Reduced Fasting Duration in Cynomolgus Monkeys Enhances Animal Welfare During Toxicology Studies.

During toxicology studies, fasting animals prior to clinical pathology blood collection is believed to reduce variability in some clinical chemistry analytes. However, fasting adds stress to animals that are already stressed from the administration of potentially toxic doses of the test article. The purpose of this study was to assess the impacts of different fasting durations on cynomolgus monkeys' welfare during toxicology studies. To this end, we assessed the cynomolgus monkeys traditional and ancillary clinical pathology endpoints at different fasting times. We showed that most clinical pathology endpoints were largely comparable between different fasting times suggesting that cynomolgus monkeys could be fasted for as little as 4 hours for toxicology studies, as longer fasting times (up to 20 hours) resulted in stress, dehydration, and significant decreases in blood glucose- changes that impacts animal welfare. Shorter fasting times were associated with higher triglycerides variability among individual animals. Therefore, we propose that shorter fasting time (i.e., 4 hours) should be adequate for most toxicology studies except when: (1) parameters that could be affected by non-fasting conditions are important for safety and pharmacodynamic assessments (i.e., glucose and lipids) and (2) fasting would be needed for the bioavailability of an orally administered test article.

Nonclinical toxicology development of a novel antibody antibiotic conjugate for treating invasive Staphylococcus Aureus infections.

Invasive Staphylococcus aureus (S. aureus) infections are a leading cause of death and not effectively treated with prolonged standard of care antibiotics. A novel THIOMAB™ antibody antibiotic conjugate (TAC) was developed that uses a bacterial-wall specific antibody to deliver the antibiotic (dmDNA31, a rifamycin analogue) to bacteria to minimize toxicities typically seen with prolonged use of traditional antibiotics. The TAC nonclinical toxicology package included repeat dose rat and cynomolgus monkey toxicology studies for 8 weekly intravenous (IV) doses, a 7-day daily repeat dose IV toxicology study of dmDNA31 and an assessment of genotoxicity, cardiovascular toxicity, neurotoxicity and sperm parameters. TAC and dmDNA31 were well tolerated in rats and monkeys, and there was no evidence of genotoxicity, cardiovascular toxicity or neurotoxicity. Non-adverse findings were observed and included blue discoloration in skin, blood, etc. due to the blue color of dmDNA31, increased globulin due to the high doses of antibodies, and abnormal sperm morphology of small heads in male rats with no histopathology correlate in testis. This is an example of antibody-mediated delivery of an antibiotic that has the potential to offer a more effective way of eradicating infection while providing a better safety profile compared to traditional antibiotics.

Bruton's Tyrosine Kinase Small Molecule Inhibitors Induce a Distinct Pancreatic Toxicity in Rats.

Bruton's tyrosine kinase (BTK) is a member of the Tec family of cytoplasmic tyrosine kinases involved in B-cell and myeloid cell signaling. Small molecule inhibitors of BTK are being investigated for treatment of several hematologic cancers and autoimmune diseases. GDC-0853 ((S)-2-(3'-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4'-bipyridin]-2'-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one) is a selective and reversible oral small-molecule BTK inhibitor in development for the treatment of rheumatoid arthritis and systemic lupus erythematosus. In Sprague-Dawley (SD) rats, administration of GDC-0853 and other structurally diverse BTK inhibitors for 7 days or longer caused pancreatic lesions consisting of multifocal islet-centered hemorrhage, inflammation, fibrosis, and pigment-laden macrophages with adjacent lobular exocrine acinar cell atrophy, degeneration, and inflammation. Similar findings were not observed in mice or dogs at much higher exposures. Hemorrhage in the peri-islet vasculature emerged between four and seven daily doses of GDC-0853 and was histologically similar to spontaneously occurring changes in aging SD rats. This suggests that GDC-0853 could exacerbate a background finding in younger animals. Glucose homeostasis was dysregulated following a glucose challenge; however, this occurred only after 28 days of administration and was not directly associated with onset or severity of pancreatic lesions. There were no changes in other common serum biomarkers assessing endocrine and exocrine pancreatic function. Additionally, these lesions were not readily detectable via Doppler ultrasound, computed tomography, or magnetic resonance imaging. Our results indicate that pancreatic lesions in rats are likely a class effect of BTK inhibitors, which may exacerbate an islet-centered pathology that is unlikely to be relevant to humans.

Nonclinical Pharmacokinetics, Pharmacodynamics, and Translational Model of RO7297089, A Novel Anti-BCMA/CD16A Bispecific Tetravalent Antibody for the Treatment of Multiple Myeloma.

RO7297089, an anti-B-cell maturation antigen (BCMA)/CD16A bispecific tetravalent antibody, is being developed as a multiple myeloma (MM) therapeutic. This study characterized nonclinical pharmacokinetics (PK), pharmacodynamics (PD), soluble BCMA (sBCMA), and soluble CD16 (sCD16) changes following administration of RO7297089 to support clinical trials. Unbound and total RO7297089 concentrations were measured in cynomolgus monkeys. RO7297089 exhibited a bi-phasic systemic concentration-time profile, similar to a typical human immunoglobulin 1 antibody. Target engagement by RO7297089 led to a robust increase (~100-fold) in total systemic sBCMA levels and relatively mild increase (~2-fold) in total sCD16 levels. To describe the relationship of nonclinical PK/PD data, we developed a target-mediated drug disposition (TMDD) model that includes the systemic target engagement of membrane BCMA (mBCMA), sBCMA, membrane CD16 (mCD16), and sCD16. We then used this model to simulate the PK/PD relationship of RO7297089 in MM patients by translating relevant PK parameters and target levels, based on the literature and newly generated data such as baseline sCD16A levels. Our model suggested that the impact of TMDD on RO7297089 exposure may be more significant in MM patients due to significantly higher expression levels of both mBCMA and sBCMA compared to healthy cynomolgus monkeys. Based on model simulations, we propose more frequent dosing of RO7297089 compared to regular monthly frequency in the clinic at the beginning of treatment to ensure sustained target engagement. This study demonstrates a translational research strategy for collecting relevant nonclinical data, establishing a TMDD model, and using simulations from this model to inform clinical dose regimens.

A BCMA/CD16A bispecific innate cell engager for the treatment of multiple myeloma.

Despite the recent progress, multiple myeloma (MM) is still essentially incurable and there is a need for additional effective treatments with good tolerability. RO7297089 is a novel bispecific BCMA/CD16A-directed innate cell engager (ICE®) designed to induce BCMA+ MM cell lysis through high affinity binding of CD16A and retargeting of NK cell cytotoxicity and macrophage phagocytosis. Unlike conventional antibodies approved in MM, RO7297089 selectively targets CD16A with no binding of other Fcγ receptors, including CD16B on neutrophils, and irrespective of 158V/F polymorphism, and its activity is less affected by competing IgG suggesting activity in the presence of M-protein. Structural analysis revealed this is due to selective interaction with a single residue (Y140) uniquely present in CD16A opposite the Fc binding site. RO7297089 induced tumor cell killing more potently than conventional antibodies (wild-type and Fc-enhanced) and induced lysis of BCMA+ cells at very low effector-to-target ratios. Preclinical toxicology data suggested a favorable safety profile as in vitro cytokine release was minimal and no RO7297089-related mortalities or adverse events were observed in cynomolgus monkeys. These data suggest good tolerability and the potential of RO7297089 to be a novel effective treatment of MM patients.

Nonclinical safety assessment of a human interleukin-22FC IG fusion protein demonstrates in vitro to in vivo and cross-species translatability.

Although Interleukin-22 (IL-22) is produced by various leukocytes, it preferentially targets cells with epithelial origins. IL-22 exerts essential roles in modulating various tissue epithelial functions, such as innate host defense against extracellular pathogens, barrier integrity, regeneration, and wound healing. Therefore, IL-22 is thought to have therapeutic potential in treating diseases associated with infection, tissue injury or chronic tissue damage. A number of in vitro and in vivo nonclinical studies were conducted to characterize the pharmacological activity and safety parameters of UTTR1147A, an IL-22 recombinant fusion protein that links the human cytokine IL-22 with the Fc portion of a human immunoglobulin. To assess the pharmacological activity of UTTR1147A, STAT3 activation was evaluated in primary hepatocytes isolated from human, cynomolgus monkey, minipig, rat, and mouse after incubation with UTTR1147A. UTTR1147A activated STAT3 in all species evaluated, demonstrating that all were appropriate nonclinical species for toxicology studies. The nonclinical safety profile of UTTR1147A was evaluated in rats, minipigs, and cynomolgus monkeys to establish a safe clinical starting dose for humans in Phase I trials and to support clinical intravenous, subcutaneous and/or topical administration treatment regimen. Results demonstrate the cross-species translatability of the biological response in activating the IL-22 pathway as well as the translatability of findings from in vitro to in vivo systems. UTTR1147A was well tolerated in all species tested and induced the expected pharmacologic effects of epidermal hyperplasia and a transient increase in on-target acute phase proteins. These effects were all considered to be clinically predictable, manageable, monitorable, and reversible.

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.