Peter Hoffmann

Scientific review and recommendations on preclinical cardiovascular safety evaluation of biologics.

Biological therapeutic agents (biologicals), such as monoclonal antibodies (mAbs), are increasingly important in the treatment of human disease, and many types of biologicals are in clinical development. During preclinical drug development, cardiovascular safety pharmacology studies are performed to assess cardiac safety in accord with the ICH S7A and S7B regulations that guide these studies. The question arises, however, whether or not it is appropriate to apply these guidelines, which were devised primarily to standardize small molecule drug testing, to the cardiovascular evaluation of biologicals. We examined the scientific literature and formed a consensus of scientific opinion to determine if there is a rational basis for conducting an in vitro hERG assay as part of routine preclinical cardiovascular safety testing for biologicals. We conclude that mAb therapeutics have very low potential to interact with the extracellular or intracellular (pore) domains on hERG channel and, therefore, are highly unlikely to inhibit hERG channel activity based on their targeted, specific binding properties. Furthermore, mAb are large molecules (>140,000 Da) that cannot cross plasma membranes and therefore would be unable to access and block the promiscuous inner pore of the hERG channel, in contrast with typical small molecule drugs. Consequently, we recommend that it is not appropriate to conduct an in vitro hERG assay as part of a preclinical strategy for assessing the heart rate corrected QT interval (QTc) prolongation risk of mAbs and other types of biologicals. It is more appropriate to assess QTc risk by integrating cardiovascular endpoints into repeat-dose general toxicology studies performed in an appropriate non-rodent species. These recommendations should help shape future regulatory strategy and discussions for the cardiovascular safety pharmacology testing of mAbs as well as other biologicals and provide guidance for the preclinical cardiovascular evaluation of such agents.

Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound.

AMP397 is a novel antiepileptic agent and the first competitive AMPA antagonist with high receptor affinity, good in vivo potency, and oral activity. AMP397 has a structural alert (aromatic nitro group) and was mutagenic in Salmonella typhimurium strains TA97a, TA98 and TA100 without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. The amino derivative of AMP397 was negative in wild-type strains TA98 and TA100. AMP397 was negative in a mouse lymphoma tk assay, which included a 24h treatment without S9. A weak micronucleus induction in vitro was found at the highest concentrations tested in V79 cells with S9. AMP397 was negative in the following in vivo studies, which included the maximum tolerated doses of 320mg/kg in mice and 2000mg/kg in rats: MutaMouse assay in colon and liver (5x320mg/kg) at three sampling times (3, 7 and 31 days after the last administration); DNA binding study in the liver of mice and rats after a single treatment with [14C]-AMP397; comet assay (1x2000mg/kg) in jejunum and liver of rats, sampling times 3 and 24h after administration; micronucleus test (2x320mg/kg) in the bone marrow of mice, sampling 24h after the second administration. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. In particular, no genotoxic metabolite is formed in mammalian cells, and, if formed by intestinal bacteria, is unable to exert any genotoxic activity in the adjacent intestinal tissue. These data were considered to provide sufficient safety to initiate clinical development of the compound.

Investigation of the Potential of Clozapine to Cause Torsade de Pointes

Antipsychotics are frequently associated with QTc interval prolongation, a proposed marker for vulnerability to fatal ventricular arrhythmias, e.g. torsade de pointes (TdP). Little has been published on this topic in relation to clozapine. The objectives of this review were to: (i) calculate the frequency of QTc interval prolongation, T-wave abnormalities, TdP, ventricular tachycardia/fibrillation and sudden unexplained death in patients treated with clozapine and thioridazine from clinical trial and post-marketing reports; (ii) to compare these data with published findings for haloperidol, risperidone, olanzapine, sertindole and ziprasidone; and (iii) to correlate these clinical data with results from preclinical tests presently considered to be of predictive value for a compound’s potential to cause QTc interval prolongation and TdP.A review of the global Novartis databases for clozapine and thioridazine and a Medline/Internet search for information on these cardiac events and for preclinical effects on the human ether-a-go-go related gene channels, action potential duration, and QT interval changes produced by the selected antipsychotics were performed.The clozapine database (2.8 million patient-years spanning 27 years) demonstrated that at therapeutic doses all but three reports of QTc interval prolongation and both of TdP were confounded by relevant co-medication/comorbidity. The literature review revealed that all antipsychotics considered except clozapine induced TdP and/or QTc interval prolongation at therapeutic doses. Preclinical in vitro tests appear to overestimate the risk of clozapine, haloperidol and risperidone to prolong QTc interval in patients and underestimate such a risk for sertindole and ziprasidone. Extrapolation of in vitro results to clinical events requires qualified interpretation.

Cardiac Safety Implications of hNav1.5 Blockade and a Framework for Pre-Clinical Evaluation

The human cardiac sodium channel (hNav1.5, encoded by the SCN5A gene) is critical for action potential generation and propagation in the heart. Drug-induced sodium channel inhibition decreases the rate of cardiomyocyte depolarization and consequently conduction velocity and can have serious implications for cardiac safety. Genetic mutations in hNav1.5 have also been linked to a number of cardiac diseases. Therefore, off-target hNav1.5 inhibition may be considered a risk marker for a drug candidate. Given the potential safety implications for patients and the costs of late stage drug development, detection, and mitigation of hNav1.5 liabilities early in drug discovery and development becomes important. In this review, we describe a pre-clinical strategy to identify hNav1.5 liabilities that incorporates in vitro, in vivo, and in silico techniques and the application of this information in the integrated risk assessment at different stages of drug discovery and development.

Nonclinical Safety Biomarkers of Drug-induced Vascular Injury Current Status and Blueprint for the Future

Better biomarkers are needed to identify, characterize, and/or monitor drug-induced vascular injury (DIVI) in nonclinical species and patients. The Predictive Safety Testing Consortium (PSTC), a precompetitive collaboration of pharmaceutical companies and the U.S. Food and Drug Administration (FDA), formed the Vascular Injury Working Group (VIWG) to develop and qualify translatable biomarkers of DIVI. The VIWG focused its research on acute DIVI because early detection for clinical and nonclinical safety monitoring is desirable. The VIWG developed a strategy based on the premise that biomarkers of DIVI in rat would be translatable to humans due to the morphologic similarity of vascular injury between species regardless of mechanism. The histomorphologic lexicon for DIVI in rat defines degenerative and adaptive findings of the vascular endothelium and smooth muscles, and characterizes inflammatory components. We describe the mechanisms of these changes and their associations with candidate biomarkers for which advanced analytical method validation was completed. Further development is recommended for circulating microRNAs, endothelial microparticles, and imaging techniques. Recommendations for sample collection and processing, analytical methods, and confirmation of target localization using immunohistochemistry and in situ hybridization are described. The methods described are anticipated to aid in the identification and qualification of translational biomarkers for DIVI.

The evaluation of drug-induced changes in cardiac inotropy in dogs: Results from a HESI-sponsored consortium.

INTRODUCTION Drug-induced effects on the cardiovascular system remain a major cause of drug attrition. While hemodynamic (blood pressure (BP) and heart rate (HR)) and electrophysiological methods have been used in testing drug safety for years, animal models for assessing myocardial contractility are used less frequently and their translation to humans has not been established. The goal of these studies was to determine whether assessment of contractility and hemodynamics, when measured across different laboratories using the same protocol, could consistently detect drug-induced changes in the inotropic state of the heart using drugs known to have clinically relevant positive and negative effects on myocardial contractility. METHODS A 4×4 double Latin square design (n=8) design using Beagle dogs was developed. Drugs were administrated orally. Arterial blood pressure, left ventricular pressure (LVP) and the electrocardiogram were assessed. Each of the six laboratories studied at least 2 drugs (one positive inotrope (pimobendan or amrinone) and one negative inotrope) (itraconazole or atenolol) at 3 doses selected to match clinical exposure data and a vehicle control. Animals were instrumented with an ITS telemetry system, DSIs D70-PCTP system or DSIs Physiotel Digital system. Data acquisition and analysis systems were Ponemah, Notocord or EMKA. RESULTS Derived parameters included: diastolic, systolic and mean arterial BP, peak systolic LVP, HR, end-diastolic LVP, and LVdP/dtmax as the primary contractility index. Blood samples were drawn to confirm drug exposures predicted from independent pharmacokinetic studies. Across the laboratories, a consistent change in LVdP/dtmax was captured despite some differences in the absolute values of some of the hemodynamic parameters prior to treatment. DISCUSSION These findings indicate that this experimental model, using the chronically instrumented conscious dog, can accurately and consistently detect changes in cardiac contractility, across multiple sites and instrumentation systems, and that data obtained in this model may also translate to clinical outcomes.

Vascular Origin of Vildagliptin-induced Skin Effects in Cynomolgus Monkeys Pathomechanistic Role of Peripheral Sympathetic System and Neuropeptide Y

The purpose of this article is to characterize skin lesions in cynomolgus monkeys following vildagliptin (dipeptidyl peptidase-4 inhibitor) treatment. Oral vildagliptin administration caused dose-dependent and reversible blister formation, peeling and flaking skin, erosions, ulcerations, scabs, and sores involving the extremities at ≥5 mg/kg/day and necrosis of the tail and the pinnae at ≥80 mg/kg/day after 3 weeks of treatment. At the affected sites, the media and the endothelium of dermal arterioles showed hypertrophy/hyperplasia. Skin lesion formation was prevented by elevating ambient temperature. Vildagliptin treatment also produced an increase in blood pressure and heart rate likely via increased sympathetic tone. Following treatment with vildagliptin at 80 mg/kg/day, the recovery time after lowering the temperature in the feet of monkeys and inducing cold stress was prolonged. Ex vivo investigations showed that small digital arteries from skin biopsies of vildagliptin-treated monkeys exhibited an increase in neuropeptide Y–induced vasoconstriction. This finding correlated with a specific increase in NPY and in NPY1 receptors observed in the skin of vildagliptin-treated monkeys. Present data provide evidence that skin effects in monkeys are of vascular origin and that the effects on the NPY system in combination with increased peripheral sympathetic tone play an important pathomechanistic role in the pathogenesis of cutaneous toxicity.

Osilodrostat (LCI699), a potent 11β-hydroxylase inhibitor, administered in combination with the multireceptor-targeted somatostatin analog pasireotide: A 13-week study in rats

The somatostatin analog pasireotide and the 11β-hydroxylase inhibitor osilodrostat (LCI699) reduce cortisol levels by distinct mechanisms of action. There exists a scientific rationale to investigate the clinical efficacy of these two agents in combination. This manuscript reports the results of a toxicology study in rats, evaluating different doses of osilodrostat and pasireotide alone and in combination. Sixty male and 60 female rats were randomized into single-sex groups to receive daily doses of pasireotide (0.3mg/kg/day, subcutaneously), osilodrostat (20mg/kg/day, orally), osilodrostat/pasireotide in combination (low dose, 1.5/0.03mg/kg/day; mid-dose, 5/0.1mg/kg/day; or high dose, 20/0.3mg/kg/day), or vehicle for 13weeks. Mean body-weight gains from baseline to Week 13 were significantly lower in the pasireotide-alone and combined-treatment groups compared to controls, and were significantly higher in female rats receiving osilodrostat monotherapy. Osilodrostat and pasireotide monotherapies were associated with significant changes in the histology and mean weights of the pituitary and adrenal glands, liver, and ovary/oviduct. Osilodrostat alone was associated with adrenocortical hypertrophy and hepatocellular hypertrophy. In combination, osilodrostat/pasireotide did not exacerbate any target organ changes and ameliorated the liver and adrenal gland changes observed with monotherapy. Cmax and AUC0-24h of osilodrostat and pasireotide increased in an approximately dose-proportional manner. In conclusion, the pasireotide and osilodrostat combination did not exacerbate changes in target organ weight or toxicity compared with either monotherapy, and had an acceptable safety profile; addition of pasireotide to the osilodrostat regimen may attenuate potential adrenal gland hyperactivation and hepatocellular hypertrophy, which are potential side effects of osilodrostat monotherapy.

Translation Strategy for the Qualification of Drug-induced Vascular Injury Biomarkers

Drug-induced vascular injury (DIVI) is a common preclinical toxicity usually characterized by hemorrhage, vascular endothelial and smooth muscle damage, and inflammation. DIVI findings can cause delays or termination of drug candidates due to low safety margins. The situation is complicated by the absence of sensitive, noninvasive biomarkers for monitoring vascular injury and the uncertain relevance to humans. The Safer And Faster Evidence-based Translation (SAFE-T) consortium is a public–private partnership funded within the European Commission’s Innovative Medicines Initiative (IMI) aiming to accelerate drug development by qualifying biomarkers for drug-induced organ injuries, including DIVI. The group is using patients with vascular diseases that have key histomorphologic features (endothelial damage, smooth muscle damage, and inflammation) in common with those observed in DIVI, and has selected candidate biomarkers associated with these features. Studied populations include healthy volunteers, patients with spontaneous vasculitides and other vascular disorders. Initial results from studies with healthy volunteers and patients with vasculitides show that a panel of biomarkers can successfully discriminate the population groups. The SAFE-T group plans to seek endorsement from health authorities (European Medicines Agency and Food and Drug Administration) to qualify the biomarkers for use in regulatory decision-making processes.

Aliskiren toxicity in juvenile rats is determined by ontogenic regulation of intestinal P-glycoprotein expression.

Juvenile rat toxicity studies with the direct renin inhibitor aliskiren were initiated to support treatment in the pediatric population. In Study 1, aliskiren was administered orally to juvenile rats at doses of 0, 30, 100 or 300 mg/kg/day with repeated dosing from postpartum day (PPD) 8 to PPD 35/36. In-life, clinical pathology, anatomic pathology, and toxicokinetics evaluations were performed. In Study 2, single oral doses of aliskiren (0, 100 or 300 mg/kg) were given to 14-, 21-, 24-, 28-, 31- or 36-day-old rats; in-life data and toxicokinetics were evaluated. Study 3 was a single dose (3 mg/kg i.v.) pharmacokinetic study in juvenile rats on PPD 8, 14, 21 and 28. In Study 4, naïve rats were used to investigate ontogenic changes of the multidrug-resistant protein 1 (MDR1) and the organic anion transporting polypeptide (OATP) mRNA in several organs. Oral administration of aliskiren at 100 and 300 mg/kg caused unexpected mortality and severe morbidity in 8-day-old rats. Aliskiren plasma and tissue concentrations were increased in rats aged 21days and younger. Expression of MDR1 and OATP mRNA in the intestine, liver and brain was significantly lower in very young rats. In conclusion, severe toxicity and increased exposure in very young rats after oral administration of aliskiren are considered to be the result of immature drug transporter systems. Immaturity of MDR1 in enterocytes appears to be the most important mechanism responsible for the high exposure.