Naloxegol, an opioid antagonist with reduced CNS penetration: Mode-of-action and human relevance for rat testicular tumours.

Naloxegol is an opioid antagonist which has been developed for the treatment of patients with opioid induced constipation. In the nonclinical safety program naloxegol was shown to have a very benign toxicity profile. In the rat, but not the mouse, 2-year carcinogenicity study a change in tumour pattern with an increase in testicular Leydig cell tumours (LCT) was observed after dosing at high (supra-pharmacological) concentrations. To establish the basis of the increase in LCT and to assess its potential relevance to humans, studies to exclude and potentially identify mode-of-action (MoA) were performed. A genotoxic mechanism was ruled out following negative results in the Ames, mouse lymphoma, and micronucleus assays. An effect on androgen metabolism was excluded since the treatment of rats with naloxegol for 14days did not result in any induction of CYP protein levels. It was demonstrated that administration of centrally restricted opioid antagonists naloxegol or methylnaltrexone at high doses induced an increase in LH release with no clear increase in testosterone, in contrast to the centrally acting opioid antagonist naloxone, which showed marked increases in both LH and testosterone. LCT due to increased LH stimulation is common in rats but not documented in humans. Collectively, the lack of genotoxicity signal, the lack of androgen effect, the increase in LH secretion in rats, which is no considered to be relevant for LCT formation in humans, and high margins to clinical exposures, the observed increase in LCT in the rat is not expected to be clinically relevant.

Time Course for Onset and Recovery from Effects of a Novel Male Reproductive Toxicant: Implications for Apical Preclinical Study Designs.

In the pharmaceutic ICH S5(R2) guidelines for reproductive toxicity testing, a premating dose duration of 14 days is considered sufficient for assessment of male fertility for compounds that are not testicular toxicants. A novel α7 subtype of nicotinic acetylcholine receptor (α7nAChR) agonist, originally intended for treatment of Alzheimer's disease, did not cause changes in sperm counts, motility, or testicular histopathology in rat toxicity studies of up to 6 months duration. However, profound decrements in male fertility (reduced pregnancy rates and litter sizes) occurred after 11 weeks of dosing in male rats. In two time-course investigations, dosed male rats were paired with undosed females after 5, 14, and 28 daily doses and again after 2 and 4 weeks off-dose. Effects on male fertility were undetectable after 5 days. After 14 days, there was no effect on pregnancy rate, but preimplantation losses were increased. Effects on both pregnancy rates and preimplantation losses were clearly detectable after 28 days, but were of lesser magnitude than after 11 weeks of dosing. Fertility recovered rapidly after dose cessation. These studies illustrate the sensitivity of a long premating dose period at revealing hazard and determining the magnitude of effect on male fertility for compounds that are intended for chronic administration and do not affect testicular histopathology.

The novel use of a heterozygous knockout mouse for embryofetal development assessment of a glucokinase activator.

Glucokinase activators (GKAs), such as AZD1656, are designed as antihyperglycemic agents for diabetics and can cause dose-limiting hypoglycemia in normal animals used in embryofetal development studies. Genetically modified heterozygous GK knockout (gkdel/wt) mice are less susceptible to severe GKA-induced hypoglycemia than wild-type mice due to their elevated baseline glucose levels. In this study, the gkdel/wt mouse was used as an alternative rodent strain for embryofetal development studies with AZD1656. Heterozygous global knockout gkdel/wt females were dosed with 20, 50, or 130 mg/kg/day of AZD1656 or vehicle for a minimum of 14 consecutive days before mating with wild-type males and throughout organogenesis. Maternal effects were confined to slightly reduced food consumption, reduced body weight gain, and the pharmacologic effect of decreased plasma glucose. Fetuses were genotyped. Fetal weights at the high dose were slightly reduced but there was no effect on fetal survival. There were two specificmajormalformations, omphalocele and right-sided aortic arch, with increased fetal incidence in mid- and high-dose fetuses (e.g., omphalocele fetal incidence of 0.6, 0.7, 4.6, and 2% across the dose groups) plus increased incidences of minor abnormalities and variants indicative of either delayed or disturbed development. Fetal weight and abnormalities were unaffected by fetal genotype. The fetal effects are considered hypoglycemia related. There was no effect on embryofetal survival in the gkdel/wt mouse at AZD1656 exposures, which were 70× higher than those causing 75% fetal death in rabbits. This illustrates the value of genetically modified animals in unraveling target versus chemistry-related effects.

The inhibin B response in male rats treated with a GnRH agonist and an endothelin receptor antagonist.

BACKGROUND: This study examined the correlation between Inhibin B and testicular pathology. METHODS: Male Han Wistar rats (approximately 10 weeks old) were administered either vehicle or an endothelin receptor antagonist (ET-An) orally for 28 days or a Gonadotropin Releasing Hormone (GnRH) agonist (GnRH-A) as a subcutaneous implant on day 1. Ten animals/group/time point were killed on days 4, 8, 15, and 29 (controls on days 15 and 29) for testes weights and histopathology. In-life blood samples were taken on days 4, 8, 15, and 29 to measure Inhibin B, Follicle-Stimulating Hormone (FSH), and Lutenising Hormone (LH), and at necropsy for the same hormones plus testosterone. RESULTS: Plasma Inhibin B showed a wide concentration range in controls (group means 76.4-184.2 pg/ml; individual animals 17.8-381 pg/ml). GnRH-A caused decreased testes weights plus degenerative testicular pathology from day 4 with partial recovery by day 29. Statistically significant reductions in Inhibin B were observed at all time points and appeared to track the development and partial recovery of the pathology (generally <50 pg/ml on days 4-15; group mean 92 pg/ml on day 29). ET-An produced an increase in testes weights and a nondegenerative lesion of minimal tubular dilatation. There was a trend for lower Inhibin B values (30-50%) at all time points, including on day 4 when tubular dilatation was not yet evident. CONCLUSION: Inhibin B showed a good correlation with testicular pathology for GnRH-A, and following ET-An administration appeared to give a signal that might reflect changes in tubular function in the absence of degenerative pathology.

Assessment of male rodent fertility in general toxicology 6-month studies.

An outcome and statistical review of male reproductive performance assessed by including a mating phase within 6-month general toxicity studies in the Han Wistar rat was undertaken. The basic study design was 16-20 animals per group dosed for approximately 9 weeks before pairing the male rats with undosed females. This design provides opportunity for remating and automatically includes general toxicity parameters. The dose levels used in the 1- and 6-month studies show that male reproduction was assessed at generally similar doses. The majority of males (compound-dosed and controls) mated within 7 days. All vehicle-dosed males mated and 98.5% of these females were pregnant. Modeling shows that a pregnancy rate of less than 14 out of 16 pregnant animals is very unlikely to occur due to biological variability. Power calculations based on vehicle control data show that group sizes of >10 males have a >80% power of detecting a decrease in median of three embryos per group compared with the control group. Even if the number of pregnancies decreased by a third, a group size of ≥12 would still detect a decrement in the median of three embryos with >80% power. Based on the statistical modeling and inherent strengths of the study design, this review indicates that decrements in male reproductive function can be successfully detected by incorporating a mating phase into a 6-month rat study and that a group size of 12-16 is generally adequate rather than the 16-20 group size indicated as a generic default within ICHS5(R2).

Incidence and nature of testicular toxicity findings in pharmaceutical development.

BACKGROUND: Testicular toxicity (TT) is a sporadic and challenging issue in pharmaceutical drug development. Efforts to develop TT screening assays or biomarkers have been overshadowed by consortium efforts to predict drug-induced toxicities such as hepatic injury, which are encountered more frequently. METHODS: To gauge the current state of the field and to prioritize future TT activities, the International Life Sciences Institute-Health and Environmental Sciences Institute Developmental and Reproductive Toxicology (DART) Technical Committee sponsored a survey to better understand the incidence and nature of TT findings encountered during drug development. RESULTS: Highlights from the 16 survey respondents include: (1) Although preclinical TT was encountered relatively infrequently, half of the participants observed repeated problems with TT during pharmaceutical development, (2) despite control measures such as use of sexually mature animals to diminish confounding effects of spurious lesions, interpretation of TT remains a challenge, (3) "traditional" evaluation tools such as hormonal monitoring and newer approaches such as -omics are utilized to investigate testicular changes, and (4) an understanding of the risk and relevance of TT findings is achieved through joint consideration of factors such as species specificity, potential mode of action, and safety margins. CONCLUSIONS: TT remains a relatively uncommon but persistent challenge in pharmaceutical development. Although current preclinical TT approaches appear to be effective in limiting the occurrence of pharmaceutical candidate attrition in clinical trials, improved biomarker or screening platforms would allow companies to identify TT at an earlier stage, thus decreasing the time and resources expended on safety evaluation of pharmaceutical candidates.

The cynomolgus monkey as a model for developmental toxicity studies: variability of pregnancy losses, statistical power estimates, and group size considerations.

BACKGROUND: This work evaluates pregnancy and infant loss in 1,069 vehicle-treated cynomolgus monkeys from 78 embryo-fetal development (EFD) studies and 14 pre-postnatal development (PPND) studies accrued during 1981-2007. METHODS: Losses were analysed by survival function and hazard ratio using logistic regression for influence of year, study type (e.g., dose duration), and test item route of administration (ig, im, iv, sc). RESULTS: Neither study type nor route of dosing affected pregnancy outcome. Losses were higher pre-1990 (104 losses/347 pregnancies) compared to 1990 onwards (94 losses/722 pregnancies). Losses were greatest before gestation day 50 and at parturition. Using post-1989 data, Monte-Carlo simulations of pregnancy outcomes were created. The power associated with the comparison of vehicle survival curves and simulated adverse survival curves was examined. This showed that EFD studies with initial vehicle group sizes of 16 and 20 have an 80% probability of having 13 and 16 ongoing pregnancies at gestational day 100, respectively. For PPND studies with initial vehicle group sizes of 16, 20, or 28, there is an 80% likelihood of having 9, 11, or 16 infants at day 7 post-partum, respectively. A PPND study initiated with group size 20 could detect a threefold increase of test item-related pregnancy or infant loss. CONCLUSIONS: For designing and managing primate developmental toxicity studies, this type of analysis provides an objective tool to facilitate decisions either by supplementing groups with additional pregnant animals or stopping a group because an adverse effect on offspring survival has already been adequately revealed.

Investigation into the effect of microsampling on mouse fetuses and pregnant mice in the embryofetal development study design.

The effects on fetal weights and maternal health of taking 32µL blood microsamples at the end of organogenesis in a mouse embryofetal development (EFD) study design was investigated with the aim of reducing satellite animal usage. The effects of warming, handling and sampling either 3 or 6 times on gestation day 16 was evaluated. Maternal body weight gain was transiently reduced when animals underwent warming and handling with or without microsampling. Fetal weights on gestation day 18 were reduced after 6 occasion warming and handling alone or taking samples, but not when sampling was limited to 3 timepoints. Taking 3 microsamples of 32µL had no permanent adverse effects on maternal health or toxicologically significant effects on fetal development (measured by fetal weights). This regimen could be used to generate composite toxicokinetic profiles using only 6 main test animals in mouse EFD studies provided sampling procedures were matched across groups.

Reproductive senescence, fertility and reproductive tumour profile in ageing female Han Wistar rats.

A study using vehicle administration in 104 female rats investigated reproductive aging in Han Wistar rats as a useful tool to interprete carcinogenicity studies where hormonal patterns are perturbated. From 16 weeks of age oestrous cycles were monitored every 6 weeks to investigate reproductive ageing. A subset of 20 females was used to assess fertility at 21 months of age. The animals were necropsied after 106-107 weeks on study and female reproductive organs, mammary glands and pituitary glands were examined for hyperplasias and/or tumours. The majority of rats had regular oestrous cycles up to 6 months of age. After this age, there was a rapid decline in the number of rats with regular oestrous cycles and an increase in irregular cycles and cycles in persistent di-oestrus with an occasional pro-oestrus. By the end of the study, the majority of animals were acyclic and the few remaining cyclic animals had irregular cycles. In the fertility assessment, 19/20 animals mated but only four animals became pregnant. These pregnant animals had normal numbers of corpora lutea of pregnancy but had high pre-implantation losses and could not sustain a viable pregnancy. 65 animals (62.5%) showed adenomas and/or pituitary hyperplasia in the pituitary gland at necropsy. The pituitary tumours were likely to be prolactin secreting that give rise to pseudopregnancy and mammary tumours, demonstrated by the fact that 43/65 (66%) of the affected animals had histopathological signs of these conditions. Multiple corpora lutea were found in 61% of all animals at time of termination. Only one uterine tumour was seen in this study probably due to lack of persistent oestrus seen in these animals.

Reduced post-natal versus pre-natal incidence of bent long bones and scapulae in a preliminary investigation using the Han Wistar rat.

There is a "chondrodystrophy" syndrome in the Han Wistar rat fetus that manifests as characteristic skeletal abnormalities such as bent and/or short long bones, and is classified as permanent detrimental abnormalities (major malformations). This pilot study investigated whether these defects resolve after birth. Han Wistar rats were dosed during organogenesis either with vehicle or test article. Examination of gestation day 20 fetuses showed a slightly increased incidence (11%; 11/101) of skeletal abnormalities in the high dose fetuses compared with 6% (4/67) in control fetuses, whereas no skeletal abnormalities were present in the 205 pups examined on post-natal day 21. The probability of having zero litters containing pups with skeletal abnormalities was p<0.0000001. This very low probability suggests that these defects recover by weaning and supports the hypothesis that these fetal findings in the Han Wistar are probably not permanent abnormalities and therefore are potentially reclassifiable as minor malformations.