Petra Vinken

Tissue Kim-1 and Urinary Clusterin as Early Indicators of Cisplatin-Induced Acute Kidney Injury in Rats

The kidney is one of the main targets of drug toxicity, and early detection of renal damage is critical in preclinical drug development. A model of cisplatin-induced nephrotoxicity in male Sprague Dawley rats treated for 1, 3, 5, 7, or 14 days at 1 mg/kg/day was used to monitor the spatial and temporal expression of various indicators of kidney toxicity during the progression of acute kidney injury (AKI). As early as 1 day after cisplatin treatment, positive kidney injury molecule-1 (Kim-1) immunostaining, observed in the outer medulla of the kidney, and changes in urinary clusterin indicated the onset of proximal tubular injury in the absence of functional effects. After 3 days of treatment, Kim-1 protein levels in urine increased more than 20-fold concomitant with a positive clusterin immunostaining and an increase in urinary osteopontin. Tubular basophilia was also noted, while serum creatinine and blood urea nitrogen levels were elevated only after 5 days, together with tubular degeneration. In conclusion, tissue Kim-1 and urinary clusterin were the most sensitive biomarkers for detection of cisplatin-induced kidney damage. Thereafter, urinary Kim-1 and osteopontin, as well as clusterin immunostaining accurately correlated with the histopathological findings. When AKI is suspected in preclinical rat studies, Kim-1, clusterin, and osteopontin should be part of urinalysis and/or IHC can be performed.

Evaluation of miR-122 and Other Biomarkers in Distinct Acute Liver Injury in Rats

The detection of drug-induced hepatotoxicity remains an important safety issue in drug development. A liver-specific microRNA species, microRNA-122 (miR-122), has recently shown potential for predicting liver injury in addition to the standard hepatic injury biomarkers. The objective of this study was to measure miR-122 together with several other liver markers in distinct settings of acute liver toxicity in rats to determine the value of miR-122 as a biomarker for liver injury in this species. Rats were exposed to 3 well-established liver toxicants (acetaminophen, allyl alcohol, and α-naphthyl isothiocyanate), a liver-enzyme inducer (phenobarbital), or a cardiotoxicant (doxorubicin). There was a clear increase in plasma miR-122 following administration of acetaminophen, allyl alcohol, and α-naphthyl isothiocyanate. The response of miR-122 paralleled that of other markers and was consistent with liver injury as indicated by histopathological evaluation. Furthermore, the changes in miR-122 were detected earlier than standard liver injury markers and exhibited a wide dynamic range. In contrast, miR-122 responses to phenobarbital and doxorubicin were low. Based on these findings, miR-122 shows significant promise and may provide added value for assessing liver toxicity in drug development.

Carcinogenicity in rats of the SGLT2 inhibitor canagliflozin

The carcinogenicity potential of canagliflozin, an inhibitor of SGLT2, was evaluated in a 2-year rat study (10, 30, and 100 mg/kg). Rats showed an increase in pheochromocytomas, renal tubular tumors, and testicular Leydig cell tumors. Systemic exposure multiples at the highest dose relative to the maximum clinical dose were 12- to 21-fold. Pheochromocytomas and renal tubular tumors were noted in both sexes at 100 mg/kg. Leydig cell tumors were observed in males in all dose groups and were associated with increased luteinizing hormone levels. Hyperplasia was increased in the adrenal medulla at 100 mg/kg, but only a limited increase in simple tubular hyperplasia was observed in the kidney of males at 100 mg/kg. Hyperostosis occurred and was accompanied by substantial effects on calcium metabolism, including increased urinary calcium excretion and decreased levels of calcium regulating hormones (1,25-dihydroxyvitamin D and parathyroid hormone). A separate study with radiolabeled calcium confirmed that increased urinary calcium excretion was mediated via increased calcium absorption from the gastrointestinal tract. It was hypothesized that, at high doses, canagliflozin might have inhibited glucose absorption in the intestine via SGLT1 inhibition that resulted in glucose malabsorption, which increased calcium absorption by stimulating colonic glucose fermentation and reducing intestinal pH. Pheochromocytomas and adrenal medullary hyperplasia were attributed to altered calcium homeostasis, which have a known relationship in the rat. In conclusion, Leydig cell tumors were associated with increased luteinizing hormone levels and pheochromocytomas were most likely related to glucose malabsorption and altered calcium homeostasis. Renal tubular tumors may also have been linked to glucose malabsorption.

Efferent Duct Toxicity with Secondary Testicular Changes in Rats Following Administration of a Novel Leukotriene A4 Hydrolase Inhibitor

The efferent ducts represent an important site of toxicity in the male reproductive tract but are not routinely examined in toxicity studies. This article describes a primary efferent duct toxicity that resulted in secondary testicular changes in rats. Male rats were administered LTI-1, a leukotriene A4 hydrolase inhibitor, at doses up to 250 mg/kg/d for 3 month or 150 mg/kg/d for 6 month. At the highest dose levels, testicular changes were predominantly unilateral and characterized by diffuse dilation or atrophy of the seminiferous tubules. These testicular changes correlated with granulomatous inflammation in the corresponding efferent ducts, suggesting that the mechanism for the testicular changes involves obstruction and impaired fluid reabsorption in the efferent ducts. Subsequent buildup in fluid volume and back-pressure upstream of the blockage cause dilation of the seminiferous tubules, which, in its late stages, progress to tubular atrophy. There are important differences in efferent duct anatomy between rats and larger mammals, including humans, such that the latter are less susceptible to testicular injury by this mechanism. Because of the limited relevance of this rat-specific finding to humans, it is important to distinguish testicular changes secondary to efferent duct toxicity from primary drug-induced testicular toxicity.

Carbohydrate malabsorption mechanism for tumor formation in rats treated with the SGLT2 inhibitor canagliflozin

Canagliflozin is an SGLT2 inhibitor used for the treatment of type 2 diabetes mellitus. Studies were conducted to investigate the mechanism responsible for renal tubular tumors and pheochromocytomas observed at the high dose in a 2-year carcinogenicity study in rats. At the high dose (100mg/kg) in rats, canagliflozin caused carbohydrate malabsorption evidenced by inhibition of intestinal glucose uptake, decreased intestinal pH and increased urinary calcium excretion. In a 6-month mechanistic study utilization of a glucose-free diet prevented carbohydrate malabsorption and its sequelae, including increased calcium absorption and urinary calcium excretion, and hyperostosis. Cell proliferation in the kidney and adrenal medulla was increased in rats maintained on standard diet and administered canagliflozin (100mg/kg), and in addition an increase in the renal injury biomarker KIM-1 was observed. Increased cell proliferation is considered as a proximal event in carcinogenesis. Effects on cell proliferation, KIM-1 and calcium excretion were inhibited in rats maintained on the glucose-free diet, indicating they are secondary to carbohydrate malabsorption and are not direct effects of canagliflozin.

Analytic Evaluation of a Human ELISA Kit for Measurement of Inhibin B in Rat Samples

BACKGROUND A cross-laboratory analytic evaluation of a commercially available human inhibin B ELISA for measuring inhibin B in rat serum and plasma has been undertaken. METHODS Dilution linearity, spiked recovery, intra- and inter-assay precision, functional sensitivity, matrix effects, and frozen stability were assessed across five laboratories. Reference ranges were generated for male Sprague Dawley and Han Wistar rats. RESULTS Acceptable performance was defined as an overall assay coefficient of variation ≤ 20% with an intraday LLOQ ≤ 20 pg/ml. Intra- and inter-assay precision and functional sensitivity (≤6.4 pg/ml) generally met these criteria, but with occasional evidence of greater variability, particularly at lower concentrations. Dilution linearity was acceptable with occasional low recovery. Acceptable recovery of kit calibrators from rat serum confirmed the absence of matrix effects. Matched serum and plasma samples gave comparable results. The signal increased on freezing, remained constant for ≥3 freeze-thaw cycles and was generally stable for at least 8 weeks. Mean inhibin B ranged from 33.5 to 140.6 pg/ml in adult rats across laboratories, with some evidence for a decline from 6 to 9 weeks of age. Power calculations using preliminary reference range data indicated 10 animals/group would generally detect a 40% decrease in inhibin B at AstraZeneca, but laboratories with lower control values would require larger groups. CONCLUSIONS The assay meets the analytical performance criteria; however, precision at the low end of the standard curve, biological variability, and low control values observed in some laboratories indicate that the utility of the assay may be limited in some laboratories.

The Inhibin B Response to the Testicular Toxicant 1, 3 Dinitrobenzene in Male Rats

BACKGROUND This study was conducted as part of an ILSI-HESI International Life Sciences Institute-Health & Environmental Sciences Institute consortium effort to assess the utility of circulating Inhibin B as an early biomarker of Sertoli cell-specific testicular toxicity in rats. 1, 3-Dinitrobenzene (1,3-DNB) was selected as a testicular toxicant in this study as it is known to target Sertoli cells. METHODS 1,3-DNB (2 and 6 mg/kg/day) or control (corn oil) was administered orally to male rats for two or five consecutive days. Blood was collected from rats treated for 2 days on days 1 and 2 and from rats treated for 5 days on days 1, 3, and 5. The resulting serum was evaluated for Inhibin B and follicle stimulating hormone. At the end of the treatment periods, the testes were removed, weighed, and examined histopathologically. RESULTS Daily administration of 1,3-DNB resulted in decreased testis weight only on day 5 and only at the high dose (6 mg/kg/day). There was a time-dependent increase in incidence and severity of testicular findings characterized by degeneration of the germinal epithelium with loss of pachytene spermatocytes and vacuolization of the Sertoli cells in the seminiferous tubules at the high dose. Inhibin B levels in 1,3-DNB-treated animals were decreased with treatment only on day 5 at the high dose; there were no associated changes in follicle stimulating hormone. CONCLUSIONS Changes in serum Inhibin B levels were detected only in association with moderate or severe testicular toxicity as evidenced by histopathology and is therefore considered to be of limited value as a biomarker for Sertoli cell toxicity.

Hemorrhagic Cardiomyopathy in Male Mice Treated with an NNRTI: The Role of Vitamin K

Dietary dosing of the non-nucleoside reverse transcriptase inhibitor (NNRTI) TMC125, under development for treatment of HIV-1, resulted in a syndrome in male mice in a previous experiment that was termed hemorrhagic cardiomyopathy. In literature, this syndrome, which was described in rodent species only, was linked to vitamin K deficiency. Two mechanistic studies were conducted, one with dietary administration and a second with gavage. The syndrome was reproduced in only 1 male mouse after continuous dietary dosing, and TMC125 was demonstrated to affect coagulation parameters (prothrombin time [PT], activated partial thromboplastin time [APTT], clotting factors II, VII and XI), particularly in males. This was counteracted by vitamin K supplementation, supporting the hypothesis that the effects were mediated via a vitamin K deficiency. It is therefore concluded that the observed cardiac changes were not caused by a direct cardiotoxic effect but occurred after a state of disabled clotting ability with subsequent effects on mouse cardiac muscle. Therefore, clotting times can be used as adequate safety biomarkers in clinical trials. To date, no changes have been observed at therapeutic doses of TMC125, following human monitoring of PT and APTT. One other NNRTI, Efavirenz (Sustiva), has been reported to cause prolongation of coagulation times in rats and monkeys.

Chronic drug-induced effects on contractile motion properties and cardiac biomarkers in human induced pluripotent stem cell derived cardiomyocytes.

In the pharmaceutical industry risk assessments of chronic cardiac safety liabilities are mostly performed during late stages of preclinical drug development using in vivo animal models. Here, we explored the potential of human induced pluripotent stem cell‐derived cardiomyocytes (hiPS‐CMs) to detect chronic cardiac risks such as drug‐induced cardiomyocyte toxicity.

Cross-laboratory analytical validation of the cardiac biomarker NT-proANP in rat.

INTRODUCTION Natriuretic peptides, including N-terminal-proatrial natriuretic peptide (NT-proANP) are cardiac hormones that are produced in response to myocardial stretch and have been used in rats and humans as blood based functional cardiac biomarkers. There are limited validation data of these assays in rats and therefore the Predictive Safety Testing Consortium, Cardiac Hypertrophy Working Group (PSTC-CHWG) performed a cross-laboratory (5 laboratories) analytical evaluation of a commercially available NT-proANP ELISA for use with rat samples. METHODS Serum samples were collected from normal Sprague Dawley (SD) rats and were spiked with kit calibrator material or rat heart tissue extracts to provide specimens for the validation. In addition, the cardiotoxicant, isoproterenol, was used to induce elevated endogenous NT-proANP levels in a subgroup of rats for additional validation specimens. The Biomedica™ (BI-20892, Vienna, Austria) proANP (1-98) enzyme-linked immunoabsorbent assay (ELISA) kit was used to measure NT-proANP. Intra-assay and inter-assay precisions, accuracy, sample linearity, recovery, limit of detection, upper and lower limits of quantitation (ULOQ and LLOQ, respectively), sample-freeze/thaw stability and stored sample stability were assessed and compared to pre-determined acceptance criteria. RESULTS The majority of the experimental assessments met the established validation criteria, however there were individual results that did not meet these standards. Overall, acceptable intra- and inter-assay precisions and accuracies as well as inter-laboratory precision and accuracy were demonstrated. Linearity and recovery values fell within the pre-determined acceptance criteria, samples remained stable for up to three freeze-thaw cycles and frozen samples were stable at ~-70 °C for 12 months. The limit of detection (LOD) and LLOQ and ULOQ were similar to those specified by the manufacturer. DISCUSSION Overall, the assay was demonstrated to be technically adequate for the detection of NT-proANP serum levels in SD rats.