Is there a role for the no observed adverse effect level in safety pharmacology?

In nonclinical toxicology the highest dose or exposure without test article-related adverse effects, known as the No Observed Adverse Effect Level (NOAEL), is a variable that may be determined. In safety pharmacology the vast majority of the endpoints measured are quantitative numeric functional endpoints such as changes in heart rate, blood pressure or respiratory frequency, endpoints that are usually not assessed using a defined framework of adversity. Therefore, we asked the question: is there a role for the NOAEL in safety pharmacology? To help answer this question, we conducted a survey via the Safety Pharmacology Society. We found that within safety pharmacology there is no formal definition of adversity and no guidance on defining NOAEL. We also found, perhaps unsurprisingly, there is no agreed rubric for using a NOAEL in safety pharmacology and we learned that the NOAEL is not a requirement in order to progress a new investigational drug through the regulatory process. Thus, a summary label such as NOAEL lacks nuance and disregards context in relation to the nature and the severity of the safety pharmacology findings. Consequently, defining 'adversity' and determining a NOAEL in safety pharmacology studies are not recommended since the range of functional endpoints investigated do not conform to a binary 'toxic/non-toxic' rubric. Focusing on describing test article-related effects on safety pharmacology endpoints, using reasoned arguments as part of an integrated risk assessment, will ensure that the clinical pharmacologists and regulatory bodies see a clear description of relevant findings at each dose or exposure level.

Evaluation of the PhysioTel™ Digital M11 cardiovascular telemetry implant in socially housed cynomolgus monkeys up to 16weeks after surgery.

INTRODUCTION: The novel PhysioTel™ Digital M11 telemetry implant was evaluated in socially housed monkeys with respect to both safety pharmacological cardiovascular (arterial blood pressure (BP), heart rate (HR) and electrocardiogram (ECG)) and toxicological (clinical pathology and histopathology) endpoints. METHODS: Telemetry and clinical pathology data were obtained repeatedly up to 16weeks after surgery in four female cynomolgus monkeys, followed by necropsy. Due to postsurgical complications, one spare animal was included and only toxicological endpoints from the affected (fifth animal) were reported. Continuous telemetry recordings were conducted at periods without dosing and after ascending doses of moxifloxacin (0, 10, 30, 100mg/kg) and L-NAME (0, 0.1, 1, 10mg/kg). Additionally, a retrospective power analysis was conducted based on baseline M11 implant data from 32 other animals. RESULTS: During periods without dosing, the cardiovascular endpoints were stable over time and within normal ranges. Moxifloxacin and L-NAME elicited the expected pharmacological responses with dose-dependent increase in QTca (8, 17, 22ms) and BP (mean BP: 12, 21, 34mmHg), respectively. Expected intravascular and tissue reactions were observed at the sites of the BP catheter and the transmitter. Signs of infection (localised to the transmitter implantation site with associated systemic effects) was noted in the fifth animal. No systemic pathologies were seen in any animals. Power analysis (80% power) indicated that the minimal differences which can be detected in a parallel group design (n=6) are 7mmHg (mean BP), 16bpm (HR), 12ms (QTca). DISCUSSION: The M11 implant provided stable, high quality ECG and BP data for a duration covering the length of sub-chronic repeated dose toxicity studies without important impact on toxicological endpoints. Adequate power in order to elucidate major treatment-related cardiovascular effects was demonstrated. However to avoid post-surgical complications the implantation procedures should be carefully considered before using the method.

The impact of different blood sampling methods on laboratory rats under different types of anaesthesia.

Rats with implanted telemetry transponders were blood sampled by jugular puncture, periorbital puncture or tail vein puncture, or sampled by jugular puncture in carbon dioxide (CO2), isoflurane or without anaesthesia in a crossover design. Heart rate, blood pressure and body temperature were registered for three days after sampling. Initially blood pressure increased, but shortly after sampling it decreased, which led to increased heart rate. Sampling induced rapid fluctuations in body temperature, and an increase in body temperature. Generally, rats recovered from sampling within 2-3 h, except for rats sampled from the tail vein, which showed fluctuations in body temperature in excess of 30 h after sampling. Increases in heart rate and blood pressure within the first hours after sampling indicated that periorbital puncture was the method that had the largest acute impact on the rats and that it might take an extra hour to recover from it. CO2 anaesthesia seemed unable to prevent the increase in blood pressure and the fluctuations in body temperature induced by blood sampling, and up to 10 h after sampling, the rats were still affected by CO2 anaesthesia. Rats anaesthetized with isoflurane showed lower increases in blood pressure after, and fewer fluctuations in body temperature during sampling, and the post-anaesthetic effects of isoflurane, if any, seemed to disappear immediately after sampling. It is, therefore, concluded that blood sampling in rats by jugular puncture seems to be the method from which rats most rapidly recover when compared with periorbital puncture and tail vein puncture, and that for anaesthesia, isoflurane is recommended in preference to CO2.

The impact of cage ventilation on rats housed in IVC systems.

Today the use of individually ventilated cage systems (IVC systems) is common, especially for housing transgenic rodents. Typically, in each cage a ventilation rate of 40 to 50 air changes per hour is applied, but in some systems even up to 120 air changes per hour is applied. To reach this rate, the air is blown into the cage at a relatively high speed. However, at the animal's level most systems ventilate with an air speed of approximately 0.2 m/s. In the present paper, two studies were conducted, one analysing whether an air speed below 0.2 m/s or just above 0.5 m/s affects the rats, and another study analysing whether air changes of 50, 80 and 120 times per hour affect the rats. In both studies, monitoring of preferences as well as physiological parameters such as heart rate and blood pressure, was used to show the ability of the animals to register the different parameters and to avoid them if possible. Air speeds inside the cage of as high as 0.5 m/s could not be shown to affect the rats, while the number of air changes in each cage should be kept below 80 times per hour to avoid impacts on physiology (heart rate and systolic blood pressure). Also the rats prefer cages with air changes below 80 times per hour if they have the opportunity of choosing, as shown in the preference test.

The impact of low levels of carbon dioxide on rats.

The widespread use of individually ventilated cage (IVC) systems today has made the impact of CO(2) on rodents a highly important matter. Leaving cages from these systems without ventilation increases CO(2) concentrations inside the cages, as CO(2) generated from the animals is no longer removed actively. In modern IVC systems the CO(2) levels may reach 3-5% within a very short time, as the cages are very tightly sealed. The aim of the present study was to investigate the effects of 1%, 3%, and 5% CO(2) by studying the preferences of the animals as well as changes in the heart rate and systolic blood pressure as measured by telemetry. The rats avoided the cages, which contained 3% CO(2). In the telemetric study an anaesthetic effect on the rats were seen at 3% as a drop in the heart rate, and at 5% CO(2) a drop in the systolic blood pressure was also seen. The results from the present study could indicate that CO(2) levels of up to 3% do not affect the animals, or at least only to a minor extent, but that if the animals are exposed to CO(2) levels of higher than 3% they are affected directly as seen by changes in physiological parameters and preferences.

6-Chloro-3-alkylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide derivatives potently and selectively activate ATP sensitive potassium channels of pancreatic beta-cells.

6-Chloro-3-alkylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide derivatives were synthesized and characterized as activators of adenosine 5'-triphosphate (ATP) sensitive potassium (K(ATP)) channels in the beta-cells by measuring effects on membrane potential and insulin release in vitro. The effects on vascular tissue in vitro were measured on rat aorta and small mesenteric vessels. Selected compounds were characterized as competitive inhibitors of [(3)H]glibenclamide binding to membranes of HEK293 cells expressing human SUR1/Kir6.2 and as potent inhibitors of insulin release in isolated rat islets. 6-Chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (54) was found to bind and activate the SUR1/Kir6.2 K(ATP) channels in the low nanomolar range and to be at least 1000 times more potent than the reference compound diazoxide with respect to inhibition of insulin release from rat islets. Several compounds, e.g., 3-propylamino- (30), 3-isopropylamino- (34), 3-(S)-sec-butylamino- (37), and 3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (53), which were found to be potent and beta-cell selective activators of K(ATP) channels in vitro, were found to inhibit insulin secretion in rats with minimal effects on blood pressure and to exhibit good oral pharmacokinetic properties.