Andreas Mahl

Urinary clusterin, cystatin C, β2-microglobulin and total protein as markers to detect drug-induced kidney injury

Earlier and more reliable detection of drug-induced kidney injury would improve clinical care and help to streamline drug-development. As the current standards to monitor renal function, such as blood urea nitrogen (BUN) or serum creatinine (SCr), are late indicators of kidney injury, we conducted ten nonclinical studies to rigorously assess the potential of four previously described nephrotoxicity markers to detect drug-induced kidney and liver injury. Whereas urinary clusterin outperformed BUN and SCr for detecting proximal tubular injury, urinary total protein, cystatin C and β2-microglobulin showed a better diagnostic performance than BUN and SCr for detecting glomerular injury. Gene and protein expression analysis, in-situ hybridization and immunohistochemistry provide mechanistic evidence to support the use of these four markers for detecting kidney injury to guide regulatory decision making in drug development. The recognition of the qualification of these biomarkers by the EMEA and FDA will significantly enhance renal safety monitoring.

A panel of urinary biomarkers to monitor reversibility of renal injury and a serum marker with improved potential to assess renal function

The Predictive Safety Testing Consortiums first regulatory submission to qualify kidney safety biomarkers revealed two deficiencies. To address the need for biomarkers that monitor recovery from agent-induced renal damage, we scored changes in the levels of urinary biomarkers in rats during recovery from renal injury induced by exposure to carbapenem A or gentamicin. All biomarkers responded to histologic tubular toxicities to varied degrees and with different kinetics. After a recovery period, all biomarkers returned to levels approaching those observed in uninjured animals. We next addressed the need for a serum biomarker that reflects general kidney function regardless of the exact site of renal injury. Our assay for serum cystatin C is more sensitive and specific than serum creatinine (SCr) or blood urea nitrogen (BUN) in monitoring generalized renal function after exposure of rats to eight nephrotoxicants and two hepatotoxicants. This sensitive serum biomarker will enable testing of renal function in animal studies that do not involve urine collection.

Poly(ethylene carbonate)s, part II: degradation mechanisms and parenteral delivery of bioactive agents.

The degradation and drug carrier properties of poly(ethylene carbonate) (PEC) were investigated in vitro and in rats and rabbits. PEC was found to be specifically degraded in vivo and in vitro by superoxide radical anions O2-*, which are, in vivo, mostly produced by inflammatory cells. No degradation of PEC was observed in the presence of hydrolases, serum or blood. PEC is biodegraded by surface erosion without significant change in the molecular weight of the residual polymer mass. The non-hydrolytic biodegradation by cells producing O2-* is unique among the polymers used as biodegradable drug carriers. The main degradation product of PEC in aqueous systems is ethylene glycol, formed presumably by hydrolysis of ethylene carbonate. The splitting off of a five-membered ring structure from the polymer chain indicates a chain reaction mechanism for the biodegradation. PEC is a suitable drug carrier, particularly for labile drugs. Using human interleukin-3 and octreotide as model drugs, surface erosion of the PEC formulations was indicated by a 1:1 correlation between drug release and polymer mass loss.

Comparison of clinical pathology parameters with two different blood sampling techniques in rats: retrobulbar plexus versus sublingual vein

Blood samples were taken from the retrobulbar venous plexus or the sublingual vein of male HanIbm:Wist rats to compare clinical pathology parameters between the two sampling techniques. By analogy with a pharmacokinetic study, blood was sampled six times during one day from unfasted animals. After 3 weeks of recovery, blood was taken from fasted animals on a single occasion. In addition, prolactin and corticosterone levels were determined to compare stress-related effects between the two sampling methods. Body weight development and food consumption were similar after single as well as after repeated blood sampling for the two blood sampling techniques. Haemotological evaluation showed a gradual decrease in erythrocyte count, haemoglobin concentration and haematocrit after repeated blood sampling. Repeated withdrawal of blood samples over 24 h corresponding to approximately 22% of the total blood volume resulted in a decrease in red blood cell parameters by up to 30%. The withdrawal of approximately 10% of the total blood volume was associated with a decrease in these parameters by up to 10% and should not be exceeded for animal welfare reasons and to allow a reliable evaluation of data in a study. Repeated blood sampling was associated with an initial decrease in the number of white blood cells, mainly due to a reduction in lymphocytes; white blood cell counts were slightly increased one day after. The decrease in lymphocytes and the increase in neutrophils after repeated sampling were generally slightly more pronounced in the blood from the retrobulbar plexus than from the sublingual vein. Comparison of serum clinical chemistry data showed significantly higher activities of creatine kinase and aspartate aminotransferase in samples from the retrobulbar plexus. These findings suggest a higher degree of tissue damage with blood sampling from the retrobulbar plexus than from the sublingual vein. Despite a large inter-individual variability, higher mean values of prolactin on each occasion and corticosterone after a single sample in fasted animals indicate a higher stress associated with blood sampling from the retrobulbar plexus.

Preclinical evaluation of potential nilotinib cardiotoxicity

In vitro, concentrations ≥ 10 μM of nilotinib were needed to induce markers of cytotoxicity, apoptosis, and endoplasmic reticulum stress in both neonatal rat ventricular myocytes, a putative target tissue, and non-target heart fibroblasts, indicating a lack of cardiomyocyte-specific nilotinib toxicity in vitro. In rats, oral nilotinib treatment at 80 mg/kg for 4 weeks induced increased heart weight; however, this was not associated with relevant histopathological changes or effects on heart function. Thus, nilotinib at and above clinically relevant concentrations (4.27 μM) did not induce overt cardiovascular pathologies or heart failure in vitro or in vivo under study conditions.

LAV694, a new antiproliferative agent showing improved skin tolerability vs. clinical standards for the treatment of actinic keratosis

The skin tolerability of the tubulin polymerisation inhibitor LAV694 was compared to that of 5% 5-fluorouracil (5-FU) and 0.5% podophyllotoxin in vitro using a human reconstructed epidermis (HRE), and in vivo using minipigs. Topical treatment of HRE for 1 or 3 days with a 0.2, 0.6 or 1% LAV694 cream or the placebo showed no signs of irritation in terms of morphology, cell viability (lactate dehydrogenase leakage) or interleukin-8 mRNA expression and release. 5-FU increased interleukin-8 production and induced morphological signs of irritation. The substances were also applied under occlusion to the back of two minipigs, twice daily, for 9 days to allow intraindividual comparison of skin effects and tolerability. Skin reactions were monitored by visual scoring, chromometry, pro-inflammatory activity, cell cycle and apoptosis by RT-PCR, laser scanning cytometry and histopathological examination of biopsies. Application of podophyllotoxin and 5-FU had to be stopped on days 4 and 8, respectively, due to severe skin lesions. LAV694 (1%) induced only moderate skin reddening after 9 days. 5-FU and podophyllotoxin, but not LAV694, increased mRNA expression of pro-inflammatory cytokines. LAV694 arrested keratinocytes in the M phase of the cell cycle and apoptosis was detected histologically in the basal layer. LAV694 increased the expression of pro-apoptotic genes in both experimental models. In conclusion, LAV694 selectively induced apoptosis, rather than necrosis, of growth-arrested keratinocytes, thus avoiding the occurrence of extensive inflammation. This resulted in an improved skin tolerability in comparison with 5-FU and podophyllotoxin.

Editor’s Highlight: Comparative Renal Safety Assessment of the Hepatitis B Drugs, Adefovir, Tenofovir, Telbivudine and Entecavir in Rats

The aim of this study was to determine the relative safety of 4 antiviral drugs (telbivudine, tenofovir, adefovir, and entecavir) against hepatitis B virus with respect to kidney function and toxicity in male Sprague Dawley rats. The antiviral drugs were administered once daily for 4 weeks by oral gavage at ∼10 and 25–40 times the human equivalent dose. Main assessments included markers of renal toxicity in urine, magnetic resonance imaging (MRI) of kidney function, histopathology, and electron microscopic examination. Administration of adefovir at 11 and 28 mg/kg for 4 weeks caused functional and morphological kidney alterations in a time- and dose-dependent manner, affecting mainly the proximal tubules and suggesting a mechanism of toxicity related to mitochondrial degeneration/depletion. Of note, the observed adefovir-induced reduction of kidney function was not detected by the standard method of glomerular filtration rate (GFR) measurements (clearance rate of the endogenous marker, creatinine), thereby emphasizing the superiority of MRI in terms of sensitive detection of GFR in rats. For the low dose of 300 mg/kg of tenofovir, minor kidney effects such as nuclear enlargement in the tubular epithelium, and hyaline droplets accumulation were detected, which was also observed for the low dose (11 mg/kg) of adefovir. No assessments could be done at the higher dose of 600/1000 mg/kg tenofovir due to gastrointestinal tract toxicity which prevented treatment of the animals for longer than 1 week. Entecavir at 1 and 3 mg/kg and telbivudine at 600 and 1600 mg/kg caused no toxicologically relevant effects on the kidney.

Spontaneous and Drug-induced Arteritis/Polyarteritis in the Göttingen Minipig—Review

Arteritis/polyarteritis occurs spontaneously in many species used in preclinical toxicology studies. In Göttingen minipigs, arteritis/polyarteritis is an occasionally observed background change. In the minipig, this finding differs in frequency and nature from age-related polyarteritis nodosa in rats or monkeys, and Beagle pain syndrome in dogs. In minipigs, it can be present in a single small- or medium-sized artery of an organ or a few organs and is most commonly recorded in the cardiac and extracardiac blood vessels, vagina, oviduct, rectum, epididymis, spinal cord, pancreas, urinary bladder, kidneys, and stomach. The etiology is unknown although it has been considered in minipigs as well as in rats, dogs, and monkeys to be possibly immune mediated. This background change is important with respect to its nature and distribution in the minipig in order to distinguish it from drug-induced vascular changes, which might occur in similar locations and have similar morphologic features. This review summarizes the morphology, incidence, and predilection sites of arteritis as a spontaneously occurring background change and as a drug-induced vasculopathy in the minipig, and also describes the main aspects to consider when evaluating vascular changes in Göttingen minipig toxicity studies and their human relevance.

The Potential of Minipigs in the Development of Anticancer Therapeutics: Species Comparison and Examples of Special Applications

Minipigs are increasingly being used as an alternative to dog or monkey in nonclinical safety testing of pharmaceuticals since they share similar anatomical and physiological characteristics to humans. Integrative assessment of pharmacodynamic and pharmacokinetic data sets of drug candidates from in silico, in vitro, and in vivo investigations form the basis for selecting the most relevant nonrodent species for toxicology studies. Developing anticancer therapeutics represents a special challenge for species selection due to their effects on multiple organ systems. The toxicological profile of anticancer drugs can be associated with steep dose–response curves, especially due to dose-limiting toxicity on the alimentary, hematopoietic, and immune systems. Selection of an appropriate species for toxicology studies is of importance to avoid an inappropriately low (without benefit for the late-stage cancer patient) or high clinical starting dose (with a risk of unexpected adverse reactions). Although the minipig has been the preferred species to develop drugs applied topically, it is only rarely used in anticancer drug development compared to dog and monkey. In this context, we discuss the potential of minipigs in anticancer drug development with examples of programs for oral and dermal administration, intravascular application in drug-eluting stents, and local chemotherapy (chemoembolization).