Assessing the risk of drug crystallization in vivo.

INTRODUCTION: Low intrinsic solubility leading to poor oral bioavailability is a common challenge in drug discovery that can often be overcome by formulation strategies, however, it remains a potential limitation that can pose challenges for early risk assessment and represent a significant obstacle to drug development. We identified a selective inhibitor (BMS-986126) of the IL-1 receptor-associated kinase 4 (IRAK4) with favorable properties as a lead candidate, but with unusually low intrinsic solubility of <1 µg/mL. METHODS: Conventional histopathology identified the issue of crystal formation in vivo. Subsequent investigative work included confocal Raman micro-spectroscopy, MALDI-MS, polarized light microscopy of fresh wet-mount tissue scrapings and transmission electron microscopy. RESULTS: BMS-986126 was advanced into a 2-week toxicology study in rats. The main finding in this study was minimal granulomatous inflammation in the duodenum, associated with the presence of birefringent crystals at the highest dosage of 100 mg/kg/day. Considering the safety margin, and the single location of the lesion, BMS-986126 was further progressed into IND-enabling toxicology studies where tolerability deteriorated with increasing dosing duration. Birefringent crystals and granulomatous inflammation were detected in multiple organs at dosages ≥20 mg/kg/day. Raman spectroscopy confirmed the identity of the crystals as BMS-986126. Therefore, follow up investigations were conducted to further characterize drug crystallization and to evaluate detection methods for their potential to reliably detect in vivo crystallization early. DISCUSSION: The purpose of our efforts was to identify critical factors influencing in vivo drug crystallization and to provide a preliminary assessment (based on one compound) which method would be best suited for identifying crystals. Results indicated a combination of methods was required to provide a complete assessment of drug crystallization and that a simple technique, scraping of freshly collected tissue followed by evaluation under polarizing light was suitable for detecting crystals. However, dosing for 2 weeks was required for crystals to grow to a clearly detectable size.

From the Cover: Investigative Nonclinical Cardiovascular Safety and Toxicology Studies with BMS-986094, an NS5b RNA-Dependent RNA Polymerase Inhibitor.

BMS-986094, a 2'-C-methylguanosine prodrug that was in development for treatment of chronic hepatitis C infection was withdrawn from Phase 2 clinical trials because of unexpected cardiac and renal adverse events. Investigative nonclinical studies were conducted to extend the understanding of these findings using more comprehensive endpoints. BMS-986094 was given orally to female CD-1 mice (25 and 150 mg/kg/d) for 2 weeks (53/group) and to cynomolgus monkeys (15 and 30 mg/kg/d) for up to 6 weeks (2-3/sex/group for cardiovascular safety, and 5/sex/group for toxicology). Endpoints included toxicokinetics; echocardiography, telemetric hemodynamics and electrocardiography, and tissue injury biomarkers (monkey); and light and ultrastructural pathology of heart, kidney, and skeletal muscle (mouse/monkey). Dose-related and time-dependent findings included: severe toxicity in mice at 150 mg/kg/d and monkeys at 30 mg/kg/d; decreased left ventricular (LV) ejection fraction, fractional shortening, stroke volume, and dP/dt; LV dilatation, increased QTc interval, and T-wave flattening/inversion (monkeys at ≥ 15 mg/kg/d); cardiomyocyte degeneration (mice at 150 mg/kg/d and monkeys at ≥ 15 mg/kg/d) with myofilament lysis/myofbril disassembly; time-dependent proteinuria and increased urine ß-2 microglobulin, calbindin, clusterin; kidney pallor macroscopically; and tubular dilatation (monkeys); tubular regeneration (mice 150 mg/kg/d); and acute proximal tubule degeneration ultrastructurally (mice/monkeys); and skeletal muscle degeneration with increased urine myoglobin and serum sTnI. These studies identified changes not described previously in studies of BMS-986094 including premonitory cardiovascular functional changes as well as additional biomarkers for muscle and renal toxicities. Although the mechanism of potential toxicities observed in BMS-986094 studies was not established, there was no evidence for direct mitochondrial toxicity.

Toxicity of Hexamoll(®) DINCH(®) following intravenous administration.

Alternative plasticizers to di(2-ethylhexyl) phthalate (DEHP) for blood bags have been sought for many years. Cyclohexane-1,2-dicarboxylic acid, diisononylester (Hexamoll(®) DINCH(®)) is an alternative that has been evaluated in preliminary studies for compatibility and efficacy to preserve whole blood. While Hexamoll(®) DINCH(®) has an extensive database for mammalian toxicity via oral administration, data were needed to evaluate toxicity from intravenous (IV) administration to support the use of the plasticizer Hexamoll(®) DINCH(®) in blood bags. A series of studies was performed by slow IV injection or IV infusion of Hexamoll(®) DINCH(®), a highly viscous, hydrophobic substance, suspended in Intralipid(®) 20% (20% intravenous fat emulsion). Rats were injected once, followed by 14 days of recovery; injected daily for 5 days followed by 5 days of recovery, or infused for 29 days (4h/day) followed by 14 days of recovery. Dose levels were 0, 62, 125, and 250-300mg/kg body weight/day. These dose levels represent the limits of suspension and far exceed any anticipated exposures from migration out of plasticized blood bags. Animals were observed for signs of toxicity; body weight and feed consumption were measured; blood collected for clinical chemistry and hematology; and tissues collected and processed for histopathology. Special emphasis was placed on evaluating endpoints and tissues that are commonly associated with plasticizer exposure in rodents. Urine was collected during the 4-week study to quantify urinary metabolites of Hexamoll(®) DINCH(®). The results of the studies indicate that no substance-related toxicity occurred: no effects on behavior, no effects on organ weight, no effect on serum chemistry including thyroid hormones; and no effect on major organs, especially no testicular toxicity and no indication for peroxisome proliferation in the liver. The only effects seen were petechia and granulomas related to dissipation of suspended Hexamoll(®) DINCH(®) in the aqueous environment of the blood. However, the results of metabolite analyses demonstrate that Hexamoll(®) DINCH(®) was bioavailable. Therefore, based on the lack of Hexamoll(®) DINCH(®)-related systemic toxicity with the exception of the physical limitations, the no-observed-adverse-effect level for parenterally administered Hexamoll(®) DINCH(®) is considered to be 300mg/kg bw/day.

Pharmacodynamics of selective inhibition of γ-secretase by avagacestat.

A hallmark of Alzheimer's disease (AD) pathology is the accumulation of brain amyloid ß-peptide (Aß), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aß and offer a potential new approach to treat AD. As γ-secretase also cleaves Notch proteins, GSIs can have undesirable effects due to interference with Notch signaling. Avagacestat (BMS-708163) is a GSI developed for selective inhibition of APP over Notch cleavage. Avagacestat inhibition of APP and Notch cleavage was evaluated in cell culture by measuring levels of Aß and human Notch proteins. In rats, dogs, and humans, selectivity was evaluated by measuring plasma blood concentrations in relation to effects on cerebrospinal fluid (CSF) Aß levels and Notch-related toxicities. Measurements of Notch-related toxicity included goblet cell metaplasia in the gut, marginal-zone depletion in the spleen, reductions in B cells, and changes in expression of the Notch-regulated hairy and enhancer of split homolog-1 from blood cells. In rats and dogs, acute administration of avagacestat robustly reduced CSF Aß40 and Aß42 levels similarly. Chronic administration in rats and dogs, and 28-day, single- and multiple-ascending-dose administration in healthy human subjects caused similar exposure-dependent reductions in CSF Aß40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aß levels without causing Notch-related toxicities. Our results demonstrate the selectivity of avagacestat for APP over Notch cleavage, supporting further evaluation of avagacestat for AD therapy.

Brain injury from a first nations' perspective: teachings from elders and traditional healers.

BACKGROUND: There is a lack of knowledge about how cultural ideas affect First Nations peoples' perception of rehabilitation needs and the ability to access services. PURPOSE: The study explored the perceptions of treating and healing brain injury from First Nations elders and traditional healers in the communities served by Wassay-Gezhig-Na-Nahn-Dah-We-lgamig (Kenora Area Health Access Centre). METHODS: A participatory action approach was used, leading to a focus group with elders and traditional healers. Findings, established through a framework analysis method, were member checked prior to dissemination. FINDINGS: Four themes arose from the data: pervasiveness of spirituality, "fixing" illness or injury versus living with wellness, working together in treating brain injury, and financial support needed for traditional healing. IMPLICATIONS: Funding is required for traditional healing services to provide culturallysafe and responsive occupational therapy services to First Nations individuals with brain injury.

From health care to home community: an Aboriginal community-based ABI transition strategy.

PRIMARY OBJECTIVE: To explore the barriers and enablers surrounding the transition from health care to home community settings for Aboriginal clients recovering from acquired brain injuries (ABI) in northwestern Ontario. RESEARCH DESIGN: Participatory research design using qualitative methods. METHODS: Focus groups conducted with clients with ABI, their caregivers and hospital and community health-care workers. The Framework Method of analysis was used to uncover emerging themes. FINDINGS: Six main categories emerged: ABI diagnosis accuracy, acute service delivery and hospital care, transition from hospital to homecare services, transition from hospital to community services, participant suggestions to improve service delivery and transition, and views on traditional healing methods during recovery. DISCUSSION: A lack of awareness, education and resources were acknowledged as key challenges to successful transitioning by clients and healthcare providers. Geographical isolation of the communities was highlighted as a barrier to accessibility of services and programmes, but the community was also regarded as an important source of social support. The development of educational and screening tools and needs assessments of remote communities were identified to be strategies that may improve transitions. CONCLUSIONS: Findings demonstrate that the structure of rehabilitation and discharge processes for Aboriginal clients living on reserves or in remote communities are of great concern and warrants further research.

Rehabilitation challenges for Aboriginal clients recovering from brain injury: a qualitative study engaging health care practitioners.

PRIMARY OBJECTIVE: To explore the experiences of health care practitioners working with Aboriginal clients recovering from acquired brain injury (ABI). RESEARCH DESIGN: Participatory research design using qualitative methods. METHODS: Fourteen in-depth, semi-structured interviews were conducted. The Framework Method of analysis was used to uncover emerging themes. FINDINGS: Five main categories emerged: practitioners' experience with brain injury, practitioners' experience with Aboriginal clients, specialized needs of Aboriginal clients recovering from brain injury, culturally sensitive care and traditional healing methods. These categories were then further divided into emergent themes and sub-themes where applicable, with particular emphasis on the specialized needs of Aboriginal clients. DISCUSSION: Each emergent theme highlighted key challenges experienced by Aboriginal peoples recovering from ABI. A key challenge was that protocols for rehabilitation and discharge planning are often lacking for clients living on reserves or in remote communities. Other challenges included lack of social support; difficulty of travel and socio-cultural factors associated with post-acute care; and concurrent disorders. CONCLUSIONS: Results suggest that developing reasonable protocols for discharge planning of Aboriginal clients living on reserves and/or remote communities should be considered a priority.

Itraconazole IV nanosuspension enhances efficacy through altered pharmacokinetics in the rat.

The goal of this research was to evaluate an intravenous itraconazole nanosuspension dosage form, relative to a solution formulation, in the rat. Itraconazole was formulated as a nanosuspension by a tandem process of microcrystallization followed by homogenization. Acute toxicity, pharmacokinetics, and distribution were studied in the rat, and compared with a solution formulation of itraconazole. Efficacy was studied in an immunocompromised rat model, challenged with a lethal dose of either itraconazole-sensitive or itraconazole-resistant C. albicans. Itraconazole nanosuspension was tolerated at significantly higher doses compared with a solution formulation. Pharmacokinetics of the nanosuspension were altered relative to the solution formulation. C(max) was reduced and t(1/2) was much prolonged. This occurred due to distribution of the nanosuspension to organs of the monocyte phagocytic system (MPS), followed by sustained release from this IV depot. The higher dosing of the drug, enabled in the case of the nanosuspension, led to higher kidney drug levels and reduced colony counts. Survival was also shown to be superior relative to the solution formulation. Thus, formulation of itraconazole as a nanosuspension enhances efficacy of this antifungal agent relative to a solution formulation, because of altered pharmacokinetics, leading to increased tolerability, permitting higher dosing and resultant tissue drug levels.

Evaluation of reproductive development following intravenous and oral exposure to DEHP in male neonatal rats.

Di-(2-ethylhexyl)phthalate (DEHP) was administered to 3- to 5-day-old male Sprague-Dawley rats by daily intravenous injections of 60, 300, or 600 mg/kg/day or by daily oral gavage of 300 or 600 mg/kg/day for 21 days. Histopathological evaluation and organ weight measurements were performed on some animals after 21 days of dosing (primary group) and later on the recovery group animals that were held without further treatment until sexual maturity at approximately 90 days of age. No effects of any type were observed in animals treated intravenously with 60 mg/kg/day. Testicular changes, consisting of a partial depletion of the germinal epithelium and/or decrease in diameter of seminiferous tubules, were present in all animals of the 300- and 600-mg/kg/day groups after the 21-day dosing period. Testes weight decreased and liver weight increased in these animals. Testes changes were dose-related and generally more severe among animals dosed orally versus intravenously. In the recovery animals, a residual DEHP-induced decrease in seminiferous tubule diameter was present in the testis of several animals dosed orally at 300 and 600 mg/kg/day, but not in animals dosed intravenously. There was no germinal cell depletion or Sertoli cell alteration observed in any dose group at any time. Notably, no effects on sperm count, sperm morphology, or sperm motility were observed at 90 days of age in any of the groups.