The effect of angiotensin receptor neprilysin inhibitor, sacubitril/valsartan, on central nervous system amyloid-ß concentrations and clearance in the cynomolgus monkey.

Sacubitril/valsartan (LCZ696) is the first angiotensin receptor neprilysin inhibitor approved to reduce cardiovascular mortality and hospitalization in patients with heart failure with reduced ejection fraction. As neprilysin (NEP) is one of several enzymes known to degrade amyloid-ß (Aß), there is a theoretical risk of Aß accumulation following long-term NEP inhibition. The primary objective of this study was to evaluate the potential effects of sacubitril/valsartan on central nervous system clearance of Aß isoforms in cynomolgus monkeys using the sensitive Stable Isotope Labeling Kinetics (SILK™)-Aß methodology. The in vitro selectivity of valsartan, sacubitril, and its active metabolite sacubitrilat was established; sacubitrilat did not inhibit other human Aß-degrading metalloproteases. In a 2-week study, sacubitril/valsartan (50mg/kg/day) or vehicle was orally administered to female cynomolgus monkeys in conjunction with SILK™-Aß. Despite low cerebrospinal fluid (CSF) and brain penetration, CSF exposure to sacubitril was sufficient to inhibit NEP and resulted in an increase in the elimination half-life of Aß1-42 (65.3%; p=0.026), Aß1-40 (35.2%; p=0.04) and Aßtotal (29.8%; p=0.04) acutely; this returned to normal as expected with repeated dosing for 15days. CSF concentrations of newly generated Aß (AUC(0-24h)) indicated elevations in the more aggregable form Aß1-42 on day 1 (20.4%; p=0.039) and day 15 (34.7%; p=0.0003) and in shorter forms Aß1-40 (23.4%; p=0.009), Aß1-38 (64.1%; p=0.0001) and Aßtotal (50.45%; p=0.00002) on day 15. However, there were no elevations in any Aß isoforms in the brains of these monkeys on day 16. In a second study cynomolgus monkeys were administered sacubitril/valsartan (300mg/kg) or vehicle control for 39weeks; no microscopic brain changes or Aß deposition, as assessed by immunohistochemical staining, were present. Further clinical studies are planned to address the relevance of these findings.

Osilodrostat (LCI699), a potent 11ß-hydroxylase inhibitor, administered in combination with the multireceptor-targeted somatostatin analog pasireotide: A 13-week study in rats.

The somatostatin analog pasireotide and the 11ß-hydroxylase inhibitor osilodrostat (LCI699) reduce cortisol levels by distinct mechanisms of action. There exists a scientific rationale to investigate the clinical efficacy of these two agents in combination. This manuscript reports the results of a toxicology study in rats, evaluating different doses of osilodrostat and pasireotide alone and in combination. Sixty male and 60 female rats were randomized into single-sex groups to receive daily doses of pasireotide (0.3mg/kg/day, subcutaneously), osilodrostat (20mg/kg/day, orally), osilodrostat/pasireotide in combination (low dose, 1.5/0.03mg/kg/day; mid-dose, 5/0.1mg/kg/day; or high dose, 20/0.3mg/kg/day), or vehicle for 13weeks. Mean body-weight gains from baseline to Week 13 were significantly lower in the pasireotide-alone and combined-treatment groups compared to controls, and were significantly higher in female rats receiving osilodrostat monotherapy. Osilodrostat and pasireotide monotherapies were associated with significant changes in the histology and mean weights of the pituitary and adrenal glands, liver, and ovary/oviduct. Osilodrostat alone was associated with adrenocortical hypertrophy and hepatocellular hypertrophy. In combination, osilodrostat/pasireotide did not exacerbate any target organ changes and ameliorated the liver and adrenal gland changes observed with monotherapy. Cmax and AUC0-24h of osilodrostat and pasireotide increased in an approximately dose-proportional manner. In conclusion, the pasireotide and osilodrostat combination did not exacerbate changes in target organ weight or toxicity compared with either monotherapy, and had an acceptable safety profile; addition of pasireotide to the osilodrostat regimen may attenuate potential adrenal gland hyperactivation and hepatocellular hypertrophy, which are potential side effects of osilodrostat monotherapy.

Vascular origin of vildagliptin-induced skin effects in Cynomolgus monkeys: pathomechanistic role of peripheral sympathetic system and neuropeptide Y.

The purpose of this article is to characterize skin lesions in cynomolgus monkeys following vildagliptin (dipeptidyl peptidase-4 inhibitor) treatment. Oral vildagliptin administration caused dose-dependent and reversible blister formation, peeling and flaking skin, erosions, ulcerations, scabs, and sores involving the extremities at ≥5 mg/kg/day and necrosis of the tail and the pinnae at ≥80 mg/kg/day after 3 weeks of treatment. At the affected sites, the media and the endothelium of dermal arterioles showed hypertrophy/hyperplasia. Skin lesion formation was prevented by elevating ambient temperature. Vildagliptin treatment also produced an increase in blood pressure and heart rate likely via increased sympathetic tone. Following treatment with vildagliptin at 80 mg/kg/day, the recovery time after lowering the temperature in the feet of monkeys and inducing cold stress was prolonged. Ex vivo investigations showed that small digital arteries from skin biopsies of vildagliptin-treated monkeys exhibited an increase in neuropeptide Y-induced vasoconstriction. This finding correlated with a specific increase in NPY and in NPY1 receptors observed in the skin of vildagliptin-treated monkeys. Present data provide evidence that skin effects in monkeys are of vascular origin and that the effects on the NPY system in combination with increased peripheral sympathetic tone play an important pathomechanistic role in the pathogenesis of cutaneous toxicity.

Preclinical Characterization and Phase I Study of an Anti-HER2-TLR7 Immune-Stimulator Antibody Conjugate in Patients with HER2+ Malignancies.

Immune-stimulator antibody conjugates (ISAC) combining tumor-targeting monoclonal antibodies with immunostimulatory agents allow targeted delivery of immune activators into tumors. NJH395 is a novel, first-in-class ISAC comprising a Toll-like receptor 7 (TLR7) agonist conjugated to an anti-HER2 antibody via a noncleavable linker payload. Preclinical characterization showed ISAC-mediated activation of myeloid cells in the presence of antigen-expressing cancer cells, with antigen targeting and TLR7 agonism contributing to antitumor activity. Safety, efficacy, immunogenicity, pharmacokinetics, and pharmacodynamics were investigated in a phase I, multicenter, open-label study in patients with HER2+ non-breast advanced malignancies (NCT03696771). Data from 18 patients enrolled in single ascending dose escalation demonstrated delivery of the TLR7-agonist payload in HER2+ tumor cells and induction of type I IFN responses, which correlated with immune modulation in the tumor microenvironment. Cytokine release syndrome was a common, but manageable, drug-related adverse event. Antidrug antibodies and neuroinflammation at high doses represented significant clinical challenges. Data provide proof-of-mechanism and critical insights for novel immunotherapies.

Self-regulation strategies of children with emotional disturbance.

Children with emotional disturbance frequently have difficulty regulating their classroom behaviors. Many have co-occurrence of other disabilities, such as sensory processing problems, which compound difficulties in school participation. This exploratory project evaluated the 8-week-long use of the Alert Program within the classroom setting for seven children with emotional disturbance. Five children with emotional disturbance served as a control group. Self-regulation, behavioral adjustments, and sensory processing skills as reported by the children and teachers were evaluated. Changes from pretest to posttest indicated that children who received the Alert Program demonstrated a small improvement on all measures while performance of the control group remained relatively constant or decreased. These preliminary results indicate programs that target self-regulation skills may be useful in helping to improve self-regulation of children with emotional disturbance.

Gonadotropin-releasing hormone antagonist (Cetrorelix) therapy fails to protect nonhuman primates (Macaca arctoides) from radiation-induced spermatogenic failure.

Treatment of men of reproductive age with radiation or alkylating agents often produces prolonged azoospermia. We previously demonstrated that suppression of testosterone (T) with gonadotropin-releasing hormone (GnRH) analogs restored spermatogenesis following atrophy induced by radiation or chemotherapy in rats. This study tested whether GnRH antagonist therapy could reverse radiation-induced testicular injury in primates with a similar protocol. Adult male stump-tailed macaques were given either 6.7 Gy radiation to the testis alone, 6.7 Gy radiation combined with GnRH-antagonist treatment starting on the day of exposure, or daily injections of the GnRH antagonist Cetrorelix for 3 months alone and were monitored for 18 months. Cetrorelix alone produced a 20-40-week fully reversible suppression of serum T, but although spermatogenic recovery was incomplete, 40%-90% of tubules contained differentiating germ cells. Following radiation alone, testis volumes were reduced to approximately 28% and sperm counts to less than 1% of pretreatment values. A biopsy at 18 months after radiation showed that only 3.0% of seminiferous tubule cross sections had germ cells. In irradiated animals that received GnRH antagonist, testis volumes were reduced to 18% of pretreatment volume, and at 18 months, only 1.9% of seminiferous tubule cross sections contained germ cells. Inhibin B values were reduced to 10% and 3% of pretreatment levels in the radiation-only and the radiation plus GnRH antagonist groups, respectively. Species differences exist in the testicular response to radiation, GnRH antagonist therapy, or both, so that rescue protocols that were successful in rodents might not work in primates.

Defining the cellular and molecular mechanisms of toxicant action in the testis.

A symposium was held at the 41st annual meeting of the Society of Toxicology with presentations that emphasized novel molecular and cellular pathways that modulate the response to testicular toxicants. The first two presentations described cellular alterations after exposure to the Sertoli cell toxicant, mono-(2-ethylhexyl) phthalate (MEHP). The expression of flamingo1, a G protei coupled receptor family member that may couple cell-cell adhesion to G protein-dependent signaling in Sertoli cells, was rapidly altered after MEHP exposure. Sertoli cell associated flamingo1 immunostaining was redistributed early (within 2 h) after MEHP exposure and disappeared by 12 h, suggesting that flamingo1 is a proximal phthalate target. MEHP was also described to alter the expression and activity of the newly identified death receptors DR4, 5 and 6 in the testis. The differential cellular changes in the levels of DR4, 5 and 6 after phthalate exposure suggested that they may act as surrogates or in concert with the widely described Fas-signaling pathway in the initiation of germ cell apoptosis after MEHP exposure. The next two presentations focused on revealing mechanisms that may explain the persistent post-exposure testicular atrophy that is observed in rodents after a variety of chemical or physical insults (radiation, chemotherapeutics, toxicants) and possible strategies to re-initiate spermatogenesis in the atrophic testis. Hormonal manipulations that lower testosterone and serum FSH levels allow for re-initiation of spermatogonial development. Recent investigation of additional models of persistent atrophy such as mutant mice, the aged Brown Norway rat, EDS-induced Leydig cell deficient rat, and primates, have broadened insight into the mechanisms responsible for persistent atrophy. The last presentation described the use of cDNA arrays in the investigation of cellular elements and mechanisms responsible for disruption of spermatogenesis by the drinking water disinfectant bromochloroacetic acid (BCA). A custom mouse testis cDNA array interrogating 950 genes was used for analysis of testis mRNA. BCA altered the expression of 53 of the 950 genes, including two encoding sperm proteins known to be significant for male fertility, and other genes involved in spermatogenesis, stress response, and cell communication/adhesion. These observations strengthen the hypothesis that BCA disrupts spermatogenesis by altering the process of spermiogenesis.

Expression of a K48R mutant ubiquitin protects mouse testis from cryptorchid injury and aging.

Testis injury models can be useful for determining the in vivo function of genes. In this study, ubiquitin, a tag for 26S-proteasome degradation, was mutated at lysine 48 (K48R) to inhibit ubiquitin chain assembly. K48R transgenic mice had testes with delayed germ cell loss following the acute injury of experimental cryptorchidism, and were resistant to the chronic injury of aging-associated testicular atrophy. After 4 days of cryptorchid-mediated heat stress, the average weight of cryptorchid testes in wild-type ubiquitin mice was significantly lower (P < 0.05) than in K48R mutant ubiquitin mice, indicating that altered ubiquitination delayed germ cell death. Light microscopy confirmed that the testicular injury, in both wild-type and K48R ubiquitin mice, was due to germ cell death. In addition, wild-type ubiquitin mice aged 19 to 22 months showed greater testicular atrophy and decreased average seminiferous tubule diameter when compared with K48R-aged testes. These results demonstrate a resistance to testicular injury conferred by the K48R mutation, suggesting that ubiquitin-mediated protein degradation is involved in the processing or modulation of testicular insults.

2,5-hexanedione-induced testicular injury.

Now in its third decade of mechanistic investigation, testicular injury caused by 2,5-hexanedione (2,5-HD) exposure is a well-studied model with a rich database. The development of this model reflects the larger changes that have moved biology from a branch of chemistry into the molecular age. Critically examined in this review is the proposed mechanism for 2,5-HD-induced testicular injury in which germ cell maturation is disrupted owing to alterations in Sertoli cell microtubule-mediated functions. The goal is to evaluate the technical and conceptual approaches used to assess 2,5-HD-induced testicular injury, to highlight unanswered questions, and to identify fruitful avenues of future research.