Translation of nonclinical to clinical safety findings for 27 biotherapeutics.

Human adverse drug reactions (ADRs), and in vivo nonclinical adverse and nonadverse findings, were identified in 27 biotherapeutic programs and placed into organ categories to determine translation. The sensitivity of detecting human ADRs was 30.8% with a positive predictive value (PPV) of 53.3% for nonclinical adverse findings; sensitivity increased to 67.3% and PPV fell to 35.0% when including nonadverse findings. Nonclinical findings were associated with a greater likelihood of a human ADR in that organ category, especially for adverse findings [positive likelihood ratio (LR+) >10 (lower 95% confidence interval [CI] of >5)]. The specificity and negative predictive value (NPV) were very high (>85%). A lack of nonclinical findings in an organ category was associated with a lower likelihood of a human ADR in that organ category. About 40-50% of human ADRs and nonclinical adverse findings, and about 30% of nonclinical nonadverse findings, were attributed to pharmacology. Slightly more than half of the human ADRs with a translating nonclinical finding had findings in animals that could be considered very similar. Overall, 38% of nonclinical findings translated to a human ADR at the organ category level. When nonclinical findings did not translate to humans, the cause was usually higher exposures or longer dosing in animals. All programs with human ADRs attributed to immunogenicity also had nonclinical adverse or nonadverse findings related to immunogenicity. Overall, nonclinical adverse and nonadverse findings were useful in predicting human ADRs, especially at an organ category level, and the majority of human ADRs were predicted by nonclinical toxicity studies.

Development challenges associated with rAAV-based gene therapies.

The number of gene therapies in development continues to increase, as they represent a novel method to treat, and potentially cure, many diseases. Gene therapies can be conducted with an in vivo or ex vivo approach, to cause gene augmentation, gene suppression, or genomic editing. Adeno-associated viruses are commonly used to deliver gene therapies, but their use is associated with several manufacturing, nonclinical and clinical challenges. As these challenges emerge, regulatory agency expectations continue to evolve. Following administration of rAAV-based gene therapies, nonclinical toxicities may occur, which includes immunogenicity, hepatotoxicity, neurotoxicity, and the potential risks for insertional mutagenesis and subsequent tumorgenicity. The mechanism for these findings and translation into the clinical setting are unclear at this time but have influenced the nonclinical studies that regulatory agencies are increasingly requesting to support clinical trials and marketing authorizations. These evolving regulatory expectations and toxicities, as well as future nonclinical considerations, are discussed herein.

Central nervous system tumors in 2-year rat carcinogenicity studies: perspectives on human risk assessment.

Rodent in vivo carcinogenicity bioassays are required for human risk assessment and have been utilized in this capacity for decades. Accordingly, there is an abundance of data that could be accessed and analyzed to better understand the translatability of xenobiotic-induced rodent tumors to human risk assessment. In the past decade, various groups have published assessments of the value garnered by these life-time rodent studies. Results and recommendations from the International Council for Harmonization Expert Working Group (ICH-S1 EWG) on the predictability of the current testing paradigm and proposal for an integrated approach to human carcinogenicity risk assessment are pending. Central nervous system (CNS) tumors in rats are rare and translatability to human remains unknown. This review focuses on microglial cell tumors (MCT) of the CNS in rats including its classification, nomenclature, incidence and translatability to human risk assessment. Based on emerging immunohistochemistry (IHC) characterization, glial tumors previously thought of astrocytic origin are more likely MCTs. These may be considered rodent specific and glucose dysregulation may be one component contributing to their formation. Based on review of the literature, MCTs are rarely diagnosed in humans, thus this tumor type may be rat-specific. We propose to include MCTs as a tumor type in revised International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) classification and all glial tumors to be classified as MCTs unless proven otherwise by IHC.

Cardio-renal safety of non-steroidal anti-inflammatory drugs.

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely used therapeutic class in clinical medicine. These are sub-divided based on their selectivity for inhibition of cyclooxygenase (COX) isoforms (COX-1 and COX-2) into: (1) non-selective (ns-NSAIDs), and (2) selective NSAIDs (s-NSAIDs) with preferential inhibition of COX-2 isozyme. The safety and pathophysiology of NSAIDs on the renal and cardiovascular systems have continued to evolve over the years following short- and long-term treatment in both preclinical models and humans. This review summarizes major learnings on cardiac and renal complications associated with pharmaceutical inhibition of COX-1 and COX-2 with focus on preclinical to clinical translatability of cardio-renal data.

Inflammatory Cell Findings in the Female Rabbit Heart and Stress-associated Exacerbation with Handling and Procedures Used in Nonclinical Studies.

Despite the use of rabbits in biomedical research, including regulatory toxicology and cardiovascular studies, little data exist on heart findings in this species. This study was designed to document myocardial findings in female rabbits and the impact of study-related procedures typical for vaccine toxicology studies. One hundred and forty 6- to 8-month-old female New Zealand White rabbits were divided equally into 2 groups, high and low study procedure groups (group 1 and group 2, respectively). All animals received intramuscular (IM) injections of sterile saline every 2 weeks for 5 times and were necropsied 2 days after the final IM injection. Clinical chemistry, hematology, and urinalysis were evaluated. Blood for stress biomarkers (norepinephrine, epinephrine, cortisol, and corticosterone), C-reactive protein, cardiac troponin I, and creatine kinase were collected at time 0 (just before dose administration) and then at 4, 24, and 48 hr after dose administration in group 1 only. Hearts were assessed histologically. Focal to multifocal minimal inflammatory cell infiltrates were common (∼80%), particularly in the left ventricle and interventricular septum, and were similar to the types of infiltrates identified in other laboratory animal species. Additionally, study-related procedures elevated serum stress biomarkers and exacerbated the frequency and severity of myocardial inflammatory cell infiltrates.

Screening New Drugs for Immunotoxic Potential: I. Assessment of the Effects of Conventional Nonsteroidal Anti-Inflammatory Drugs and Selective COX-2 Inhibitors on In Vitro and In Vivo Phagocytic Activity.

Although both experimental and clinical literature contain reports suggestive of associations between enhanced susceptibility to soft tissue infections and nonsteroidal anti-inflammatory drug (NSAID) use, the immunotoxicological potential of this class of therapeutic agents has not been thoroughly investigated. In consideration of the widespread clinical use of these agents, we have initiated studies of the interaction between NSAIDs (both nonselective and selective COX-2 inhibitors) and the immune system. This communication describes the conduct and results of assessments of the effects of NSAIDs on the in vitro phagocytic activity of rat macrophages and canine neutrophils and on the functional activity of the intact murine mononuclear phagocytic system. During in vitro experiments 0.1 to 10 muM concentrations of naproxen, indomethacin, and experimental selective COX-2 inhibitors, SC-236, SC-245 and SC-791, caused marginal, but statistically significant, reductions in phagocytic activity of resident rat peritoneal macrophages. The effects were consistently small and there was no evidence of concentration-response relationships. An in vitro concentration of 10 muM of either SC-236 or SC-791 was required to decrease phagocytosis by dog neutrophils. Repeated oral doses of either naproxen or SC-236 (3, 10, or 30 mg/kg twice daily for 3 days) were without effect on the intact phagocytic system of the mouse. A potential immunotoxicologic effect based on direct impairment of phagocytic processes seems an unlikely explanation for drug-induced susceptibility to infection reported earlier. However, the results of these experiments do not support an unequivocal conclusion relative to immunotoxicological potential of either conventional NSAIDs or selective COX-2 inhibitors. Further studies on other components of the immune system are needed to fully explore possible immunomodulatory effects of NSAIDs.

Screening New Drugs for Immunotoxic Potential: II. Assessment of the Effects of Selective and Nonselective COX-2 Inhibitors on Complement Activation, Superoxide Anion Production and Leukocyte Chemotaxis and Migration Through Endothelial Cells.

Results from earlier experiments in our laboratories revealed that both selective and nonselective inhibitors of cyclooxygenase-2 possess little potential for decreasing in vitro phagocytosis by rat macrophages or canine neutrophils and no potential for decreasing in vivo phagocytosis by the intact murine immune system. We now report the results of studies to assess in vitro and ex vivo effects of the drugs on 1) canine complement activation, 2) generation of superoxide anion and hydrogen peroxide (oxidative burst) by canine neutrophils, and 3) leukocytic chemotaxis and transmigration through endothelial cell monolayers. In vitro concentrations of naproxen sodium, SC-236, SC-245, and SC-791 ranging from 0.1 to 10 muM were tested for their abilities to inhibit canine complement-mediated hemolysis of opsonized sheep erythrocytes and to block phorbol myristate acetate-induced oxidative burst in canine neutrophils. Both models responded to known inhibitory agents, leupeptin in the complement activation test and staurosporine in the superoxide anion assay. In contrast, tested nonsteroidal anti-inflammatory drugs produced only trivial changes in complement activation and superoxide anion production. Experiments on plasma and neutrophils isolated from dogs administered an experimental selective COX-2 inhibitor during a 28-day toxicology study revealed no evidence of drug-associated changes in complement activation or formation of superoxide anion. SC-791 reduced chemotaxis of canine leukocytes toward zymosan-activated dog plasma, but not toward leukotriene B(4). None of the other drugs tested significantly affected leukocytic chemotaxis. Ibuprofen, SC-245 and SC-791 but not SC-236, reduced transmigration of canine leukocytes through endothelial cell monolayers. Based on the results of these experiments and our earlier studies we have concluded that, although high (suprapharmacologic) concentrations of the drugs may induce in vitro evidence of apparent immunomodulation of the innate immune system, the findings are unlikely to represent a significant human health risk.

Screening new drugs for immunotoxic potential: III. assessment of the effects of selective and nonselective COX-2 inhibitors on T-cell-dependent antibody and natural killer cell responses in the rat.

Results from earlier experiments in our laboratories revealed that both selective and non-selective inhibitors of cyclooxygenase-2 possess little potential for decreasing in vitro phagocytosis by rat macrophages or canine neutrophils, and no potential for decreasing in vivo phagocytosis by the intact murine immune system. We have also demonstrated that pharmacologically relevant doses and concentrations of these drugs do not reduce canine complement activation, superoxide anion generation, leukocytic chemotaxis or transmigration of leukocytes through endothelial monolayers. We now report the results of immunotoxicology studies to assess the effects of the drugs on cell-mediated immunity. Male and female Sprague-Dawley rats were administered daily oral gavage doses of naproxen (10 mg/kg), SC-791 (2.5 mg/kg), or SC-245 (17 mg/kg) for 28 consecutive days or treated with cyclophosphamide or anti-asialo GM1 antibody as positive immunomodulatory controls (for T-dependent antibody response and natural killer cell assay, respectively). All rats, except those treated with GM1 antibody or used in toxicokinetic analyses, were immunized on study day 25 with sheep red blood cells to induce a primary T-dependent antibody response. The doses of test agents were chosen to be either supra-pharmacologic or limited by anticipated systemic toxicity. Hematologic changes consistent with gastrointestinal (GI) blood loss due to mild GI mucosal toxicity were seen with naproxen and SC-791. Both positive control agents produced anticipated immunomodulatory effects confirming the validity of the assay system. In the antibody-forming cell assay, naproxen, SC-791 and SC-245 were without effects on splenic cellularity, splenocyte viability or the number of sheep red blood cell antibody-forming cells. Cyclophosphamide reduced both splenic cellularity and antibody-forming cell counts. In the natural killer cell assay, naproxen, SC-791 and SC-245 were all without effects on natural killer cell activity, while anti-asialo antibody reduced natural killer cell activity up to 85%. In considering the sum total of scientific information relative to the immunotoxicological potential of non-selective and selective cyclooxygenase-2 inhibitors, we conclude that, although high (supra-pharmacologic) concentrations of these drugs may induce some in vitro immunomodulatory effects on the innate immune system, the findings are of doubtful predictive significance with respect to human health implications.

Interspecies differences in the nephrotoxic response to cyclooxygenase inhibition.

In contrast to cyclooxygenase-1 (COX-1), the basal expression of renal cyclooxygenase-2 (COX-2) varies among species. High basal levels of COX-2 in the renal cortex and papilla in dogs compared with monkeys suggest that COX-2 inhibition may lead to distinct nephrotoxic responses. In this study, we compared the renal effects of COX inhibition between dogs and cynomolgus monkeys (n = 6/group) following the administration of naproxen sodium, a non-selective COX-1/COX-2 inhibitor. Dogs and monkeys were treated with 50 or 150 mg/kg/day naproxen sodium, respectively, for 2 to 6 weeks. Naproxen doses used in this study resulted in equivalent inhibition of COX activity in both species as measured by reductions in urinary prostaglandin E2 (PGE2) and 6-keto-PGF1-alpha levels. There was prominent reduction in renal blood flow (43%) and urinary sodium excretion (62%) in dogs but no alterations in renal blood flow and only minimal change (19%) in urinary sodium excretion in monkeys. The canine but not monkey kidney showed prominent COX-2 expression in the macula densa, thick ascending limb of Henle and papillary interstitial cells by immunohistochemistry. After treatment, the canine but not monkey kidneys had mild to moderate renal tubular atrophy and interstitial fibrosis and renal papillary necrosis. Obstructive nephropathy secondary to intra-tubular drug accumulation was seen in monkeys but not in dogs. Collectively, these data demonstrate species differences in the renal response to COX inhibition. The nature of functional and morphologic changes suggests a more prominent role of COX-2 in renal hemodynamics and natriuresis in dogs than in monkeys.

Enhanced expression of cyclooxygenase-2 in glaucomatous dog eyes.

OBJECTIVE: Cyclooxygenase-2 (COX-2)-derived prostaglandins (PGs) are shown to play important pathophysiologic roles in various disease states. Recently, the effectiveness of topical PGs in reducing intraocular pressure (IOP) has stimulated further interest in the physiologic function of COX-2 and PGs in normal and glaucomatous eyes. Therefore, we investigated the cell-type distribution and expression of COX-2 in normal and glaucomatous dog eyes. PROCEDURES: Using isoform-specific antibodies, we immunohistochemically evaluated COX-2 expression in formalin-fixed and paraffin-embedded normal (n = 5) and glaucomatous (n = 17) dog eyes. RESULTS: In the normal eyes, only minimal COX-2 immunoreactivity was observed in the ciliary epithelium. In the glaucomatous eyes, COX-2 expression was further observed in the cornea and corneoscleral limbus. In the cornea, moderate to strong COX-2 expression was observed in all corneal layers (epithelium, stromal cells and endothelium), with the greatest expression present in the epithelial layer. In the corneoscleral limbus area, COX-2 immunoreactivity was noted in the stromal cells of sclera, trabecular meshwork and endothelial cells of the angular aqueous plexus. CONCLUSIONS: Increased expression of COX-2 in dog glaucomatous eyes suggests that COX-2-derived PGs may have a potential role in the pathogenesis of canine glaucoma.

Expression of cyclooxygenase-2 in embryonic and fetal tissues during organogenesis and late pregnancy.

BACKGROUND: Cyclooxygenase (COX) catalyzes the committed step in prostaglandin biosynthesis and exists as two related but unique isoforms, COX-1 (constitutive) and COX-2 (inducible). Prostaglandins (PGs) are known to have many important functions in reproduction, such as placentation and decidualization. Studies with the COX-1 and COX-2 knockout mice have demonstrated that COX-2, but not COX-1, is crucial for normal ovulation, implantation, and decidualization, suggesting that COX-2-derived PGs are important during the initial stages of pregnancy. Although the COX-2 knockout mice did not exhibit any abnormalities at birth, relatively little information exists with regard to the expression of COX-2 in the fetus during development. METHODS: In order to understand the role of COX-2 throughout pregnancy, we characterized the cell type and the temporal expression of inducible COX-2 throughout embryonic and fetal development in the rat (n = 22) by immunohistochemistry and in situ hybridization. RESULTS: High levels of COX-2 expression were seen in decidualized uterine tissue on gestation days 7-13 and then in the fetal membranes on gestation days 17-20. Cyclooxygenase-2 expression was not detectable in any tissues from developing embryos during gestation days 7-13, but was observed in the fetal growth period (gestation days 15-20) in the skin, heart, cartilage, and the kidney. CONCLUSIONS: No COX-2 expression was seen in fetal tissues at days 7-13 of gestation, but was seen in various tissues at days 15-17 of gestation. These observations suggest that COX-2 may be important in mid to late pregnancy through an effect on fetal organ growth, but not in the organogenetic phase of fetal development.

Localization of COX-2 in the male reproductive tract during sexual maturation.

Prostaglandins (PGs) are shown to influence sperm motility, contractility of the smooth muscle layers surrounding the seminiferous tubules and growth of both the seminal vesicle and the ventral prostate. PGs are produced by two distinct isoforms of cyclooxygenase (COX), including constitutively expressed COX-1 and inducible COX-2. To investigate the potential role of COX-2 in male reproductive tract maturation, we evaluated its expression in rats at pre-pubertal (14 days old), peri-pubertal (21, 28 and 35 days old) and post-pubertal (62 days old) stages. COX-2 was constitutively expressed in the initial segment of the epididymis, caput epididymidis and vas deferens at all stages of maturation. Its expression was mild in 14-day-old rats but its intensity markedly increased at 28 days and remained elevated afterwards. There was no COX-2 staining in the testis, rete testis, efferent ducts or cauda epididymidis. These data suggest that COX-2 derived PGs may be involved in the pubertal development of the epididymis, but not in the more apical regions of the excurrent duct system, including the rete testis and efferent ductules.