Smarca2 genetic ablation is phenotypically benign in a safety assessment of tamoxifen-inducible conditional knockout rats.

SMARCA2 and SMARCA4 are the ATPases of the SWI/SNF chromatin remodeling complex, which play a significant role in regulating transcriptional activity and DNA repair in cells. SMARCA2 has become an appealing synthetic-lethal, therapeutic target in oncology, as mutational loss of SMARCA4 in many cancers leads to a functional dependency on residual SMARCA2 activity. Thus, for therapeutic development, an important step is understanding any potential safety target-associated liabilities of SMARCA2 inhibition. To best mimic a SMARCA2 therapeutic, a tamoxifen-inducible (TAMi) conditional knockout (cKO) rat was developed using CRISPR technology to understand the safety profile of Smarca2 genetic ablation in a model system that avoids potential juvenile and developmental phenotypes. As the rat is the prototypical rodent species utilized in toxicology studies, a comprehensive toxicological and pathological assessment was conducted in both heterozygote and homozygous knockout rats at timepoints up to 28 days, alongside relevant corresponding controls. To our knowledge, this represents the first TAMi cKO rat model utilized for safety assessment evaluations. No significant target-associated phenotypes were observed when Smarca2 was ablated in mature (11- to 15-week-old) rats; however subsequent induction of SMARCA4 was evident that could indicate potential compensatory activity. Similar to mouse models, rat CreERT2-transgene and TAMi toxicities were characterized to avoid confounding study interpretation. In summary, a lack of significant safety findings in Smarca2 cKO rats highlights the potential for therapeutics targeting selective SMARCA2 ATPase activity; such therapies are predicted to be tolerated in patients without eliciting significant on-target toxicities.

Intravitreal Administration of Acetyl Triethyl Citrate and Benzyl Benzoate Is Retinotoxic in Rabbits but Not in Cynomolgus Monkeys.

Sustained drug delivery formulations are developed to reduce dose frequency while maintaining efficacy of intravitreal (ITV) administered therapeutics. Available safety data for components novel to the eye's posterior segment may be limited, requiring preclinical assessments to identify potential toxicities. We evaluated the in vivo and in vitro safety of two solvents, acetyl triethyl citrate (ATEC) and benzyl benzoate (BB), as novel sustained delivery formulations for ITV administration. In vivo tolerability was assessed following ITV administration of ATEC and BB to rabbits and cynomolgus monkeys. In rabbits, ITV solvent administration resulted in moderate to severe retinal toxicity characterized by focal retinal necrosis and/or degeneration, sometimes accompanied by inflammation, with a clear association between the physical presence of the solvent and areas of retinal damage. In contrast, solvent administration in monkeys appeared well tolerated, producing no histologic abnormalities. Toxicity in primary human retinal pigment epithelial cells, characterized by cellular toxicity and mitochondrial injury, corroborated the retinal toxicity in rabbits. In conclusion, ITV solvent depots of ATEC or BB result in chemical and focal retinal toxicity in rabbits, but not monkeys. Additional investigation is needed to demonstrate a sufficient margin of safety prior to use of ATEC or BB in ITV drug products.

Retinal and Lens Degeneration in New Zealand White Rabbits Administered Intravitreal TSG-6 Link Domain-Rabbit FAb Fusion Proteins.

Fusion of biologic therapeutics to hyaluronic acid binding proteins, such as the link domain (LD) of Tumor necrosis factor (TNF)-Stimulated Gene-6 (TSG-6), is expected to increase vitreous residence time following intravitreal injection and provide for long-acting delivery. The toxicity of a single intravitreal dose of free TSG-6-LD and fusion proteins of TSG-6-LD and a nonbinding rabbit antibody fragment (RabFab) were assessed in New Zealand White rabbits. Animals administered free TSG-6-LD exhibited extensive lens opacities and variable retinal vascular attenuation, correlated with microscopic findings of lens and retinal degeneration. Similar but less severe findings were present in animals dosed with the RabFab-TSG-6-LD fusion proteins. In-life ocular inflammation was noted in all animals from 7-days postdose and was associated with high anti-RabFab antibody titers in animals administered fusion proteins. Inflammation and retinal degeneration were multifocally associated with evidence of retinal detachment, and hypertrophy and migration of vimentin, glial fibrillary acidic protein, and glutamine synthetase positive Müller cells to the outer nuclear layer. Further assessment of alternative hyaluronic acid binding protein fusions should consider the potential for retinal degeneration and enhanced immune responses early in development.

Preclinical Safety Assessment of a Highly Selective and Potent Dual Small-Molecule Inhibitor of CBP/P300 in Rats and Dogs.

Cyclic adenosine monophosphate-response element (CREB)-binding protein (CBP) and EP300E1A-binding protein (p300) are members of the bromodomain and extraterminal motif (BET) family. These highly homologous proteins have a key role in modulating transcription, including altering the status of chromatin or through interactions with or posttranslational modifications of transcription factors. As CBP and p300 have known roles for stimulating c-Myc oncogenic activity, a small-molecule inhibitor, GNE-781, was developed to selectively and potently inhibit the CBP/p300 bromodomains (BRDs). Genetic models have been challenging to develop due to embryonic lethality arising from germline homozygous mutations in either CBP or P300. Hence, the purpose of this study was to characterize the role of dual inhibition of these proteins in adult rats and dogs. Repeat dose toxicity studies were conducted, and toxicologic and pathologic end points were assessed. GNE-781 was generally tolerated; however, marked effects on thrombopoiesis occurred in both species. Evidence of inhibition of erythroid, granulocytic, and lymphoid cell differentiation was also present, as well as deleterious changes in gastrointestinal and reproductive tissues. These findings are consistent with many preclinical (and clinical) effects reported with BET inhibitors targeting BRD proteins; thus, the current study findings indicate a likely important role for CBP/p300 in stem cell differentiation.

Principles and procedures for handling out-of-domain and indeterminate results as part of ICH M7 recommended (Q)SAR analyses.

The International Council for Harmonization (ICH) M7 guideline describes a hazard assessment process for impurities that have the potential to be present in a drug substance or drug product. In the absence of adequate experimental bacterial mutagenicity data, (Q)SAR analysis may be used as a test to predict impurities' DNA reactive (mutagenic) potential. However, in certain situations, (Q)SAR software is unable to generate a positive or negative prediction either because of conflicting information or because the impurity is outside the applicability domain of the model. Such results present challenges in generating an overall mutagenicity prediction and highlight the importance of performing a thorough expert review. The following paper reviews pharmaceutical and regulatory experiences handling such situations. The paper also presents an analysis of proprietary data to help understand the likelihood of misclassifying a mutagenic impurity as non-mutagenic based on different combinations of (Q)SAR results. This information may be taken into consideration when supporting the (Q)SAR results with an expert review, especially when out-of-domain results are generated during a (Q)SAR evaluation.

Individual serum bile acid profiling in rats aids in human risk assessment of drug-induced liver injury due to BSEP inhibition.

Drug-induced liver injury (DILI) has been the most frequent cause of post-marketing drug withdrawals in the last 50years. The multifactorial nature of events that precede severe liver injury in human patients is difficult to model in rodents due to a variety of confounding or contributing factors that include disease state, concurrent medications, and translational species differences. In retrospective analyses, a consistent risk factor for DILI has been the inhibition of the Bile Salt Export Pump (BSEP). One compound known for potent BSEP inhibition and severe DILI is troglitazone. The purpose of the current study is to determine if serum profiling of 19 individual bile acids by liquid chromatography-mass spectrometry (LC/MS) can detect perturbations in bile acid homeostasis in rats after acute intravenous (IV) administration of vehicle or 5, 25, or 50mg/kg troglitazone. Minimal serum transaminase elevations (approximately two-fold) were observed with no evidence of microscopic liver injury. However, marked changes in individual serum bile acids occurred, with dose-dependent increases in the majority of the bile acids profiled. When compared to predose baseline values, tauromuricholic acid and taurocholic acid had the most robust increase in serum levels and dynamic range, with a maximum fold increase from baseline of 34-fold and 29-fold, respectively. Peak bile acid increases occurred within 2hours (h) after dosing and returned to baseline values before 24h. In conclusion, serum bile acid profiling can potentially identify a mechanistic risk of clinical DILI that could be poorly detected by traditional toxicity endpoints.

A proof-of-concept rat toxicity study highlights the potential utility and challenges of virtual control groups.

The virtual control group (VCG) concept provides a potential opportunity to reduce animal use in drug development by replacing concurrent control groups (CCGs) in nonclinical toxicity studies. This work investigated the feasibility and reliability of using VCGs in place of CCGs. A historical control database (HCD), constructed from Genentech Inc. rat toxicity study data, was reviewed to under­stand trends and sources of variability in control animals over time, and to identify data curation requirements for assembling VCGs, e.g., alignment of units of measurement. Several endpoints were investigated and stratified against different study design parameters. Sex, route of administration, fasting status, and body weight at study initiation were among the parameters that were indicated as key matching criteria. With a high-level understanding of potential sources of variability, a retro­spective proof-of-concept (POC) study was designed, evaluating a historical rat pilot toxicity study for test article-related changes. A masked interpretation of the study was conducted using its CCG and two unique VCGs that were constructed from individual animal data pulled from our HCD. While the results of the microscopic pathology assessment and most endpoints were similar across the different control groups, the POC revealed the risk of using VCGs to interpret subtle test article-related changes in clinical pathology parameters. Within the context of our POC, it appears the use of a VCG is not completely equivalent to the CCG, especially with clinical pathology parameters. Additional work is needed to understand the potential utility, and thus, viability of VCGs in other contexts.

This study explored the potential of virtual control groups (VCGs) to reduce the number of living control animals in drug development. The process involves using historical control animal data instead of live control animals in toxicity studies. Several parameters were identified as crucial factors that must be aligned in assembling VCGs. The VCG concept was tested using a historical rat toxicity study by comparing results against the conventional control group as well as two different VCGs. Although results were similar in most cases, interpreting subtle changes in clinical pathology parameters was almost impossible when using the VCGs. Further work is needed to fully optimize and assess the potential of VCGs. The significance of this work lies in the possibility of reducing the number of animals used in testing, in support of the 3Rs (replace, reduce, and refine).

Balancing Blood Sample Volume with 3Rs: Implementation and Best Practices for Small Molecule Toxicokinetic Assessments in Rats.

Improved small molecule bioanalytical sensitivity and concomitant decreased sample volume requirements provide an opportunity to reconsider how toxicokinetic (TK) data are collected in rat toxicity studies. Often, satellite groups of rats are designated to separate procedural effects of TK blood collection from the primary toxicity evaluation. Blood microsampling (i.e., ≤50 µL) decreases the blood volume collected such that TK samples can be collected from toxicity groups without impacting toxicity assessment. Small plasma sampling uses slightly higher blood volumes (i.e., 200 µL) with comparable technical feasibility and, importantly, allows multiple analyses with no negative impact on study interpretation. Our "base case" study designs utilize sparse TK sampling from sample toxicity group rats (1-2 samples/rat). Alternate designs with satellite animals may still be warranted based on study objectives (e.g., biomarkers), intolerability, or smaller rat strains; however, we propose these as exceptions rather than standard practice and with a focus to use the fewest animals possible. We review the state of knowledge in bioanalytical and blood sampling techniques and support the paradigm whereby TK sampling of main study animals significantly decreases the overall number of rats required for toxicity assessments and refines study interpretation with additional data options. These efforts maintain a commitment to the 3Rs (replacement, reduction, and refinement) while maintaining high-quality TK evaluations on toxicity studies.