Improving target assessment in biomedical research: the GOT-IT recommendations.

Academic research plays a key role in identifying new drug targets, including understanding target biology and links between targets and disease states. To lead to new drugs, however, research must progress from purely academic exploration to the initiation of efforts to identify and test a drug candidate in clinical trials, which are typically conducted by the biopharma industry. This transition can be facilitated by a timely focus on target assessment aspects such as target-related safety issues, druggability and assayability, as well as the potential for target modulation to achieve differentiation from established therapies. Here, we present recommendations from the GOT-IT working group, which have been designed to support academic scientists and funders of translational research in identifying and prioritizing target assessment activities and in defining a critical path to reach scientific goals as well as goals related to licensing, partnering with industry or initiating clinical development programmes. Based on sets of guiding questions for different areas of target assessment, the GOT-IT framework is intended to stimulate academic scientists' awareness of factors that make translational research more robust and efficient, and to facilitate academia-industry collaboration.

Reprint of: Nonclinical species sensitivity to convulsions: An IQ DruSafe consortium working group initiative.

Clinical development of compounds that carry a convulsion liability is typically limited by safety margins based on the most sensitive nonclinical species. To better understand differences in sensitivity to drug-induced convulsion of commonly used preclinical species, a survey was distributed amongst pharmaceutical companies through an IQ consortium (International Consortium for Innovation and Quality in Pharmaceutical Development) resulting in convulsion-related data on 80 unique compounds from 11 companies. The lowest free drug plasma concentration at which convulsions were observed and the no observed effect level for convulsions were compared between species to determine their relative sensitivity. Additionally, data were collected on other endpoints including use of electroencephalography, premonitory signs, convulsion type, the reason why development was stopped, and the highest development phase reached. The key outcomes were: (1) the dog was most often determined to be the most sensitive species by both non-exposure and exposure-based analyses, (2) there was not a clear sensitivity ranking of other species (NHP, rat and mouse), (3) CNS symptoms were frequently present at exposures that were not associated with convulsions, but no single reliable premonitory indicator of convulsion was identified, and (4) the lack of convulsions in the compounds that were tested in humans in this dataset may suggest that convulsion liability is well mitigated via current drug development strategies.

Nonclinical species sensitivity to convulsions: An IQ DruSafe consortium working group initiative.

Clinical development of compounds that carry a convulsion liability is typically limited by safety margins based on the most sensitive nonclinical species. To better understand differences in sensitivity to drug-induced convulsion of commonly used nonclinical species, a survey was distributed amongst pharmaceutical companies through an IQ consortium (International Consortium for Innovation and Quality in Pharmaceutical Development) resulting in convulsion-related data on 80 unique compounds from 11 companies. The lowest free drug plasma concentration at which convulsions were observed and the no observed effect level for convulsions were compared between species to determine their relative sensitivity. Additionally, data were collected on other endpoints including use of electroencephalography, premonitory signs, convulsion type, the reason why development was stopped, and the highest development phase reached. The key outcomes were: (1) the dog was most often determined to be the most sensitive species by both non-exposure and exposure-based analyses, (2) there was not a clear sensitivity ranking of other species (NHP, rat and mouse), (3) CNS symptoms were frequently present at exposures that were not associated with convulsions, but no single reliable premonitory indicator of convulsion was identified, and (4) the lack of convulsions when compounds were tested in humans in this dataset may suggest that convulsion liability is well mitigated via current drug development strategies.

Refinement of the rodent pentylenetetrazole proconvulsion assay, which is a good predictor of convulsions in repeat-dose toxicology studies.

INTRODUCTION: The pentylenetetrazole (PTZ)-induced seizure assay in rodents is an established method for investigating drug-induced alterations in seizure threshold such as proconvulsant effects. The standard procedure in our laboratory was to administer the test item prior to 75-120 mg/kg subcutaneous PTZ. However, this dose range is associated with a high incidence of mortality, including approximately 40% or greater deaths of control animals. METHODS: The predictivity of the PTZ-induced seizure assay was retrospectively evaluated by relating drug plasma levels associated with proconvulsant effects to exposures observed during convulsions in repeat-dose toxicology studies. Margins to estimated efficacious doses were also considered. To investigate potential refinements, a high PTZ dose (80 mg/kg, subcutaneously) was compared to two lower doses (40 and 60 mg/kg), and a range of doses of theophylline was orally administered as positive control. RESULTS: The PTZ-induced proconvulsion assay proved to be a good predictor of convulsions in toxicology studies. In the refinement study, theophylline potentiated PTZ-induced seizures over all doses tested. At 60 mg/kg PTZ, the proconvulsant dose-dependency of theophylline was best observed. At both 40 and 60 mg/kg PTZ, mortality in control animals was significantly reduced. DISCUSSION: Risk assessment at an early stage of drug development supports candidate selection and, along that approach, the PTZ proconvulsion assay was proven to be a good predictor of convulsions in subsequent toxicology studies. It was also demonstrated that a relatively lower PTZ dose (60 mg/kg) improved the dose-response-curve of the positive control tested, decreased mortality overall and, therefore, contributes to refining this standard procedure for CNS safety evaluation.

Exploratory EEG studies for the assessment of neurological liabilities in conscious dogs in early drug development.

INTRODUCTION: Convulsions in toxicology studies can be the first indication of seizure liability. Drug levels during convulsions are not usually evaluated. This, and exposure variability after oral administration, complicates estimation of safety margins. The electroencephalogram (EEG) enables symptoms to be attributed to seizures and to collect samples during epileptiform activity without clinical convulsion. We evaluated an EEG-study design for optimized detection of neurological symptoms. Additionally, we assessed whether EEG- based anticonvulsive treatment is feasible, to prevent progression to convulsions and if dogs have higher sensitivity towards neurological symptoms than non-human-primates. METHODS: Three compounds that previously were tested in non-human-primates were selected to evaluate the dog EEG-study design. Two substances were administered in escalating intravenous doses; the third was given as single oral dose. Per compound, one male and one female telemetered dog were evaluated; males also had cerebrospinal-fluid-ports. Drug levels, video-EEG and clinical symptoms were evaluated and compared to previous studies. RESULTS: While similar neurological symptoms were induced, intravenous administration reduced experimental time compared to standard toxicology studies. EEG analysis could link animal behavior to seizures but did not allow convulsion prevention. This was due to artefacts and the short latency between onset of epileptiform EEG activity and clinical convulsions. Free plasma concentrations during convulsions were comparable between dogs and non-human-primates. DISCUSSION: The findings suggest that infusion studies provide a possibility to investigate neurological adverse effects in few animals in a short time period. For candidates with a high risk for seizures, such studies can guide dose selection for longer regulatory studies and improve safety margin definition.

Measuring drug absorption improves interpretation of behavioral responses in a larval zebrafish locomotor assay for predicting seizure liability.

INTRODUCTION: Unanticipated effects on the central nervous system are a concern during new drug development. A larval zebrafish locomotor assay can reveal seizure liability of experimental molecules before testing in mammals. Relative absorption of compounds by larvae is lacking in prior reports of such assays; having those data may be valuable for interpreting seizure liability assay performance. METHODS: Twenty-eight reference drugs were tested at multiple dose levels in fish water and analyzed by a blinded investigator. Responses of larval zebrafish were quantified during a 30min dosing period. Predictive metrics were calculated by comparing fish activity to mammalian seizure liability for each drug. Drug level analysis was performed to calculate concentrations in dose solutions and larvae. Fifteen drug candidates with neuronal targets, some having preclinical convulsion findings in mammals, were tested similarly. RESULTS: The assay has good predictive value of established mammalian responses for reference drugs. Analysis of drug absorption by larval fish revealed a positive correlation between hyperactive behavior and pro-convulsive drug absorption. False negative results were associated with significantly lower compound absorption compared to true negative, or true positive results. The predictive value for preclinical toxicology findings was inferior to that suggested by reference drugs. DISCUSSION: Disproportionately low exposures in larvae giving false negative results demonstrate that drug exposure analysis can help interpret results. Due to the rigorous testing commonly performed in preclinical toxicology, predicting convulsions in those studies may be more difficult than predicting effects from marketed drugs.

A retrospective evaluation of species-specific sensitivity for neurological signs in toxicological studies: Is the dog more sensitive than the non-human primate?

Selection of the appropriate non-rodent species in preclinical programs is crucial for good translatability and human safety. There is no data available in the literature which provides exact comparison of dog and non-human primate (NHP) sensitivity regarding neurological signs in toxicological studies. We performed a retrospective analysis of 174 toxicity studies with 15 neuroscience substances. Neurological signs in dogs and NHPs were evaluated in correlation to exposure data. Overall incidence of substance induced convulsions was similar in both species and no gender differences were observed. The reported liability of beagles to spontaneous convulsions was not confirmed in our studies. The symptom tremor showed the best inter-species translatability. The current toxicological study design does not include exposure assessment at the time-point of neurological signs, therefore, we propose to include additional toxicokinetic samples. Our analysis revealed factors including housing, handling, and behavior, which prevents direct species comparison. In addition only one non-rodent species is routinely tested in development programs, therefore data for both species is rare. We however, had sufficient data which enabled comparison for one compound. In the spirit of 3Rs further examples should be evaluated.

Soluplus® as an effective absorption enhancer of poorly soluble drugs in vitro and in vivo.

As many new active pharmaceutical ingredients are poorly water soluble, solubility enhancers are one possibility to overcome the hurdles of drug dissolution and absorption in oral drug delivery. In the present work a novel solubility enhancing excipient (Soluplus®) was tested for its capability to improve intestinal drug absorption. BCS class II compounds danazol, fenofibrate and itraconazole were tested both in vivo in beagle dogs and in vitro in transport experiments across Caco-2 cell monolayers. Each drug was applied as pure crystalline substance, in a physical mixture with Soluplus®, and as solid solution of the drug in the excipient. In the animal studies a many fold increase in plasma AUC was observed for the solid solutions of drug in Soluplus® compared to the respective pure drug. An effect of Soluplus® in a physical mixture with the drug could be detected for fenofibrate. In vitro transport studies confirm the strong effect of Soluplus® on the absorption behavior of the three tested drugs. Furthermore, the increase of drug flux across Caco-2 monolayer is correlating to the increase in plasma AUC and C(max)in vivo. For these poorly soluble substances Soluplus® has a strong potential to improve oral bioavailability. The applicability of Caco-2 monolayers as tool for predicting the in vivo transport behavior of the model drugs in combination with a solubility enhancing excipient was shown. Also the improvement of a solid dispersion compared to physical mixtures of the drugs and the excipient was correctly reflected by Caco-2 experiments. In the case of fenofibrate the possible improvement by a physical mixture was demonstrated, underscoring the value of the used tool as alternative to animal studies.

Refinement and reduction of acute oral toxicity testing: a critical review of the use of cytotoxicity data.

Acute oral toxicity testing is still required for the classification and labelling of chemicals, agrochemicals and related formulations. There have been increasing efforts over the last two decades to reduce the number of animals needed for this testing, according to the Three Rs concept. To evaluate the utility of an in vitro cytotoxicity test in our routine testing for acute oral toxicity, we have implemented in our laboratory the neutral red uptake (NRU) method, with Balb/c 3T3 fibroblasts after a 48-hour exposure, which was recommended in ICCVAM Report 07-4519, 2006. Initially, we tested 16 substances that had existing in vivo and in vitro data available, to prove our technical proficiency with the in vitro test. Then, testing was performed with 187 test substances, including a broad variety of chemicals, agrochemicals and formulations. The starting dose for acute oral systemic toxicity assays in rats (LD50) was estimated by using the prediction model presented in the ICCVAM validation study, and subsequently compared to the results obtained by in vivo testing performed according to, or similar to, OECD Test Guideline 423. Comparison of all of the 203 predicted LD50 values that were deduced from the in vitro IC50 values, with the in vivo results from oral toxicity studies in rats, resulted in a low overall concordance of 35%. The in vitro cytotoxicity assay achieved a good concordance of 74%, only for the weakly toxic substances (EU-GHS Cat. 4). However, it must be noted that 71% of the substances tested (i.e. 145/203) were classified as being weakly toxic in vitro. We further analysed the utility of the in vitro test for predicting the starting dose for an in vivo study, and the potential reduction in animal usage that this would engender. In this regard, the prediction by the cytotoxicity test was useful for 59% of the substances. However, the use of a standard starting dose of 300 mg/kg bw by default (without previous cytotoxicity testing) would have been almost as useful (50%). In contrast, the prediction by an experienced toxicologist was correct for 95% of the substances. However, this was only performed for 40% of the substances, mainly those of no to low toxicity. Calculating the theoretical animal numbers needed in several scenarios supported these results. The additional analysis, considering some physicochemical data (solubility, molecular weight, log POW), substance class and mode of action, revealed no specific applicability domains. In summary, the use of the 3T3 NRU cytotoxicity data alone did not sufficiently contribute to refinement and reduction in the acute oral toxicity testing of the substance portfolio tested routinely in our laboratory.