrate the mechanism of the protective role of MT against AFB1-induced hepatotoxicity, as well as to explore the feasibility of enhancing the tolerance of poultry to AFB1 by upregulating the expression of hepatic MT. After being exposed to AFB1 (50 ng/kg) primary duckling hepatocytes, the cell viability, the antioxidant index (SOD and GPx) and the mRNA levels of MT downstream genes (PTGR, p53, TrxR, AR and Bcl-2) significantly (p < 0.05) decreased, while the intracellular formation of (AFBO)-DNA adduct content, apoptosis, and MDA content significantly (p < 0.05) increased. Interestingly, overexpression of MT in primary duckling hepatocytes markedly (p < 0.05) reversed the detrimental impact of AFB1 and increased the expression of MT downstream genes. HepG2 cells were applied to study the mechanism how MT works to relieve the hepatic toxicity of AFB1. The ZnO-NPs (20 µg/mL) + AFB1 (20 µg/mL) group significantly (p < 0.05) increased the cell viability, the expression of NRF2, NQO1 and SOD, and expression of MT and MTF-1, as well as significantly (p < 0.05) decreased LDH, ROS and apoptotic rate, comparing with the AFB1 group. While joint treatment with AFB1 and ZnO-NPs, the hepatic toxicity exerted by AFB1 alone was reversed, along with the translocation of MTF-1 from the cytoplasm to the nucleus and upregulated its expression. Duckling trails were further carried out. A total number of 96 1-day-old healthy Cherry Valley commercial ducklings were randomly allocated according to a 2 by 2 factorial arrangement of treatments with the main factors including oral administration of AFB1 (0 vs. 40 µg/kg) and dietary supplementation of ZnO-NPs (0 vs. 60 mg/kg) for 7 days. It showed that AFB1 exposure caused body weight loss (p < 0.05), impaired liver structure and failure in hepatic function (activity of ALT, AST and concentration of TP and GLU) (p < 0.05), and decreases in antioxidant capacity(activity of SOD, CAT and concentration of GSH) (p < 0.05), along with the decrease in hepatic concentration of Zn, increase in expression of apoptosis-related genes and protein CAS3 and mRNA Bcl-2 expression (p < 0.05), and suppressed mRNA levels of antioxidant-related genes MT, SOD1, NRF2, and NQO1 (p < 0.05). In accordance with the cell test, dietary supplementation with ZnO-NPs mitigated the toxicity exerted by AFB1. In conclusion, ZnO-NPs has the protective effects against AFB1-induced hepatocyte injury by activating the expression of MTF-1 and the ectopic induction of MT expression, providing detailed information on the detoxification ability of MT on AFB1.

Risk Assessment for Hepatobiliary Toxicity Liabilities in Drug Development.

Risk assessment of hepatobiliary toxicities represents one of the greatest challenges and, more often than not, one of the most rewarding activities in which toxicologic pathologists can partake, and often times lead. This is in part because each liver toxicity picture is a bit different, informed by a broad range and diversity of relevant data, and also in part because the heavily relied upon animal models are imperfect regarding predictivity of hepatic effects in humans. Following identification and characterization of a hepatotoxicity hazard, typically in nonclinical toxicology studies, a holistic and integrated assessment of liver-relevant endpoints is conducted that typically incorporates ADME (absorption, distribution, metabolism, and excretion) information (ideally, including extensive transporter data, exposure margins, and possibly concentration of parent/metabolite at region of injury), target expression/function, in silico prediction data, in vitro hepatocyte data, liver/circulating biomarkers, and importantly, species specificity of any of these data. Of course, a thorough understanding, developed in close partnership with clinical colleagues, of the anticipated liver disease status of intended patient populations is paramount to hepatic risk assessment. This is particularly important since the likelihood of translatable determinant hepatic events observed in nonclinical models to occur in humans has been reasonably well established.

Sophorae tonkinensis radix et rhizome-induced pulmonary toxicity: A study on the toxic mechanism and material basis based on integrated omics and bioinformatics analyses.

The root and rhizome of Sophora tonkinensis Gagnep. (ST) are widely used for the treatment of tonsillitis, sore throats, and heat-evil-induced diseases in traditional Chinese medicine. However, the clinical application of ST is relatively limited due to its toxicity. The mechanism and material basis of ST-induced pulmonary toxicity are still unclear. In the present research, integrated omics and bioinformatics analyses were used to investigate the toxic mechanism and material basis of ST in lung tissue. Proteomics and metabonomics were integrated to analyze the differentially expressed proteins and metabolites. Joint pathway analysis was used to analyze the significantly dysregulated pathways. PubChem and the Comparative Toxicogenomics Database were applied for the screen of toxic targets and compounds. Integrated omics revealed that 323 proteins and 50 metabolites were differentially expressed after treating with ST, out of which 19 proteins and 1 metabolite were significantly enriched in seven pathways. Bioinformatics showed that 15 compounds may indirectly affect the expression of 9 toxic targets of ST. Multiple toxic targets of ST-induced pulmonary injury were found in the study, whose dysregulation may trigger pulmonary cancer, dyspnea, and oxidative stress. Multiple compounds may be the toxic material basis in response to these effects.

The use of emerging safety biomarkers in nonclinical and clinical safety assessment - The current and future state: An IQ DruSafe industry survey.

Pharmaceutical and biotechnology companies rarely disclose their use of translational emerging safety biomarkers (ESBs) during drug development, and the impact of ESB use on the speed of drug development remains unclear. A cross-industry survey of 20 companies of varying size was conducted to understand current trends in ESB use and future use prospects. The objectives were to: (1) determine current ESB use in nonclinical and clinical drug development and impact on asset advancement; (2) identify opportunities, gaps, and challenges to greater ESB implementation; and (3) benchmark perspectives on regulatory acceptance. Although ESBs were employed in only 5-50% of studies/programs, most companies used ESBs to some extent, with larger companies demonstrating greater nonclinical use. Inclusion of ESBs in investigational new drug applications (INDs) was similar across all companies; however, differences in clinical trial usage could vary among the prevailing health authority (HA). Broader implementation of ESBs requires resource support, cross-industry partnerships, and collaboration with HAs. This includes generating sufficient foundational data, demonstrating nonclinical to clinical translatability and practical utility, and clearly written criteria by HAs to enable qualification. If achieved, ESBs will play a critical role in the development of next-generation, translationally-tailored standard laboratory tests for drug development.

Pumpless, unidirectional microphysiological system for testing metabolism-dependent chemotherapeutic toxicity.

Drug development is often hindered by the failure of preclinical models to accurately assess and predict the efficacy and safety of drug candidates. Body-on-a-chip (BOC) microfluidic devices, a subset of microphysiological systems (MPS), are being created to better predict human responses to drugs. Each BOC is designed with separate organ chambers interconnected with microfluidic channels mimicking blood recirculation. Here, we describe the design of the first pumpless, unidirectional, multiorgan system and apply this design concept for testing anticancer drug treatments. HCT-116 colon cancer spheroids, HepG2/C3A hepatocytes, and HL-60 promyeloblasts were embedded in collagen hydrogels and cultured within compartments representing "colon tumor", "liver," and "bone marrow" tissue, respectively. Operating on a pumpless platform, the microfluidic channel design provides unidirectional perfusion at physiologically realistic ratios to multiple channels simultaneously. The metabolism-dependent toxic effect of Tegafur, an oral prodrug of 5-fluorouracil, combined with uracil was examined in each cell type. Tegafur-uracil treatment induced substantial cell death in HCT-116 cells and this cytotoxic response was reduced for multicellular spheroids compared to single cells, likely due to diffusion-limited drug penetration. Additionally, off-target toxicity was detected by HL-60 cells, which demonstrate that such systems can provide useful information on dose-limiting side effects. Collectively, this microscale cell culture analog is a valuable physiologically-based pharmacokinetic drug screening platform that may be used to support cancer drug development.

Ameliorative Effects of Honey, Propolis, Pollen, and Royal Jelly Mixture against Chronic Toxicity of Sumithion Insecticide in White Albino Rats.

Sumithion (Fenitrothion) (SUM) is an organophosphorus insecticide used to combat a wide variety of plant pests. Exposure to SUM causes significant toxicity to the brain, liver, kidney, and reproductive organs through, for example, binding to DNA, and it induces DNA damage, which ends with oxidative stress. Therefore, the present study aimed to examine the protective role of bee products: a mixture of honey, propolis, palm pollen, and royal jelly (HPPJ) against SUM-induced toxicity. Twenty-four male albino rats (Rattus norvegicus) were classified into four groups, each containing six rats: control (corn oil), SUM (85 mg/kg; 1/20 LD50), HPPJ, and SUM + HPPJ once daily for 28 consecutive days. Blood samples were gently collected in sterilized ethylenediaminetetraacetic acid (EDTA) tubes for blood picture analyses and tubes without anticoagulant for serum isolation. Serum was used for assays of enzymatic and biochemical characteristics. The results revealed that SUM increased the weights of the liver, kidney, and brain as well as the enzymatic activity of glutathione peroxidase (GP), serum superoxide dismutase (SOD), and glutathione-S-transferase (GST). Additionally, SUM significantly increased the activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and γ-glutamyltransferase (γ-GT) and glucose, uric acid, and creatinine contents, while decreasing the acetylcholine esterase (AChE) activity and total lipids and total protein content. Furthermore, because of the inclusion of phenolic, flavonoids, terpenoids, and sugars, the HPPJ mixture counteracted the hematological, renal, and hepatic toxicity of SUM exposure.

Relationship of MATE1 Inhibition and Cytotoxicity in Nephrotoxicity: Application for Safety Evaluation in Early Drug Discovery.

Accumulation of toxic endogenous and/or exogenous substances can trigger tissue injury. Multidrug and toxin extrusion proteins (MATEs) are transporters at renal proximal tubules involved in the secretion of hydrophilic substances into urine. Multidrug and toxin extrusion protein inhibition can lead to nephrotoxicity via accumulation of toxic substances; however, case studies demonstrating causality are rare, except for drug-drug interaction studies. To explore the involvement of MATE inhibition in nephrotoxicity, MATE1 inhibition, cytotoxicity, and mitochondrial toxicity (MT) of 38 in-house compounds that showed toxicity were assessed in in vivo safety evaluations using rats, dogs, and monkeys and compared considering unbound exposures at minimal steady-state concentration (C24h,u) between nephrotoxicity positive and negative compounds. Logarithmic-corrected means of C24h,u normalized by MATE1 IC50 or cytotoxicity EC50 (C24h,u/IC50 and C24h,u/EC50) were higher for nephrotoxic compounds. An exposure cutoff of C24h,u/IC50 > 0.01 filtered nephrotoxicity with a 54% positive predictive value. Of 7 cases filtered with this cutoff, all the cases showed pathological changes at renal proximal tubules expressing MATE1. Furthermore, all cases with > 0.01 reliable exposure for MATE1 inhibition and cytotoxicity exhibited nephrotoxicity. Although compounds potent for MATE1 inhibition and cytotoxicity without and with MT (potentials of 10, 30, and 40 µM, respectively) were correctly classified as nephrotoxic by evaluation of in vitro potency alone, without considering exposures, these results suggest that MATE1 inhibition potency and cytotoxicity can be used to assess nephrotoxicity, especially at proximal tubules, and could be used for safety assessment in early drug discovery.

Predictive Value of Cellular Accumulation of Hydrophobic Bile Acids As a Marker of Cholestatic Drug Potential.

Drug-induced cholestasis is mostly intrahepatic and characterized by alterations of bile canaliculi dynamics and morphology as well as accumulation of bile acids (BAs) in hepatocytes. However, little information exists on first changes in BA content and profile induced by cholestatic drugs in human liver. In this study, we aimed to analyze the effects of a large set of cholestatic and noncholestatic drugs in presence of physiological serum concentrations and 60-fold higher levels of 9 main BAs on cellular accumulation of BAs using HepaRG hepatocytes. BAs were measured in cell layers (cells + bile canaliculi) and culture media using high-pressure liquid chromatography coupled with tandem mass spectrometry after 24 h-treatment. Comparable changes in total and individual BA levels were observed in cell layers and media from control and noncholestatic drug-treated cultures: unconjugated BAs were actively amidated and lithocholic acid (LCA) was entirely sulfated. In contrast, cellular accumulation of LCA and in addition, of the 2 other hydrophobic BAs, chenodeoxycholic acid and deoxycholic acid, was evidenced only with cholestatic compounds in presence of BA mixtures at normal and 60-fold serum levels, respectively, suggesting that LCA was the first BA to accumulate. Cellular accumulation of hydrophobic BAs was associated with inhibition of their amidation and for LCA, its sulfation. In conclusion, these results demonstrated that cellular accumulation of unconjugated hydrophobic BAs can be caused by various cholestatic drugs in human hepatocytes and suggest that their cellular detection, especially that of LCA, could represent a new strategy for evaluation of cholestatic potential of drugs and other chemicals.

Wild edible onions - Allium flavum and Allium carinatum - successfully prevent adverse effects of chemotherapeutic drug doxorubicin.

The objective of this study was to evaluate potential of two chemically characterized edible wild onion species, Allium flavum and Allium carinatum, to reduce side effects of cytostatic doxorubicin (Dox). Since Dox application is mainly limited due to its high cardiotoxicity, while there are no approved cardioprotective agents for the prevention of Dox adverse effects, new co-treatments are urgently needed. Here, we showed that methanol extracts expressed high antioxidant activity and synergistically increased Dox anticancer activity against human hepatoma (HepG2) and lung carcinoma (A549) cells, while protected normal human fibroblasts (MRC-5) from Dox cytotoxicity. Analysis of the antioxidative enzymes level (catalase and superoxide dismutases) showed that the catalase level was differently altered in cancer cells compared to normal cells upon applied treatments. In vivo toxicity evaluation in the zebrafish model revealed significantly lower toxicity of extracts compared to Dox, and no teratogenic effects at applied doses. We found that extracts successfully rescued the Dox-treated embryos of life-threating cardiomyopathy, while at the same time reduced developmental toxicity and neutropenia. Further analysis demonstrated that extracts had higher anti-angiogenic activity than sunitinib or auranofin, clinically used antiangiogenic drugs. In addition, angiogenesis was markedly more suppressed in Dox-extract cotreatments than upon single treatments.

Curcumin as an anti-inflammatory agent: Implications to radiotherapy and chemotherapy.

Cancer is the second cause of death worldwide. Chemotherapy and radiotherapy are the most common modalities for the treatment of cancer. Experimental studies have shown that inflammation plays a central role in tumor resistance and the incidence of several side effects following both chemotherapy and radiotherapy. Inflammation resulting from radiotherapy and chemotherapy is responsible for adverse events such as dermatitis, mucositis, pneumonitis, fibrosis, and bone marrow toxicity. Chronic inflammation may also lead to the development of second cancer during years after treatment. A number of anti-inflammatory drugs such as nonsteroidal anti-inflammatory agents have been proposed to alleviate chronic inflammatory reactions after radiotherapy or chemotherapy. Curcumin is a well-documented herbal anti-inflammatory agents. Studies have proposed that curcumin can help management of inflammation during and after radiotherapy and chemotherapy. Curcumin targets various inflammatory mediators such as cyclooxygenase-2, inducible nitric oxide synthase, and nuclear factor κB (NF-κB), thereby attenuating the release of proinflammatory and profibrotic cytokines, and suppressing chronic production of free radicals, which culminates in the amelioration of tissue toxicity. Through modulation of NF-κB and its downstream signaling cascade, curcumin can also reduce angiogenesis, tumor growth, and metastasis. Low toxicity of curcumin is linked to its cytoprotective effects in normal tissues. This protective action along with the capacity of this phytochemical to sensitize tumor cells to radiotherapy and chemotherapy makes it a potential candidate for use as an adjuvant in cancer therapy. There is also evidence from clinical trials suggesting the potential utility of curcumin for acute inflammatory reactions during radiotherapy such as dermatitis and mucositis.

Serum retinyl esters are positively correlated with analyzed total liver vitamin A reserves collected from US adults at time of death.

Background: Minimal human data exist on liver vitamin A (VA) compared with serum biomarkers. Cutoffs of 5% and 10% total serum VA as retinyl esters (REs) suggest a VA intoxication diagnosis. Objectives: We compared total liver VA reserves (TLRs) with the percentage of total serum VA as REs to evaluate hypervitaminosis with the use of US adult autopsy samples. Secondary objectives evaluated serum retinol sensitivity, TLRs among lobes, and hepatic α-retinol concentrations, an α-carotene cleavage product. Design: Matched serum and liver samples were procured from cadavers (n = 27; mean ± SD age: 70.7 ± 14.9 y; range: 49-101 y). TLRs and α-REs were quantified by ultra-performance liquid chromatography. Pearson correlations showed liver and serum associations. Sensitivity and specificity were calculated for >5%, 7.5%, and 10% total serum VA as REs to predict TLRs and for serum retinol <0.7 and 1 µmol/L to predict deficiency. Results: Serum RE concentrations were correlated with TLRs (r = 0.497, P < 0.001). Nine subjects (33%) had hypervitaminosis A (≥1.0 µmol VA/g liver), 2 of whom had >7.5% total serum VA as REs; histologic indicators corroborated toxicity at 3 µmol/g liver. No subject had >10% total serum VA as REs. Serum retinol sensitivity to determine deficiency (TLRs <0.1 µmol VA/g) was 83% at 0.7 and 1 µmol/L. Hepatic α-retinol was positively correlated with age (P = 0.047), but removing an outlier nullified significance. Conclusions: This study evaluated serum REs as a biomarker of VA status against TLRs (gold standard), and abnormal histology suggested that 7.5% total serum VA as REs is diagnostic for toxicity at the individual level in adults. The long-term impact of VA supplements and fortificants on VA status is currently unknown. Considering the high prevalence of hypervitaminotic TLRs in this cohort, and given that many countries are adding preformed VA to processed products, population biomarkers diagnosing hypervitaminosis before toxicity are urgently needed. This trial was registered at clinicaltrials.govas NCT03305042.

Toxicologic Pathology Forum Opinion Paper*: Recommendations for a Tiered Approach to Nonclinical Mechanistic Nephrotoxicity Evaluation.

Nephrotoxicity is one of the more common causes of attrition in nonclinical drug development. Like most tissues, the kidney has a limited number of ways of responding to toxicological insults from diverse mechanistic pathways, which can limit the ability to determine mechanisms of renal injury using the assays routinely performed in preclinical toxicologic studies. In situations where the renal injury is unusual in morphology or if a therapeutic margin is low, additional investigative techniques may be needed to identify a potential mechanism of toxicity in order to inform clinical risk assessment or establish human relevance and translatability of the toxicity. While routine microscopic evaluation can suggest a specific pathogenesis, understanding the mechanism of renal injury often requires additional hypothesis-driven investigations and specialized techniques to obtain the data necessary to identify a nephrotoxic mechanism. Nonclinical mechanistic investigations can be resource-intensive and often yield limited new information. Although there are multiple avenues to investigate renal toxicity, no single mechanistic study or prescriptive battery of tests will identify the pathophysiologic basis for every potential mechanism of renal injury. To aid the nonclinical investigator, we outline a tiered approach for prioritizing investigations to provide a rational and linear road map for the exploration of mechanisms of drug-induced kidney injury. [Box: see text].

A predictive index of biomarkers for ictogenesis from tier I safety pharmacology testing that may warrant tier II EEG studies.

Three significant contributions to the field of safety pharmacology were recently published detailing the use of electroencephalography (EEG) by telemetry in a critical role in the successful evaluation of a compound during drug development (1] Authier, Delatte, Kallman, Stevens & Markgraf; JPTM 2016; 81:274-285; 2] Accardi, Pugsley, Forster, Troncy, Huang & Authier; JPTM; 81: 47-59; 3] Bassett, Troncy, Pouliot, Paquette, Ascaha, & Authier; JPTM 2016; 70: 230-240). These authors present a convincing case for monitoring neocortical biopotential waveforms (EEG, ECoG, etc) during preclinical toxicology studies as an opportunity for early identification of a central nervous system (CNS) risk during Investigational New Drug (IND) Enabling Studies. This review is about "ictogenesis" not "epileptogenesis". It is intended to characterize overt behavioral and physiological changes suggestive of drug-induced neurotoxicity/ictogenesis in experimental animals during Tier 1 safety pharmacology testing, prior to first dose administration in man. It is the presence of these predictive or comorbid biomarkers expressed during the requisite conduct of daily clinical or cage side observations, and in early ICH S7A Tier I CNS, pulmonary and cardiovascular safety study designs that should initiate an early conversation regarding Tier II inclusion of EEG monitoring. We conclude that there is no single definitive clinical marker for seizure liability but plasma exposures might add to set proper safety margins when clinical convulsions are observed. Even the observation of a study-related full tonic-clonic convulsion does not establish solid ground to require the financial and temporal investment of a full EEG study under the current regulatory standards. PREFATORY NOTE: For purposes of this review, we have adopted the FDA term "sponsor" as it refers to any person who takes the responsibility for and initiates a nonclinical investigations of new molecular entities; FDA uses the term "sponsor" primarily in relation to investigational new drug application submissions.

Protective effects of persian honey, Apis Mellifera Meda Skorikov on side effects of chemotherapy and ischemia/reperfusion induced testicular injury.

Introduction The aim of the present study was to survey the protective effect of pretreatment with Persian honey on amelioration of side effects of chemotherapy and ischemia/reperfusion induced testicular injury. Materials and methods Forty adult's male wistar rats were divided into four groups of ischemia-reperfusion (IR), honey + ischemia-reperfusion (HIR), Busulfan (B) and Busulfan intraperitoneally+ honey (BH). The seminiferous tubules were rated for their modified spermatogenesis index (SI) by Johnsons score. Detection of single- and double-stranded DNA breaks at the early stages of apoptosis was performed using the in-situ cell death detection kit. Total serum concentration of Follicle-stimulating hormone (FSH) , Luteinizing hormone (LH) and testosterone was measured using ELISA. All data were expressed as mean ± SD and significance was set at p≤0.05. Results Honey improved SI in the HIR and BH groups and serum levels of FSH and LH in the BH and HIR groups (p<0.001). Also, serum levels of testosterone were significantly higher in BH and HIR groups. But, apoptotic cells in IR and B groups significantly increased (p<0.001), while in HIR and BH groups, the number of apoptotic cells decreased and the positive cells of TUNEL (TdT-mediated dUTP-X nick end labelling) staining were detected in spermatocytes and spermatid. Discussion Pretreatment with honey protect testis against chemotherapy and testicular IR injury, increase FSH and LH and testosterone and decrease the cellular damage and apoptosis. Honey can decrease the side effects of chemotherapy on reproductive system and prevent sterility.

Predicting Drug Safety and Communicating Risk: Benefits of a Bayesian Approach.

Drug toxicity is a major source of attrition in drug discovery and development. Pharmaceutical companies routinely use preclinical data to predict clinical outcomes and continue to invest in new assays to improve predictions. However, there are many open questions about how to make the best use of available data, combine diverse data, quantify risk, and communicate risk and uncertainty to enable good decisions. The costs of suboptimal decisions are clear: resources are wasted and patients may be put at risk. We argue that Bayesian methods provide answers to all of these problems and use hERG-mediated QT prolongation as a case study. Benefits of Bayesian machine learning models include intuitive probabilistic statements of risk that incorporate all sources of uncertainty, the option to include diverse data and external information, and visualizations that have a clear link between the output from a statistical model and what this means for risk. Furthermore, Bayesian methods are easy to use with modern software, making their adoption for safety screening straightforward. We include R and Python code to encourage the adoption of these methods.