Novel methods for the assessment of safety pharmacology and toxicology parameters in anesthetized and ventilated dogs receiving inhaled drugs.

INTRODUCTION: For nonclinical drug development, it is optimal if safety pharmacology and toxicology studies are performed in a model that reasonably represents the patient the drug is intended to treat. To simulate prolonged inhalation therapy in ventilated patients, GLP inhalation toxicology methods, including safety pharmacology endpoints, in anesthetized, intubated and mechanically ventilated dogs were developed. This model required establishment of a canine intensive care unit (ICU) capable of providing prolonged anesthesia (propofol infusion and morphine titration) and partial parenteral nutrition (dextrose, amino acids and lipids) while safety parameters were monitored. METHOD: Telemetry was used to continuously monitor heart rate, ECG and blood pressure. Blood gas parameters were periodically measured while oxygen saturation and core temperature were reported continuously. Glucose was measured hourly while other standard clinical pathology (hematology, coagulation, clinical chemistry) samples were evaluated approximately every 12 h. Aerosols were administered continuously over 48 h by inhalation using a mesh nebulizer (Aerogen Solo) fed by a syringe pump into a humidified circuit of a critical care ventilator (LTV® 1000) ending in an endotracheal tube placed in the trachea. Animals were ventilated with pressure control ventilation targeting a respiratory minute volume of 2.0-3.5 l per minute (LPM). Peak inspiratory pressure (PIP) was maintained between 10 and 17 cm H2O and inspiratory time was set to 1 s with an inspiratory:expiratory (I:E) ratio of 1:2. Ventilator parameters and anesthesia were adjusted to maintain normal PaCO2 levels and adequate sedation, respectively. Novel methods were developed to determine dose and particle size in vitro as on-line measurements were not feasible during in vivo aerosol delivery. RESULTS AND DISCUSSION: Acceptable baseline measurements were established for all parameters over the 48-h evaluation period, qualifying the method as appropriate for assessment of GLP safety pharmacology and toxicology studies.

Citrulline as a Biomarker for Gastrointestinal-Acute Radiation Syndrome: Species Differences and Experimental Condition Effects.

Animal models of hematopoietic and gastrointestinal acute radiation syndromes (ARS) have been characterized to develop medical countermeasures. Acute radiation-induced decrease of intestinal absorptive function has been correlated to a decrease in the number of intestinal crypt cells resulting from apoptosis and enterocyte mass reduction. Citrulline, a noncoded amino acid, is produced almost exclusively by the enterocytes of the small intestine. Citrullinemia has been identified as a simple, sensitive and suitable biomarker for radiation-induced injury associated with gastrointestinal ARS (GI-ARS). Here we discuss the effect of radiation on plasma citrulline levels in three different species, C57BL/6 mice, Göttingen minipigs and rhesus nonhuman primates (NHPs), measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). The effects of experimental study conditions such as feeding and anesthesia were also examined on plasma citrulline levels in the NHPs. Both the mice and Göttingen minipigs were partial-body irradiated (PBI) with doses from 13-17 Gy and 8-16 Gy, respectively, whereas NHPs were total-body irradiated (TBI) with doses from 6.72-13 Gy. Blood samples were taken at different time points and plasma citrulline levels were measured in the three species at baseline and after irradiation. Basal plasma citrulline concentrations (mean ± SEM) in mice and minipigs were 57.8 ± 2.8 µM and 63.1 ± 2.1 µM, respectively. NHPs showed a basal plasma citrulline concentration of 32.6 ± 0.7 µM, very similar to that of humans (∼40 µM). Plasma citrulline progressively decreased after irradiation, reaching nadir values between day 3.5 and 7. The onset of citrulline recovery was observed earlier at lower radiation doses, while only partial citrulline recovery was noted at higher radiation doses in minipigs and NHPs, complete recovery was noted in mice at all doses. Plasma citrulline levels in NHPs anesthetized with ketamine and acepromazine significantly decreased by 35.5% (P = 0.0017), compared to unanesthetized NHPs. In the postprandial state, citrulline concentrations in NHPs were slightly but significantly decreased by 12.2% (P = 0.0287). These results suggest that plasma citrulline is affected by experimental conditions such as anesthesia and feeding.