Sandra De Jonghe

An Analysis of Pharmaceutical Experience with Decades of Rat Carcinogenicity Testing: Support for a Proposal to Modify Current Regulatory Guidelines

Data collected from 182 marketed and nonmarketed pharmaceuticals demonstrate that there is little value gained in conducting a rat two-year carcinogenicity study for compounds that lack: (1) histopathologic risk factors for rat neoplasia in chronic toxicology studies, (2) evidence of hormonal perturbation, and (3) positive genetic toxicology results. Using a single positive result among these three criteria as a test for outcome in the two-year study, fifty-two of sixty-six rat tumorigens were correctly identified, yielding 79% test sensitivity. When all three criteria were negative, sixty-two of seventy-six pharmaceuticals (82%) were correctly predicted to be rat noncarcinogens. The fourteen rat false negatives had two-year study findings of questionable human relevance. Applying these criteria to eighty-six additional chemicals identified by the International Agency for Research on Cancer as likely human carcinogens and to drugs withdrawn from the market for carcinogenicity concerns confirmed their sensitivity for predicting rat carcinogenicity outcome. These analyses support a proposal to refine regulatory criteria for conducting a two-year rat study to be based on assessment of histopathologic findings from a rat six-month study, evidence of hormonal perturbation, genetic toxicology results, and the findings of a six-month transgenic mouse carcinogenicity study. This proposed decision paradigm has the potential to eliminate over 40% of rat two-year testing on new pharmaceuticals without compromise to patient safety.

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.

Inflammatory monocytes recruited to the liver within 24 hours after virus-induced inflammation, resemble Kupffer cells, but are functionally distinct

ABSTRACT Due to a scarcity of immunocompetent animal models for viral hepatitis, little is known about the early innate immune responses in the liver. In various hepatotoxic models, both pro- and anti-inflammatory activities of recruited monocytes have been described. In this study, we compared the effect of liver inflammation induced by the Toll-like receptor 4 ligand lipopolysaccharide (LPS) with that of a persistent virus, lymphocytic choriomeningitis virus (LCMV) clone 13, on early innate intrahepatic immune responses in mice. LCMV infection induces a remarkable influx of inflammatory monocytes in the liver within 24 h, accompanied by increased transcript levels of several proinflammatory cytokines and chemokines in whole liver. Importantly, while a single LPS injection results in similar recruitment of inflammatory monocytes to the liver, the functional properties of the infiltrating cells are dramatically different in response to LPS versus LCMV infection. In fact, intrahepatic inflammatory monocytes are skewed toward a secretory phenotype with impaired phagocytosis in LCMV-induced liver inflammation but exhibit increased endocytic capacity after LPS challenge. In contrast, F4/80high-Kupffer cells retain their steady-state endocytic functions upon LCMV infection. Strikingly, the gene expression levels of inflammatory monocytes dramatically change upon LCMV exposure and resemble those of Kupffer cells. Since inflammatory monocytes outnumber Kupffer cells 24 h after LCMV infection, it is highly likely that inflammatory monocytes contribute to the intrahepatic inflammatory response during the early phase of infection. Our findings are instrumental in understanding the early immunological events during virus-induced liver disease and point toward inflammatory monocytes as potential target cells for future treatment options in viral hepatitis. IMPORTANCE Insights into how the immune system deals with hepatitis B virus (HBV) and HCV are scarce due to the lack of adequate animal model systems. This knowledge is, however, crucial to developing new antiviral strategies aimed at eradicating these chronic infections. We model virus-host interactions during the initial phase of liver inflammation 24 h after inoculating mice with LCMV. We show that infected Kupffer cells are rapidly outnumbered by infiltrating inflammatory monocytes, which secrete proinflammatory cytokines but are less phagocytic. Nevertheless, these recruited inflammatory monocytes start to resemble Kupffer cells on a transcript level. The specificity of these cellular changes for virus-induced liver inflammation is corroborated by demonstrating opposite functions of monocytes after LPS challenge. Overall, this demonstrates the enormous functional and genetic plasticity of infiltrating monocytes and identifies them as an important target cell for future treatment regimens.

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.

The effect of macrophage and angiogenesis inhibition on the drug release and absorption from an intramuscular sustained-release paliperidone palmitate suspension

The intramuscular (IM) administration of long-acting injectable (LAI) aqueous nano-/microsuspensions elicits a chronic granulomatous injection site reaction, which recently has been hypothesized to drive the (pro)drug dissolution and systemic absorption resulting in flip-flop pharmacokinetics. The goal of this mechanistic study was to investigate the effects of the local macrophage infiltration and angiogenesis on the systemic drug exposure following a single IM administration of a paliperidone palmitate (PP) LAI nano-/microsuspension in the rat. Liposomal clodronate (CLO) and sunitinib (SNT) were co-administered to inhibit the depot infiltration and nano-/microparticle phagocytosis by macrophages, and the neovascularization of the depot, respectively. Semi-quantitative histopathology of the IM administration sites at day 1, 3, 7, 14, 21 and 28 after dosing with PP-LAI illustrated that CLO significantly decreased the rate and extent of the granulomatous inflammatory reaction. The macrophage infiltration was slowed down, but only partially suppressed by CLO and this translated in paliperidone (PAL) plasma concentration-time profiles that resembled those observed upon injection of PP-LAI only, albeit with a lower PAL input rate and delayed maximum plasma concentration (CMAX). Conversely, SNT treatment completely suppressed the granulomatous reaction, besides effectively inhibiting the neovascularization of the PP-LAI depot. This resulted in an even slower systemic PAL input with delayed and lower maximum PAL CMAX. The reduced PP-LAI lymph node retention after CLO and SNT treatment, as well as pharmacokinetic drug-drug interactions were rejected as possible sources of the observed pharmacokinetic differences. The biphasic PAL plasma concentration-time profiles could best be described by an open first-order disposition model with parallel fast (first-order) and slow (sequential zero-first-order) absorption. The correlation of the pharmacokinetic data with the histopathological findings indicated that the macrophage infiltration, with subsequent phagocytosis of an important fraction of the PP-LAI dose, actively contributed to the observed PAL plasma exposures by promoting the prodrug dissolution and conversion to the active. An initial fast PP dissolution of individual nano-/microcrystals present in the interstitium was followed by a second, slower, but dominating input process that was driven by the PAL formation rate in the infiltrated portions of the LAI depot. The present work provides new fundamental insights into the influence of the local tissue response to IM LAI (pro)drug suspensions on the systemic drug exposure. This knowledge might support the future development of predictive in vitro and in silico models, which could help guide the LAI formulation design.

Antiviral Activity of TMC353121, a Respiratory Syncytial Virus (RSV) Fusion Inhibitor, in a Non-Human Primate Model.

Background The study assessed the antiviral activity of TMC353121, a respiratory syncytial virus (RSV) fusion inhibitor, in a preclinical non-human primate challenge model with a viral shedding pattern similar to that seen in humans, following continuous infusion (CI). Methods African green monkeys were administered TMC353121 through CI, in 2 studies. Study 1 evaluated the prophylactic and therapeutic efficacy of TMC353121 at a target plasma level of 50 ng/mL (n=15; Group 1: prophylactic arm [Px50], 0.033 mg/mL TMC353121, flow rate 2.5 mL/kg/h from 24 hours pre-infection to 10 days; Group 2: therapeutic arm [Tx50], 0.033mg/mL TMC353121 from 24 hours postinfection to 8 days; Group 3: control [Vh1] vehicle, 24 hours post-infection to 8 days). Study 2 evaluated the prophylactic efficacy of TMC353121 at target plasma levels of 5 and 500 ng/mL (n=12; Group 1: prophylactic 5 arm [Px5], 0.0033 mg/mL TMC353121, flow rate 2.5 mL/kg/h from 72 hours pre-infection to 14 days; Group 2: prophylactic 500 arm [Px500], 0.33 mg/mL TMC353121; Group 3:control [Vh2] vehicle, 14 days). Bronchoalveolar lavage fluid and plasma were collected every 2 days from day 1 postinfection for pharmacokinetics and safety analysis. Findings TMC353121 showed a dose-dependent antiviral activity, varying from 1log10 reduction of peak viral load to complete inhibition of the RSV replication. Complete inhibition of RSV shedding was observed for a relatively low plasma exposure (0.39 μg/mL) and was associated with a dose-dependent reduction in INFγ, IL6 and MIP1α. TMC353121 administered as CI for 16 days was generally well-tolerated. Conclusion TMC353121 exerted dose-dependent antiviral effect ranging from full inhibition to absence of antiviral activity, in a preclinical model highly permissive for RSV replication. No new safety findings emerged from the study.

Successful integration of nonclinical and clinical findings in interpreting the clinical relevance of rodent neoplasia with a new chemical entity.

Canagliflozin, a sodium glucose co-transporter 2 (SGLT2) inhibitor, has been developed for the treatment of adults with type 2 diabetes mellitus (T2DM). During the phase 3 program, treatment-related pheochromocytomas, renal tubular tumors, and testicular Leydig cell tumors were reported in the 2-year rat toxicology study. Treatment-related tumors were not seen in the 2-year mouse study. A cross-functional, mechanism-based approach was undertaken to determine whether the mechanisms responsible for tumorigenesis in the rat were of relevance to humans. Based on findings from nonclinical and clinical studies, the treatment-related tumors observed in rats were not deemed to be of clinical relevance. Here, we describe the scientific and regulatory journey from learning of the 2-year rat study findings to the approval of canagliflozin for the treatment of T2DM.

Modeling the Time Course of the Tissue Responses to Intramuscular Long-acting Paliperidone Palmitate Nano-/Microcrystals and Polystyrene Microspheres in the Rat

Long-acting injectable (LAI) drug suspensions consist of drug nano-/microcrystals suspended in an aqueous vehicle and enable prolonged therapeutic drug exposure up to several months. The examination of injection site reactions (ISRs) to the intramuscular (IM) injection of LAI suspensions is relevant not only from a safety perspective but also for the understanding of the pharmacokinetics. The aim of this study was to perform a multilevel temporal characterization of the local and lymphatic histopathological/immunological alterations triggered by the IM injection of an LAI paliperidone palmitate suspension and an analog polystyrene suspension in rats and identify critical time points and parameters with regard to the host response. The ISRs showed a moderate to marked chronic granulomatous inflammation, which was mediated by multiple cyto-/chemokines, including interleukin-1β, monocyte Chemoattractant Protein-1, and vascular endothelial growth factor. Lymphatic uptake and lymph node retention of nano-/microparticles were observed, but the contribution to the drug absorption was negligible. A simple image analysis procedure and empirical model were proposed for the accurate evaluation of the depot geometry, cell infiltration, and vascularization. This study was designed as a reference for the evaluation and comparison of future LAIs and to support the mechanistic modeling of the formulation–physiology interplay regulating the drug absorption from LAIs.

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.