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chemicals identified by the International Agency for Research on Cancer as likely human carcinogens and to drugs withdrawn from the market for carcinogenicity concerns confirmed their sensitivity for predicting rat carcinogenicity outcome.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.

The value of juvenile animal studies "What have we learned from preclinical juvenile toxicity studies? II".

Recent changes in the regulations from the FDA and EMA have shifted the focus of juvenile toxicity studies more to the safe use of all pharmaceuticals and the absence of label or safety information for the pediatric population. Unlike other regulatory guidance, the need or design of these animal studies is not specified. Ideally these should be decided "case-by-case" based on the patient population, pharmacology, existing toxicological and clinical data, dosing regimen, and developing system impact. Following the publishing of a small intercompany survey (Bailey and Mariën, 2009), a more extensive survey was commissioned by the ILSI/HESI DART Technical Committee to clarify what has been learned for the safety assessment for pediatrics. Contributions from 24 companies totaling 241 studies (84% rat and 14% dog) were received. In 12 of 82 programs (15%) were the existing adult preclinical or clinical data considered a sufficient safety prediction for pediatric trials. Clinical/preclinical correlates were observed in 17.2% (rat) and 42.9% (dog) of the studies and a lack of predictability from the pharmacology or the adult toxicity data was seen in 25% of rat and 14.3% of dog studies. Many of the studies were large, lengthy, complex, included parameters that mirrored the adult studies and yielded no new or useful information. We should avoid conducting complex or inappropriate studies and Contract Research Organisations and regulatory agencies have a role in encouraging more targeted designs. Only with appropriate designs can we adequately identify safety or pharmacokinetic issues, suggest clinical endpoints, and contribute to the product label.

Timing is everything for sperm assessment in fertility studies.

The fertility study design recommended in the ICH S5(R2) Harmonised Guideline for Detection of Toxicity to Reproduction for Medicinal Products emphasizes the importance of histopathological endpoints next to a pairing assessment in evaluating male fertility. However, in a male rat fertility study with JNJ-26489112, a CNS-active agent, while there were no effects on histological endpoints, mating performance or pregnancy outcomes, sperm assessment was included. The high dose males presented with reversible decreases in epididymal, but not testicular, sperm concentration and motility and an increase in abnormal sperm morphology. In view of the differences in fertility between rats and humans, these types of sperm effects in rats suggest the potential for an impact on human male fertility that would be undetected if not for the sperm assessment. Therefore, the current example suggests that including semenology as a standard endpoint in nonclinical fertility studies may be warranted.

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.

Juvenile animal testing of hydroxypropyl-ß-cyclodextrin in support of pediatric drug development.

Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) is being explored as excipient for administration of poorly soluble NCE's in pediatrics. In support of pharmaceutical development, non-clinical studies were performed to investigate whether oral and intravenous administration of HP-ß-CD showed a different response in juvenile rats versus adult rats. Juvenile rats received HP-ß-CD via the intravenous route at dose levels of 50, 200 and 400mg/kg/day from postnatal day 16 to 44, or via oral gavage at 500, 1000 and 2000mg/kg/day from postnatal day 4 to 46. In addition to in vivo parameters, toxicokinetics and post-mortem evaluations were conducted. The main findings were related to the renal excretion of intact HP-ß-CD and were regarded as non-adverse transient adaptive responses. The pathogenesis of the osmotic nephrosis-like changes are discussed. With increasing age a more effective renal clearance of HP-ß-CD is present in line with the postnatal functional maturation of the kidney. In addition, following oral administration an increase in soft stools was seen which was related to osmotic water retention in the large intestine. The findings in the juvenile studies are very similar to those observed in previously performed adult rat studies at similar dose levels, same routes and similar or longer dose duration. No novel toxicity was seen in the juvenile studies.

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.

What have we learned from pre-clinical juvenile toxicity studies?

Juvenile toxicity studies have assumed a higher priority within the pharmaceutical industry following recent changes in regulations. The requirement and designs of these studies should be on a 'case-by-case' basis. Discussions have suggested that recently the regulatory agencies have requested a more standard design incorporating multiple parameters, despite the disparity in the data available and the mode of action and indication of the respective drugs. In addition, the general perception was that studies were generating nothing new; there was no clear indication of novel toxicity or sensitivity; and the findings observed were predictable from what was already known. Clarity was therefore required both in terms of the study designs used and their usefulness and ability to generate meaningful data. This paper discusses the contributions from 10 pharmaceutical companies of 39 studies to clarify what has been learned and whether this has contributed to our greater understanding. Juvenile toxicity studies should be designed to fulfil a scientific rationale, only after first deciding what useful toxicological information might be obtained. Should a study be conducted, the endpoints must be assessed for both practicality and interpretability. Only when using appropriately designed studies can we adequately identify potential safety or pharmacokinetic issues, suggest additional clinical endpoints and/or contribute to the product label.