Comparisons between in vitro whole cell imaging and in vivo zebrafish-based approaches for identifying potential human hepatotoxicants earlier in pharmaceutical development

Drug-induced liver injury (DILI) is a major cause of attrition during both the early and later stages of the drug development and marketing process. Reducing or eliminating drug-induced severe liver injury, especially those that lead to liver transplants or death, would be tremendously beneficial for patients. Therefore, developing new pharmaceuticals that have the highest margins and attributes of hepatic safety would be a great accomplishment. Given the current low productivity of pharmaceutical companies and the high costs of bringing new medicines to market, any early screening assay(s) to identify and eliminate pharmaceuticals with the potential to cause severe liver injury in humans would be of economic value as well. The present review discusses the background, proof-of-concept, and validation studies associated with high-content screening (HCS) by two major pharmaceutical companies (Pfizer Inc and Jansen Pharmaceutical Companies of Johnson & Johnson) for detecting compounds with the potential to cause human DILI. These HCS assays use fluorescent-based markers of cell injury in either human hepatocytes or HepG2 cells. In collaboration with Evotec, an independent contract lab, these two companies also independently evaluated larval zebrafish as an early-stage in vivo screen for hepatotoxicity in independently conducted, blinded assessments. Details about this model species, the need for bioanalysis, and, specifically, the outcome of the phenotypic-based zebrafish screens are presented. Comparing outcomes in zebrafish against both HCS assays suggests an enhanced detection for hepatotoxicants of most DILI concern when used in combination with each other, based on the U.S. Food and Drug Administration DILI classification list.     

Late erosions of mid-urethral tapes for stress urinary incontinence—need for long-term follow-up?

Tension-free vaginal tape (TVT) procedure has become one of the most common treatments of female urinary stress incontinence. Success rates as high as 81.3% were reported over a follow-up period of 7 years. Erosion of the synthetic mesh is a well-described complication. The mean time for the onset of erosion after sling insertion was 11.2 months. These case reports describe an erosion of a mid-urethral tape after 18 and 28 months, which is uncommon. There is a need for long-term follow-up of patients with TVT.     

Non‐invasive cardiovascular profiling using combined electrocardiogram–Doppler ultrasonography and impedance cardiography: An experimental approach

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Diurnal and position-induced variability of impedance cardiography measurements in healthy subjects

Cardiovascular (CV) parameters and their measurements are subject to variation. In this study, we evaluated the reproducibility of impedance cardiography (ICG) measurements following orthostatic and diurnal challenges for a set of 22 CV parameters in ten randomly selected healthy nonpregnant women. A standard protocol was used to record a consecutive series of measurements for each parameter before and after three position changes. This series of measurements was performed twice (AM and PM sessions). For each parameter, measurement-shift following position change was evaluated at 5% cutoff and compared between sessions. Intra- and intersession intraclass correlation (ICC) was calculated for individual measurements per position using repeated-measures analysis of variance. Intra- and intersession Pearson's correlation coefficient (PCC) was calculated for mean values per position. Intersession correlation for measurement-shift following position change was 0·42 (5/12) for pressure parameters, whereas this was 0·96 (52/54) for other parameters. Inter- and intrasession ICC for individual measurements varied between 0·02 and 1 for all parameters, however inter- and intrasession PCC for mean values was consistently >0·80 for stroke volume (SV), stroke index (SI), cardiac output (CO), acceleration and velocity index (ACI and VI), thoracic fluid content (TFC), TFC index (TFCI) and heart period duration (HPD). We conclude that in healthy subjects under standardised conditions, reproducibility of means of multiple ICG measurements is high for SV, SI, CO, ACI, VI, TFC, TFCI and HPD. From our data, we cannot draw conclusions on trends in diseased subjects.     

Hydroxypropyl-beta-cyclodextrin can modulate the activity of spinally administered sufentanil.

Hydroxypropyl-beta-cyclodextrin increased the effectiveness of sufentanil after epidural and intrathecal administration in rats, both in terms of a longer duration of analgesia after a fixed dose of sufentanil, and in a reduction of the lowest ED50s to produce analgesia. There was also an increase in specificity, as indicated by the greater dissociation between the ED50s for analgesia and for supra-spinal side-effects. Maximal activity was measured after inclusion complexation of sufentanil in 10% hydroxypropyl-beta-cyclodextrin. At higher concentrations of hydroxypropyl-beta-cyclodextrin, both the activity and the specificity were attenuated. The increased safety of sufentanil in 10% hydroxypropyl-beta-cyclodextrin after spinal administration was also confirmed in terms of opioid-induced deviations in arterial PO2, PCO2 and oxygen saturation. At a dose of twice the ED50 for deep surgical analgesia, the sufentanil/hydroxypropyl-beta-cyclodextrin complex produced no changes in these parameters. With sufentanil alone at comparable analgesic doses, significant shifts in all three parameters were present immediately after drug administration. At higher concentrations of sufentanil in hydroxypropyl-beta-cyclodextrin changes in the three blood gases were present but the deviations were always smaller than those observed with comparable doses of plain sufentanil. These results support the notion that after complexation sufentanil is present longer at the spinal level after spinal administration. As a consequence, there is less free sufentanil available for redistribution into lipid tissue and into the circulatory system, producing less systemic side-effects.     

A Study of the Percutaneous Absorption-enhancing Effects of Cyclodextrin Derivatives in Rats

Abstract— 2-Hydroxypropyl-β-cyclodextrin (HP-β-CyD) and 2,6-dimethyl-β-cyclodextrin (D-β-CyD) were studied for transdermal penetration enhancement of the cytochrome P450 inhibitor liarozole by an in-vivo transdermal absorption rat model. The mode of action of penetration enhancement was investigated by differential scanning calorimetry (DSC). In-vivo, HP-β-CyD, as a 20% aqueous solution, increased the absorption of liarozole approximately threefold and a 20% aqueous solution of D-β-CyD decreased the percutaneous absorption of liarozole in blood by a factor of 0·6. However, pretreatment with D-β-CyD (20%, 4 h) enhanced the transdermal absorption 9·4-fold. In the DSC experiments the thermal profile of human stratum corneum was practically unchanged after treatment with HP-β-CyD, but treatment with D-β-CyD revealed an interaction of D-β-CyD with the protein and lipid fraction. Thus the results from DSC and those from the permeability experiments revealed that D-β-CyD acts as a transdermal absorption enhancer by changing the stratum corneum barrier whereas HP-β-CyD influences the partitioning behaviour of the drug in the skin.     

Characteristics of heart,arteries, and veins in low and high cardiac output preeclampsia

Objective

To assess the feasibility of non-invasive measurements of maternal cardiac output in relation to birth weight percentile and cardiovascular physiology in preeclampsia.

Study design

In a cohort of 62 women with preeclampsia, impedance cardiography was used to measure cardiac output and to evaluate heart and arteries. Venous characteristics were assessed by combined electrocardiogram-Doppler ultrasonography. Statistical differences were evaluated by Mann–Whitney U-tests.

Results

Cardiac output correlated with birth weight percentile (P = .002), with more small for gestational age newborns in low cardiac output preeclampsia (<7.5 L/min) than in high cardiac output preeclampsia (≥8.9 L/min) (12/29 vs. 2/16, P = .044). This was associated with lower aortic flow indices and shorter venous pulse transit times in low than in high cardiac output preeclampsia.

Conclusion

Non-invasive impedance cardiography measurements of maternal cardiac output correlate with birth weight percentile and are associated with different functionality of heart, arteries, and veins in low and high cardiac output preeclampsia.     

Zebrafish developmental toxicity assay: A fishy solution to reproductive toxicity screening, or just a red herring?

The zebrafish embryotoxicity/teratogenicity assay is described as a useful alternative screening model to evaluate the effect of drugs on embryofoetal development.Fertilized eggs were exposed to different concentrations of 15 compounds with teratogenic (8) and non-teratogenic (7) potential until 96 h post-fertilization when 28 morphological endpoints and the level of compound uptake was assessed.The majority of drugs testing positive in mammals was also positive in zebrafish (75% sensitivity), while a relative high number of false positives were noted (43% specificity). Compound uptake determination appears useful for clarifying classifications as teratogenic or potential overdose although assay sensitivity could be improved to 71% if the exposure threshold, previously suggested as ∼50 ng/larvae, is reconsidered.The zebrafish assay shows some potential, though limited in its current form, as a screening tool for developmental toxicity within Janssen drug development. Further assay refinement with respect to endpoints and body burden threshold is required.     

Phospholipidosis in rats treated with amiodarone: serum biochemistry and whole genome micro-array analysis supporting the lipid traffic jam hypothesis and the subsequent rise of the biomarker BMP

To provide mechanistic insight in the induction of phospholipidosis and the appearance of the proposed biomarker di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate (BMP), rats were treated with 150 mg/kg amiodarone for 12 consecutive days and analyzed at three different time points (day 4, 9, and 12). Biochemical analysis of the serum revealed a significant increase in cholesterol and phospholipids at the three time points. Bio-analysis on the serum and urine detected a time-dependent increase in BMP, as high as 10-fold compared to vehicle-treated animals on day 12. Paralleling these increases, micro-array analysis on the liver of treated rats identified cholesterol biosynthesis and glycerophospholipid metabolism as highly modulated pathways. This modulation indicates that during phospholipidosis-induction interactions take place between the cationic amphiphilic drug and phospholipids at the level of BMP-rich internal membranes of endosomes, impeding cholesterol sorting and leading to an accumulation of internal membranes, converting into multilamellar bodies. This process shows analogy to Niemann-Pick disease type C (NPC). Whereas the NPC-induced lipid traffic jam is situated at the cholesterol sorting proteins NPC1 and NPC2, the amiodarone-induced traffic jam is thought to be located at the BMP level, demonstrating its role in the mechanism of phospholipidosis-induction and its significance for use as a biomarker.