Novel Bacterial Topoisomerase Inhibitor Gepotidacin Demonstrates Absence of Fluoroquinolone-Like Arthropathy in Juvenile Rats.

Fluoroquinolone use in children is limited due to its potential toxicity and negative effects on skeletal development, but the actual effects/risks of fluoroquinolones on bone growth and the mechanisms behind fluoroquinolone-driven arthropathy remain unknown. Gepotidacin is a novel, bactericidal, first-in-class triazaacenaphthylene antibiotic with a unique mechanism of action that is not anticipated to have the same risks to bone growth as those of fluoroquinolones. Gepotidacin is in phase III clinical development for uncomplicated urinary tract infections (ClinicalTrials.gov identifiers NCT04020341 and NCT04187144) and urogenital gonorrhea (ClinicalTrials.gov identifier NCT04010539) in adults and adolescents ≥12 years of age. To inform arthropathy and other potential toxicity risks of gepotidacin in pediatric studies, this nonclinical study assessed oral gepotidacin toxicity in juvenile rats from postnatal day (PND) 4 to PND 32/35 (approximately equivalent to human ages from newborn to 11 years), using both in-life assessments (tolerability, toxicity, and toxicokinetics) and terminal assessments (necropsy with macroscopic and microscopic skeletal femoral head and/or stifle joint examinations). Gepotidacin doses of ≤300 mg/kg of body weight/day were well tolerated from PND 4 to PND 21, and higher doses of ≤1,250 mg/kg/day were well tolerated from PND 22 when the dose levels were escalated to maintain systemic exposure levels up to PND 35, with no observed treatment-related clinical signs, effects on mean body weight gain, or macroscopic findings on articular surfaces. A dose of 1,000 mg/kg/day was not tolerated during the dosing period from PND 4 to 21, with effects on body weight gain, fecal consistency, and body condition. Microscopic effects on articular surfaces were evaluated after 32 days of gepotidacin treatment at the highest tolerated dose. After 32 days of treatment with the highest tolerated gepotidacin dose of 300/1,250 mg/kg/day (systemic concentrations [area under the curve {AUC} values] of 93.7 µg · h/mL [males] and 121 µg · h/mL [females]), no skeletal effects on articular surfaces of the femoral head or stifle joint were observed. The absence of treatment-related clinical signs and arthropathy in juvenile rats provides evidence to support the potential future use of gepotidacin in children.

The benefits, limitations and opportunities of preclinical models for neonatal drug development.

Increased research to improve preclinical models to inform the development of therapeutics for neonatal diseases is an area of great need. This article reviews five common neonatal diseases - bronchopulmonary dysplasia, retinopathy of prematurity, necrotizing enterocolitis, perinatal hypoxic-ischemic encephalopathy and neonatal sepsis - and the available in vivo, in vitro and in silico preclinical models for studying these diseases. Better understanding of the strengths and weaknesses of specialized neonatal disease models will help to improve their utility, may add to the understanding of the mode of action and efficacy of a therapeutic, and/or may improve the understanding of the disease pathology to aid in identification of new therapeutic targets. Although the diseases covered in this article are diverse and require specific approaches, several high-level, overarching key lessons can be learned by evaluating the strengths, weaknesses and gaps in the available models. This Review is intended to help guide current and future researchers toward successful development of therapeutics in these areas of high unmet medical need.

Dose range finding approach for rodent preweaning juvenile animal studies.

OBJECTIVES: Strategies for conducting juvenile dose ranging studies before definitive toxicity juvenile animal studies (JAS) have evolved, but the aim of demonstrating study design robustness and efficient animal use remains the same. The objective of dose selection is to identify a strategy to achieve consistent systemic exposure for the duration of the JAS while maintaining exposure separation between dose groups. For preweaning rodents this can prove challenging, as these studies typically treat animals over a broad period of considerable organ development. MATERIALS AND METHODS: In our experience, over 45 rodent juvenile studies (dose range, definitive or investigative) were conducted over 20 years to support pediatric medicine development. In most cases (86%, 12/14), preweaning rodents required decreased doses of test articles than adult rodents; the majority (83%, 10/12) were due to increased systemic exposures in immature animals at the same doses. Thus, extrapolating tolerability and exposure data from adults is not ideal and should not take the place of well-designed juvenile dose range studies. RESULTS/DISCUSSION/CONCLUSION: We propose a phased dose-range-finding approach by first conducting a tolerability phase with a few animals at a starting age corresponding to the youngest clinical starting age, spanning a wide range of doses, then a dose range phase with larger group sizes and fewer doses; both phases incorporate toxicokinetics. Often, exposure was higher in preweaning animals and decreased as animals matured postweaning (postnatal day, PND 21 and older), supporting an age-based dose adjustment strategy. Case studies demonstrate dose adjustment approaches incorporating dose increases or decreases or changes in dose frequency.

The exploration of aza-quinolines as hematopoietic prostaglandin D synthase (H-PGDS) inhibitors with low brain exposure.

GlaxoSmithKline and Astex Pharmaceuticals recently disclosed the discovery of the potent H-PGDS inhibitor GSK2894631A 1a (IC50 = 9.9 nM) as part of a fragment-based drug discovery collaboration with Astex Pharmaceuticals. This molecule exhibited good murine pharmacokinetics, allowing it to be utilized to explore H-PGDS pharmacology in vivo. Yet, with prolonged dosing at higher concentrations, 1a induced CNS toxicity. Looking to attenuate brain penetration in this series, aza-quinolines, were prepared with the intent of increasing polar surface area. Nitrogen substitutions at the 6- and 8-positions of the quinoline were discovered to be tolerated by the enzyme. Subsequent structure activity studies in these aza-quinoline scaffolds led to the identification of 1,8-naphthyridine 1y (IC50 = 9.4 nM) as a potent peripherally restricted H-PGDS inhibitor. Compound 1y is efficacious in four in vivo inflammatory models and exhibits no CNS toxicity.

Immune complex disease in a chronic monkey study with a humanised, therapeutic antibody against CCL20 is associated with complement-containing drug aggregates.

Despite the potential for the chemokine class as therapeutic targets in immune mediated disease, success has been limited. Many chemokines can bind to multiple receptors and many receptors have multiple ligands, with few exceptions. One of those exceptions is CCL20, which exclusively pairs to CCR6 and is associated with several immunologic conditions, thus providing a promising therapeutic target. Following successful evaluation in a single dose, first time in human clinical study, GSK3050002-a humanized IgG1 monoclonal antibody against human CCL20-was evaluated in a 26-week cynomolgus monkey toxicology study. A high incidence of unexpected vascular and organ inflammation was observed microscopically, leading to the decision to halt clinical development. Here we report a dose-responsive increase in the incidence and severity of inflammation in multiple organs from monkeys receiving 30 and 300 mg/kg/week by either subcutaneous or intravenous injection. Histomorphological changes resembled an immune complex-mediated pathology, which is often due to formation of anti-drug antibodies in monkeys receiving a human protein therapeutic and thus not predictive of clinical outcome. However, the presentation was atypical in that there was a clear dose response with a very high incidence of inflammation with a low incidence of ADA that did not correlate well individually. Additionally, the immunohistologic presentation was atypical in that the severity and distribution of tissue inflammation was greater than the numbers of associated immune complexes (i.e., granular deposits). An extensive ex vivo analysis of large molecular weight protein complexes in monkey serum from this study and in human serum samples demonstrated a time-dependent aggregation of GSK3050002, that was not predicted by in vitro assays. The aggregates also contained complement components. These findings support the hypothesis that immune complexes of drug aggregates, not necessarily including anti-drug antibodies, can fix complement, accumulate over time, and trigger immune complex disease. A situation which may have increased clinical relevance than typical anti-drug antibody-associated immune complex disease in monkeys administered human antibody proteins.

Absence of developmental and reproductive toxicity in animals exposed to dolutegravir.

The success of new antiretroviral medicines for HIV resulted in a change to guidelines of standard therapy where continuation of antiretroviral therapy is recommended to maintain the low viral load during pregnancy, thereby preventing transmission of the virus to the fetus. As a result, pregnancy related exposure to HIV medicines has increased. Understanding the safety of these medicines during pregnancy is of paramount importance to ensure health of mothers and their offspring; well-designed animal studies that evaluate the reproductive life cycle play a key role in this effort. As part of the medicine development program for dolutegravir (DTG), a series of reproductive and developmental toxicity studies were conducted using rats and rabbits. In a fertility study, where exposure to DTG occurred in female rats before mating through conception and up to implantation of the embryo, no effects on reproductive cycles, ovulation, fertility) or preimplantation embryonic growth were observed. In rat and rabbit embryo-fetal development studies, where exposure to DTG occurred during organogenesis, no malformations or other developmental abnormalities were observed. In a rat pre- and post-natal development study, where DTG exposure to the pups occurred during pregnancy and postnatally via milk, no malformations or other developmental abnormalities were observed. In these studies, no DTG-related effects occurred on fertility, embryonic (pre- and post-implantation loss, resorptions, abortions, and malformations) or fetal development where the multiples of exposure at the maximum recommended human dose were up to 27 times higher in rats or below the human exposure in rabbits.

Species Comparison of Postnatal Development of the Female Reproductive System.

The postnatal development of the female reproductive system in laboratory animals and humans is reviewed. To enable a meaningful species comparison of the developing female reproductive system, common definitions of developmental processes were established with a focus made on aspects that are similar across species. A species comparison of the key endocrine, morphologic, and functional (onset of ovarian cycles and ability to reproduce) features of postnatal development of the female reproductive system is provided for human, nonhuman primate, dog, rat, and also mouse, minipig, and rabbit where possible. Species differences in the timing and control of female sexual maturation are highlighted. Additionally, a species comparison of the type and timing of female reproductive ovarian cycles was compiled. Human development provided the frame of reference, and then other common laboratory species were compared. The comparison has inherent challenges because the processes involved and sequence of events can differ greatly across species. Broad strokes were taken to assign a particular average age to an event and are to be used with caution. Methods of evaluation of postnatal female reproductive development in laboratory animals are discussed. Lastly, control rodent data from one of the author's laboratory on vaginal opening, first estrus, estrous cyclicity, and the histopathology involved with the developing female rat and mouse are presented. The information provided in this review is intended to be a resource for the design and interpretation of juvenile animal toxicity testing and ultimately, the relevance of the data to characterize potential risks for women and girls. Birth Defects Research 110:163-189, 2018. © 2017 Wiley Periodicals, Inc.

Comparing rat and rabbit embryo-fetal developmental toxicity data for 379 pharmaceuticals: on systemic dose and developmental effects.

A database of embryo-fetal developmental toxicity (EFDT) studies of 379 pharmaceutical compounds in rat and rabbit was analyzed for species differences based on toxicokinetic parameters of area under the curve (AUC) and maximum concentration (Cmax) at the developmental lowest adverse effect level (dLOAEL). For the vast majority of cases (83% based on AUC of n = 283), dLOAELs in rats and rabbits were within the same order of magnitude (less than 10-fold different) when compared based on available data on AUC and Cmax exposures. For 13.5% of the compounds the rabbit was more sensitive and for 3.5% of compounds the rat was more sensitive when compared based on AUC exposures. For 12% of the compounds the rabbit was more sensitive and for 1.3% of compounds the rat was more sensitive based on Cmax exposures. When evaluated based on human equivalent dose (HED) conversion using standard factors, the rat and rabbit were equally sensitive. The relative extent of embryo-fetal toxicity in the presence of maternal toxicity was not different between species. Overall effect severity incidences were distributed similarly in rat and rabbit studies. Individual rat and rabbit strains did not show a different general distribution of systemic exposure LOAELs as compared to all strains combined for each species. There were no apparent species differences in the occurrence of embryo-fetal variations. Based on power of detection and given differences in the nature of developmental effects between rat and rabbit study outcomes for individual compounds, EFDT studies in two species have added value over single studies.

Comparison of rat and rabbit embryo-fetal developmental toxicity data for 379 pharmaceuticals: on the nature and severity of developmental effects.

Regulatory non-clinical safety testing of human pharmaceuticals typically requires embryo-fetal developmental toxicity (EFDT) testing in two species (one rodent and one non-rodent). The question has been raised whether under some conditions EFDT testing could be limited to one species, or whether the testing in a second species could be decided on a case-by-case basis. As part of a consortium initiative, we built and queried a database of 379 compounds with EFDT studies (in both rat and rabbit animal models) conducted for marketed and non-marketed pharmaceuticals for their potential for adverse developmental and maternal outcomes, including EFDT incidence and the nature and severity of adverse findings. Manifestation of EFDT in either one or both species was demonstrated for 282 compounds (74%). EFDT was detected in only one species (rat or rabbit) in almost a third (31%, 118 compounds), with 58% (68 compounds) of rat studies and 42% (50 compounds) of rabbit studies identifying an EFDT signal. For 24 compounds (6%), fetal malformations were observed in one species (rat or rabbit) in the absence of any EFDT in the second species. In general, growth retardation, fetal variations, and malformations were more prominent in the rat, whereas embryo-fetal death was observed more often in the rabbit. Discordance across species may be attributed to factors such as maternal toxicity, study design differences, pharmacokinetic differences, and pharmacologic relevance of species. The current analysis suggests that in general both species are equally sensitive on the basis of an overall EFDT LOAEL comparison, but selective EFDT toxicity in one species is not uncommon. Also, there appear to be species differences in the prevalence of various EFDT manifestations (i.e. embryo-fetal death, growth retardation, and dysmorphogenesis) between rat and rabbit, suggesting that the use of both species has a higher probability of detecting developmental toxicants than either one alone.

Early Vaginal Opening in Juvenile Female Rats Given BRAF-Inhibitor Dabrafenib Is Not Associated with Early Physiologic Sexual Maturation.

Dabrafenib (DAB), an inhibitor of BRAF kinase activity, is approved for metastatic melanoma with a BRAF V600E mutation. In support of pediatric cancer development, a nonclinical juvenile rat toxicity study was conducted in which females had early vaginal opening (VO). It was hypothesized that the early VO was not indicative of sexual maturation, but a result of a local effect on the vagina. An investigative study was conducted that mimicked the definitive study design, with rats given DAB or vehicle orally from Postnatal Day (PND) 7 to 35 and with necropsy subsets just before VO, at the first and second estrus, along with age-matched controls. Histopathology was performed on reproductive tissues, including immunohistochemistry for BRAF expression. VO occurred earlier in DAB females than in controls (PND 27.2 vs. 31.5); however, the timing of the first estrus was unaffected (PND 34.0 vs. 33.0). DAB-treated females evaluated just before VO (PND 22.0) had mostly immature reproductive tracts with no evidence of ovulation, similar to age-matched controls; however, DAB-treated females had keratinized and histologically open vaginas. Also, there was raised skin around the urogenital area, which correlated with hyperplasia/keratosis of the vulvar skin and keratinization of the distal vagina. BRAF expression (evaluated in controls) was localized to these tissues. Thus, early VO in rats given DAB likely represents a local effect accelerating vaginal keratinization to become open and not accelerated sexual maturation.

Imaging MS in Toxicology: An Investigation of Juvenile Rat Nephrotoxicity Associated with Dabrafenib Administration.

As part of an investigative nephrotoxicity study, kidney tissues from juvenile rats orally administered dabrafenib at different age intervals between postnatal day (PND) 7 to 35 were investigated by MALDI and LDI imaging mass spectrometry (IMS) to determine the chemical composition of tubular deposits. In the youngest age group (PND 7-13), MALDI IMS demonstrated that a dabrafenib carboxylic acid metabolite was diffusely localized to the regions of tubular deposits (medulla and corticomedullary junction); however, no dabrafenib-related material was detected directly from the deposits. Rather, the LDI IMS analysis determined that the deposits were composed primarily of calcium phosphate. Based on these data, the dabrafenib associated nephrotoxicity, including the formation of tubular deposits, was determined to be age dependent. Furthermore, immature renal function was hypothesized to be responsible for the susceptibility of the youngest pups.

Summary of the HESI consortium studies exploring circulating inhibin B as a potential biomarker of testis damage in the rat.

The Developmental and Reproductive Toxicity Technical Committee of the Health and Environmental Sciences Institute hosted a working consortium of companies to evaluate a new commercially available analytic assay for Inhibin B in rat serum or plasma. After demonstrating that the kit was stable and robust, the group performed a series of independent pathogenesis studies (23 different compound/investigator combinations) designed to examine the correlation between the appearance of lesions in the testis and changes in circulating levels of Inhibin B. These studies were reported individually in the previous articles in this series (this issue), and are discussed in this paper. For roughly half of these exposures, lesions appeared well before Inhibin B changed. A few of the studies showed a good correlation between seminiferous tubule damage and reduced circulating Inhibin B levels, while for seven exposures, circulating Inhibin B was reduced with no detectable alteration in testis histology. Whether this indicates a prodromal response or a false-positive signal will require further investigation. These exceptions could plausibly suggest some value of circulating Inhibin B as a useful biomarker in some circumstances. However, for roughly half of these exposures, Inhibin B appeared to be a lagging biomarker, requiring significant damage to the seminiferous tubules before a consistent and credible reduction in circulating levels of Inhibin B was observed.

The inhibin B response to a motilin receptor agonist in male rats.

BACKGROUND: In a repeat oral dose toxicity study, all of 16 male rats given 100 mg/kg/day GSK1322888 sustained testicular injury after 4 weeks of treatment; the findings were not reversible after 12 weeks off-dose. The current study was conducted to further characterize testicular toxicity and to explore the possible relationship between onset of lesions, and changes in circulating hormone levels. METHODS: Male Sprague Dawley rats were orally administered 30 or 100 mg/kg/day GSK1322888 for 2 weeks with a 4-week off-dose period. Blood was collected via tail vein twice during the treatment period (days 4 and 11) and three times during the off-dose period (days 28, 36, and 42) for measurement of serum testosterone, dihydrotestosterone, and Inhibin B, luteinizing hormone, and follicle stimulating hormone concentrations. A histopathologic examination of testes was performed at the end of the treatment and off-dose periods. RESULTS: At 100 mg/kg/day, microscopic findings of the testis (degeneration of the germinal epithelium) were evident for 9 of 10 male rats on day 14 and all 10 rats at the end of the 4-week recovery period. There was no testicular toxicity observed at 30 mg/kg/day. During all stages of evaluation, there was no apparent difference among control and treated animals in hormone concentrations. CONCLUSION: There was poor correlation between changes in serum levels of Inhibin B and testis histopathology. Based on these observations, the utility of Inhibin B as a hormonal marker for germ cell toxicity is limited.

Dihydroartemisinin (DHA) treatment causes an arrest of cell division and apoptosis in rat embryonic erythroblasts in whole embryo culture.

Within 24 hr after oral administration of the antimalarial artesunate to rats on Day 10 or 11 postcoitum (pc), there is depletion of embryonic erythroblasts (EEbs), leading to embryo malformation and death. The proximate agent is dihydroartemisinin (DHA), the primary metabolite. We investigated the causes of EEb depletion by evaluating effects of DHA on EEbs in whole embryo culture (WEC). Rat embryos cultured starting on Day 9 pc were treated with 1 or 7 µM DHA for 24 hr starting after 19 hr of culture (∼Day 10 pc) and for 2 to 12 hr starting after 43 hr of culture (∼Day 11 pc). DHA effects indicating the depletion of EEbs were paling of the visceral yolk sac and reductions in visible blood cells, H&E-stained normal (Type II or III) EEbs, and dividing (BrdU-stained) EEbs. DHA-induced abnormal cell division was indicated by increases in symmetric and asymmetric binuclear cells. DHA-induced apoptosis was indicated by increases in TUNEL- and Caspase-3-positive cells and EEbs with fragmented nuclei. In addition, although the overall number of EEbs was decreasing, DHA caused increases in the numbers of circulating early-stage (Type I or earlier) EEbs that could not be accounted for by cell division, suggesting the release of new, less sensitive erythroblasts from the yolk sac. In summary, treatment of Day 10 or 11 pc rat embryos with DHA in WEC resulted in defective and arrested cell division in EEbs followed by apoptosis, suggesting a mechanism for their depletion after artesunate treatment in vivo.

Assessing a theoretical risk of dolutegravir-induced developmental immunotoxicity in juvenile rats.

HIV-1 integrase inhibitors (INIs) are a promising class of antiretrovirals for the treatment of HIV in adults; there is interest in expanding their use into pediatric populations. A theoretical concern for developmental immunotoxicity was raised after a publication suggested that two HIV INI tool compounds inhibited in vitro cleavage activity of recombination activating genes 1 and 2 (RAG1/2) through the inhibition of their binding to recombination signal sequences. RAG1/2 are required for the development of mature B and T lymphocyte populations. The potential effects of the investigational INI dolutegravir on RAG1/2 were addressed by developing assays in juvenile rats to measure T cell receptor (TCR) Vß usage by flow cytometry as an indicator of TCR repertoire diversity and a T cell dependent antibody response (TDAR) as an indicator of immunosuppression. These endpoints were incorporated into a juvenile rat toxicity study, along with immunophenotyping, hematology, and histopathology of immunologic organs. Dose levels of 0, 0.5, 2, or 75mg/kg/day dolutegravir were given via oral gavage from postnatal day 4 through 66. At the highest dose, there was decreased body weight gain and two preweanling deaths; however, there were no treatment-related effects on developmental parameters. There were no effects on immunologic competence, as measured by TDAR, and no effects on lymphocyte subsets or CD4 and CD8 TCR Vß usage in peripheral blood. Histopathology of immunologic organs (spleen, thymus, lymph nodes) and hematology evaluation revealed no effects. The no observed adverse effect level for immunotoxicity endpoints was 75mg/kg/day.

Artesunate-induced depletion of embryonic erythroblasts precedes embryolethality and teratogenicity in vivo.

BACKGROUND: Artesunate (ART), an artemisinin antimalarial, is embryolethal and teratogenic in rats, with the most sensitive days being 10 and 11 postcoitum (pc), respectively (Clark et al.: Birth Defects Res B 71:380-394, 2004; White et al.: Birth Defects Res A 70:265, 2004). METHODS: In this study, pregnant rats were administered a single oral dose of 17 mg/kg ART on Days 10-11 pc and conceptuses were evaluated through Day 14 pc. RESULTS: Paling of visceral yolk sacs was observed within 3-6 hr after treatment. Within 24 hr, marked paling and embryonic erythroblast depletion were observed macroscopically, which preceded malformations and embryo death, and persisted through Day 14 pc. Histologically, embryonic erythroblasts were reduced and cells showed signs of necrosis within 24 hr, were maximally depleted by 48 hr, and had partially rebounded within 3-4 days after treatment (Days 13 and 14 pc). Iron accumulation was evident in treated erythroblasts as early as 6 hr after treatment, suggesting impairment of heme synthesis. Heart abnormalities (swollen or collapsed chambers) were observed within 24 hr in approximately 25-60% of embryos and within 48 hr in 100% of embryos, correlating with histologic signs of cardiac myopathy (thinned and underdeveloped heart walls and enlarged chambers). Delays in limb and tail development occurred by Day 13 pc. Embryos were viable through Day 13 pc, but approximately 77% of embryos had died by Day 14 pc, presumably due to hypoxia and/or cardiac abnormalities. CONCLUSIONS: In summary, embryonic erythroblasts are the primary target of ART toxicity in the rat embryo after in vivo treatment, preceding embryolethality and malformations.