Nonclinical Evaluation Research Articles

Current Approaches to the Preclinical Assessment of Endocrine Toxicity

Scientific relevance. Endocrine disruptors affect the functioning of endocrine organs, which leads to adverse drug reactions. Endocrine toxicity requires special attention in preclinical studies of candidate medicinal products.Aim. The study aimed to review international guidelines and approaches to assessing the risk of endocrine toxicity associated with medicinal products.Discussion. This review covers documents that provide a methodological framework for identifying and classifying a chemical compound as an endocrine disruptor. These documents include the following: Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption (Organisation for Economic Cooperation and Development, 2018), Nonclinical Evaluation of Endocrine-Related Drug Toxicity (Food and Drug Administration, 2015), and Guidance for the Identification of Endocrine Disruptors in the Context of Regulations (EU) No. 528/2012 and (EC) No. 1107/2009. The proposed algorithm for endocrine toxicity assessment involves collecting all available data on the test compound, such as the literature and previously obtained experimental data, including acute and subchronic toxicity data, and in silico predictions. Particular attention should be paid to the standard battery of preclinical chronic toxicity studies, which can identify most side effects associated with the endocrine system. The main endpoints for endocrine toxicity include changes in the mass and histopathology of the major endocrine organs (adrenal glands, testes, epididymides, ovaries, and the thyroid gland), oestrous cycle effects, reproductive toxicity, and transplacental action. A thorough assessment of the data obtained provides for the determination of unfavourable endocrine activity that requires further studies.Conclusions. The OECD guidelines offer a set of validated in vivo and in vitro tests that characterise the most important mechanisms of endocrine toxicity (oestrogen, androgen, thyroid, and steroidogenic endocrine pathways) by identified toxic effects. This approach allows researchers to identify potential endocrine disorders early in the drug development process and to optimise the scope of the required studies accordingly.

Comparing the sensitivity of cross-over and parallel study designs for QTc assessment: An analysis based on a single large study of moxifloxacin in 48 nonhuman primates

The cardiovascular safety pharmacology (SP) study conducted to satisfy ICH S7A and S7B has commonly used a cross-over study design where each animal receives all treatments. In an increasing number of cases, cross-over designs are not possible and parallel studies have to be used. These can seldom be as large as 8 animals/treatment to match an n = 8 cross-over. Animals in parallel designs receive only one treatment. Parallel studies will have a different sensitivity to detect changes. This sensitivity is a critical question in using nonclinical QTc evaluations to support an integrated proarrhythmic risk assessment under the newly released ICH E14/S7B Q&As. The current analysis used a study large enough (n = 48) to be analyzed both as a parallel and as a cross-over design to directly compare the performance of the two experimental designs coupled to different statistical models, while all other study conduct aspects were the same.A total of 48 nonhuman primates (NHP) received 2 different treatments twice: vehicle, moxifloxacin (80 mg/kg), vehicle, moxifloxacin (80 mg/kg). Post-dose QTc interval data were recorded for 48 h for each treatment. Data were analyzed using 12 animals randomly selected for each treatment in a parallel design or as an n = 48 animal cross-over study. Different statistical models were used. The primary endpoint was the residual deviation (sigma) from the models applied to hourly time intervals. The sigma was used to determine the minimal detectable difference (MDD) for the study design-statistical model combination.Two statistical models were applicable to either study design. They gave similar sigma and resulting MDD values. In cross-over designs, the individual animal identification (ID) can be used in the statistical model. This enabled the smallest MDD value. Simple statistical models for analysis were identified: Treatment + Baseline for parallel designs and Treatment + ID for cross-over designs.The statistical sensitivity of NHP parallel study designs is reasonable (MDD for n = 6 of 12.7 ms), and in combination with testing exposures higher than likely to be necessary in man could be used in an integrated risk assessment. Where sensitivity of the NHP in vivo QTc assessment is critical, the cross-over design enabled a higher sensitivity (MDD 12.2 ms for n = 4; 8 ms for n = 8).

Nonclinical evaluation of a Vitis vinifera extract towards a novel antiaging cosmetic ingredient.

Skin aging is regulated by multiple physiological processes, such as oxidative stress. Natural products have been considered as a promising source of antioxidant compounds. As a result, few innovative products on the market based on natural products tackle additional underlying mechanisms of skin aging. The present work reports the nonclinical evaluation of a novel extract from the skin of V. vinifera fruits (codified as ACH37 extract), with the aim of supporting its use as an antiaging cosmetic ingredient candidate in clinical trials. We employed enzymatic, phenotypic, and gene expression assays, both in vitro and ex vivo, to investigate the action of the ACH37 extract in different biological processes that could be related to skin aging mechanisms. The ACH37 extract was able to scavenge reactive oxygen species (DPPH, O2 - ), prevent inflammation (LPS- and UV-induced COX-2, IL-1β, and IL-8 expression), modulate extracellular matrix remodeling (inhibiting elastase, MMP-1, MMP-3, and MMP-12, as well as associated expression), increase telomere length, telomerase activity, and reverse the UV-induced suppression of genes involved in skin protection. In addition, the ACH37 extract permeated human skin explants and presented antioxidant efficacy ex vivo. The results indicated that the ACH37 extract acts on multiple targets commonly related to skin aging, being a promising antiaging active ingredient candidate to be further investigated in clinical trials.

Pharmaceutical Industry Perspective on the Non-Clinical Evaluation of Novel Excipients: Results From an Industry Survey Conducted by the IQ Novel Excipient Working Group.

Excipients are essential components within drug products that contribute significantly to their overall quality, effectiveness, and safety. There is a lack of global, harmonized guidance relating to the non-clinical testing of novel excipients which is perceived to create uncertainty and strategic risk, potentially hindering innovation and disincentivizing their use. To test these perceptions, the IQ Novel Excipient Working Group surveyed member companies regarding their main concerns and prior experience regarding the non-clinical evaluation of excipients. Of the 19 respondents, 13 provided, collectively, 33 non-clinical program examples supporting the development of novel excipients. Programs were distributed across a range of therapeutic areas and included a variety of drug modalities and administration routes. Package designs were variable, but where possible, employed the use of existing data, supplemented with new toxicology studies as appropriate. Of the programs which had submitted data to regional health authorities, only three received feedback requesting additional studies or that demonstrated differences in regional opinion. In addition, companies provided recommendations on how the current (or new) guidance related to non-clinical excipient evaluation (and other areas, such as Chemistry, Manufacturing, and Controls and databases) may be improved.

Humanized mouse models with endogenously developed human natural killer cells for in vivo immunogenicity testing of HLA class I-edited iPSC-derived cells

Human induced pluripotent stem cells (hiPSCs) genetically depleted of human leucocyte antigen (HLA) class I expression can bypass T cell alloimmunity and thus serve as a one-for-all source for cell therapies. However, these same therapies may elicit rejection by natural killer (NK) cells, since HLA class I molecules serve as inhibitory ligands of NK cells. Here, we focused on testing the capacity of endogenously developed human NK cells in humanized mice (hu-mice) using MTSRG and NSG-SGM3 strains to assay the tolerance of HLA-edited iPSC-derived cells. High NK cell reconstitution was achieved with the engraftment of cord blood-derived human hematopoietic stem cells (hHSCs) followed by the administration of human interleukin-15 (hIL-15) and IL-15 receptor alpha (hIL-15Rα). Such “hu-NK mice” rejected HLA class I-null hiPSC-derived hematopoietic progenitor cells (HPCs), megakaryocytes and T cells, but not HLA-A/B-knockout, HLA-C expressing HPCs. To our knowledge, this study is the first to recapitulate the potent endogenous NK cell response to non-tumor HLA class I-downregulated cells in vivo. Our hu-NK mouse models are suitable for the non-clinical evaluation of HLA-edited cells and will contribute to the development of universal off-the-shelf regenerative medicine.

Abstract 6647: Dostarlimab shows dose-dependent immune activation of the tumor microenvironment in a patient-derived NSCLC explant model similar to pembrolizumab

Abstract The ability to more faithfully mimic the tumor microenvironment (TME) of cancer patients offers a wide range of possibilities for non-clinical evaluation of anti-cancer therapeutics, including guidance for patient selection, dose, and combinations. We have established a patient-derived tumor explant (PDE) model to monitor immune activation within a structurally preserved TME, following ex vivo incubation with immune check point inhibitors (ICI). When testing anti-PD-1 agents, such as dostarlimab and pembrolizumab, in a cohort of NSCLC explants from stage I-III treatment-naïve patients, we observed that approximately half demonstrate release of inflammatory cytokines after two days incubation. Interestingly, responsive tumors presented with a high level of PD-L1 expression (high TPS), had elevated TMB, and were highly infiltrated by CD8+ T cells at baseline, in line with tumor baseline characteristics predictive of clinical response to anti-PD-1. When testing a dose-range of dostarlimab and pembrolizumab comparable potencies were observed when measuring cytokine release after treatment of the NSCLC explants. In conclusion, we describe here an in vitro patient-derived NSCLC model that mirrors the heterogeneity of patients’ response for anti-PD-1 agents observed in clinical settings, representing a valuable tool for comparative analysis of novel ICI assets and combinations, such as the CD226 axis members. Importantly, this system is also amenable to extensive multi-omics and genetics-based analyses, providing the opportunity to better identify predictive or pharmacodynamic characteristics within patient tumors. Citation Format: Jessica Perrin, Frank Wippich, Valeriia Sherina, Dmytro Dvornikov, Katharina Lupar, Murad M. Melhem, Patrick Hanafin, Jeremy Waight, Jong Yu, Sapna Yadavilli, Daniel Poeckel, Giovanna Bergamini. Dostarlimab shows dose-dependent immune activation of the tumor microenvironment in a patient-derived NSCLC explant model similar to pembrolizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6647.

Current regulatory requirements for non-clinical evaluation of prophylactic vaccines

Vaccines are subject to specific regulatory requirements for the evaluation of their quality, safety, and efficacy. In 2005, the World Health Organisation (WHO), as the main international organisation coordinating measures to combat infectious disease outbreaks, began developing documents on the evaluation of vaccine quality, safety, and efficacy. The world’s leading regulatory authorities (FDA, EMA, etc.) have also issued recommendations for conducting non-clinical studies of vaccines.The aim of this study was a critical review of the regulatory requirements established by foreign national and international regulatory authorities for non-clinical evaluation of the safety and efficacy of vaccines.According to the study results, since the 2000s, the WHO and the world’s leading regulatory authorities have produced more than 40 regulatory documents describing certain aspects of non-clinical studies of the safety and efficacy of vaccines. These documents can be divided into two groups: the first group addresses non-clinical studies of vaccines in general, and the second one dwells upon the evaluation of the quality, safety, and efficacy of specific types of vaccines. For the Russian guidelines on non-clinical evaluation of the quality, safety, and efficacy of immunobiologicals, the latest revision dates back to 2013 and does not provide any information on new medicinal products. Currently, work is underway to prepare the regulatory framework for medicines, including vaccines, in the Member States of the Eurasian Economic Union (EAEU). This review of regulatory documents on non-clinical safety and efficacy studies of vaccines may be useful in drafting harmonised guidelines for the relevant groups of vaccines in the EAEU. It may also be of use to developers, manufacturers, and researchers involved in the creation and non-clinical study of vaccines.

Comparison of validity of standard nonclinical group size selection versus standard clinical group sizes for nonhuman primate QTc prolongation evaluation

The number of animals used in a nonhuman primate (NHP) in vivo QTc assessment conducted as part of the safety pharmacology (SP) studies on a potential new drug is relatively small (4–8 subjects). The number is much smaller than the number of healthy volunteers in a conventional thorough QT (TQT) study (40–60 volunteers). How is it possible that such small studies could offer an equivalent sensitivity in an integrated nonclinical and clinical cardiac repolarization risk assessment?This study provided the opportunity to empirically demonstrate in a large number of NHPs the performance of a nonclinical evaluation at a similar size to a TQT study.By contrasting an analysis mimicking the sampling and aggregation of QTc interval data in a manner which is TQT-like with a more conventional SP-like analysis it was demonstrated that the SP-like analysis was more sensitive. In prospective power calculations 80% power at p = 0.05 can be achieved for a 5 ms QTc change with only n = 8 NHPs using the SP-like analysis and in a group of only 4 NHPs 80% power to detect 10 ms could be achieved. By contrast groups of 24 NHPs would be required to achieve 80% power to detect 5 ms using the TQT-like sampling and aggregation approach.Overall, this study has demonstrated that smaller safety pharmacology in vivo QTc assessments using all the available data in larger data aggregates can achieve sensitivity comparable to a human TQT study.

Establishment of multicenter COVID-19 therapeutics preclinical test system in Republic of Korea

Throughout the recent COVID-19 pandemic, South Korea led national efforts to develop vaccines and therapeutics for SARS-CoV-2. The project proceeded as follows: 1) evaluation system setup (including Animal Biosafety Level 3 (ABSL3) facility alliance, standardized nonclinical evaluation protocol, and laboratory information management system), 2) application (including committee review and selection), and 3) evaluation (including expert judgment and reporting). After receiving 101 applications, the selection committee reviewed pharmacokinetics, toxicity, and efficacy data and selected 32 final candidates. In the nonclinical efficacy test, we used golden Syrian hamsters and human angiotensin-converting enzyme 2 transgenic mice under a cytokeratin 18 promoter to evaluate mortality, clinical signs, body weight, viral titer, neutralizing antibody presence, and histopathology. These data indicated eight new drugs and one repositioned drug having significant efficacy for COVID-19. Three vaccine and four antiviral drugs exerted significant protective activities against SARS-CoV-2 pathogenesis. Additionally, two anti-inflammatory drugs showed therapeutic effects on lung lesions and weight loss through their mechanism of action but did not affect viral replication. Along with systematic verification of COVID-19 animal models through large-scale studies, our findings suggest that ABSL3 multicenter alliance and nonclinical evaluation protocol standardization can promote reliable efficacy testing against COVID-19, thus expediting medical product development.

The Regulation of Cell Therapy and Gene Therapy Products in Switzerland.

This chapter describes the regulation of cell and gene therapy products (CGTPs) in Switzerland and its legal basis. The Swiss Agency for Therapeutic Products, Swissmedic, is the lead Regulatory Authority and its ATMP Division is responsible for the regulation of these products at the level of clinical trials and marketing authorization. CGTPs are regulated similarly to medicinal products. The legal basis is set by the Therapeutic Product Act, the Transplantation Act, the Human Research Act, and associated ordinances. The ATMP Division is involved in processes such as scientific advice meetings, presubmission advice meetings, pharmacovigilance, market surveillance, import/export approvals, manufacturing license approval, and inspections. In Switzerland, guidance documents relevant for cell and gene therapy provided by PIC/S, OECD, ICH, Ph.Eur., EMA, or FDA are considered. In order to harmonize requirements for CGTPs, the ATMP Division is in constant exchange of information with foreign Regulatory Authorities and part of working groups of ICH, IPRP, and Ph.Eur. As CGTPs are biologically and technically complex, a risk-based approach is applied on a case-by-case basis for the evaluation of clinical trial and marketing applications. A substantial part of this chapter will provide requirements with respect to the manufacturing and quality, nonclinical and clinical evaluation of CGTPs. Furthermore, information will be provided regarding the use of real-world evidence in evaluation of clinical long-term efficacy and safety in case of rare diseases where the numbers of patients are too small for statistically meaningful analysis during clinical trials. Finally, the chapter will provide information on a health technology assessment (HTA) program that was launched in 2015 in Switzerland by the federal authorities.

Nonclinical Testing Evaluation of Liposomes as Drug Delivery Systems.

Various marketed drugs, as well as many in-development, have utilized liposomes, vesicles composed of one or more phospholipid bilayers, as a drug delivery system, often with the statement that they are "non-toxic" materials. This paper examined safety testing considerations and reviewed nonclinical packages used to support the safe clinical use and marketing of drugs using a liposomal drug delivery system, including liposome-only study findings. It was found that most experience has come from use of an established drug (especially in the oncology field) in a liposome formulation with known excipients. From this knowledge, it is proposed that the minimal package of studies (using an oncology indication as an example) needed to support clinical entry should include in vivo pharmacology in selected mouse xenograft models, pharmacokinetic characterization showing enhanced kinetics or disposition and including tumor exposure evaluation along with repeat-dose toxicity testing in one species. It was also found that the liposomes used in drug delivery systems are not truly "non-toxic" materials. However, the majority of findings in toxicity testing relate to macrophage processing of large amounts of lipid material, with no human known safety consequence. Of note, however, are cases of hypersensitivity for some PEGylated liposome forms which translate to the clinic.

Advances in Nanotechnology for Cancer Immunoprevention and Immunotherapy: A Review.

One of the most effective cancer therapies, cancer immunotherapy has produced outstanding outcomes in the field of cancer treatment. However, the cost is excessive, which limits its applicability. A smart way to address this issue would be to apply the knowledge gained through immunotherapy to develop strategies for the immunoprevention of cancer. The use of cancer vaccines is one of the most popular methods of immunoprevention. This paper reviews the technologies and processes that support the advantages of cancer immunoprevention over traditional cancer immunotherapies. Nanoparticle drug delivery systems and nanoparticle-based nano-vaccines have been employed in the past for cancer immunotherapy. This paper outlines numerous immunoprevention strategies and how nanotechnology can be applied in immunoprevention. To comprehend the non-clinical and clinical evaluation of these cancer vaccines through clinical studies is essential for acceptance of the vaccines.

Cross clinical-experimental-computational qualification of in silico drug trials on human cardiac purkinje cells for proarrhythmia risk prediction.

The preclinical identification of drug-induced cardiotoxicity and its translation into human risk are still major challenges in pharmaceutical drug discovery. The ICH S7B Guideline and Q&A on Clinical and Nonclinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential promotes human in silico drug trials as a novel tool for proarrhythmia risk assessment. To facilitate the use of in silico data in regulatory submissions, explanatory control compounds should be tested and documented to demonstrate consistency between predictions and the historic validation data. This study aims to quantify drug-induced electrophysiological effects on in silico cardiac human Purkinje cells, to compare them with existing in vitro rabbit data, and to assess their accuracy for clinical pro-arrhythmic risk predictions. The effects of 14 reference compounds were quantified in simulations with a population of in silico human cardiac Purkinje models. For each drug dose, five electrophysiological biomarkers were quantified at three pacing frequencies, and results compared with available in vitro experiments and clinical proarrhythmia reports. Three key results were obtained: 1) In silico, repolarization abnormalities in human Purkinje simulations predicted drug-induced arrhythmia for all risky compounds, showing higher predicted accuracy than rabbit experiments; 2) Drug-induced electrophysiological changes observed in human-based simulations showed a high degree of consistency with in vitro rabbit recordings at all pacing frequencies, and depolarization velocity and action potential duration were the most consistent biomarkers; 3) discrepancies observed for dofetilide, sotalol and terfenadine are mainly caused by species differences between humans and rabbit. Taken together, this study demonstrates higher accuracy of in silico methods compared to in vitro animal models for pro-arrhythmic risk prediction, as well as a high degree of consistency with in vitro experiments commonly used in safety pharmacology, supporting the potential for industrial and regulatory adoption of in silico trials for proarrhythmia prediction.

Production and nonclinical evaluation of an autologous iPSC–derived platelet product for the iPLAT1 clinical trial

AbstractDonor-derived platelets are used to treat or prevent hemorrhage in patients with thrombocytopenia. However, ∼5% or more of these patients are complicated with alloimmune platelet transfusion refractoriness (allo-PTR) due to alloantibodies against HLA-I or human platelet antigens (HPA). In these cases, platelets from compatible donors are necessary, but it is difficult to find such donors for patients with rare HLA-I or HPA. To produce platelet products for patients with aplastic anemia with allo-PTR due to rare HPA-1 mismatch in Japan, we developed an ex vivo good manufacturing process (GMP)–based production system for an induced pluripotent stem cell–derived platelet product (iPSC-PLTs). Immortalized megakaryocyte progenitor cell lines (imMKCLs) were established from patient iPSCs, and a competent imMKCL clone was selected for the master cell bank (MCB) and confirmed for safety, including negativity of pathogens. From this MCB, iPSC-PLTs were produced using turbulent flow bioreactors and new drugs. In extensive nonclinical studies, iPSC-PLTs were confirmed for quality, safety, and efficacy, including hemostasis in a rabbit model. This report presents a complete system for the GMP-based production of iPSC-PLTs and the required nonclinical studies and thus supports the iPLAT1 study, the first-in-human clinical trial of iPSC-PLTs in a patient with allo-PTR and no compatible donor using the autologous product. It also serves as a comprehensive reference for the development of widely applicable allogeneic iPSC-PLTs and other cell products that use iPSC-derived progenitor cells as MCB.

Towards a European harmonization of health technology assessment recommendations executive paper of European regulatory conference focused on the EU commission proposal to harmonize HTA

Health Technology Assessment (HTA) is a multidisciplinary process which supports policy “decision-making”, including those related to drug reimbursement and price negotiation based on comprehensive clinical and non-clinical evaluations. The discrepancies in HTA recommendations among European countries are related to the differences in health care systems and willingness to invest in health care, differences in assessment methodologies and appraisal practices, and variations in economic constraints, and may produce disparities in terms of patient access to medicines. The political discussion about the adoption of the European Regulation on HTA lasted for years due to concerns about its feasibility and its implications for national health care systems In this paper we highlighted the importance to harmonize HTA process in Europe and some critical issues emerged during the discussion among experts in the field carried out at the European Regulatory Conference.

Exploratory toxicology studies of 2,3-substituted imidazo[1,2-a]pyridines with antiparasitic and anti-inflammatory properties.

Trichomoniasis and amoebiasis are neglected diseases and still remain as a global health burden not only for developing countries, from where are endemic, but also for the developed world. Previously, we tested the antiparasitic activity of a number of imidazo[1,2-a]pyridine derivatives (IMPYs) on metronidazole-resistant strains of Entamoeba Hystolitica (HM1:IMSS), and Trichomonas Vaginalis (GT3). Their anti-inflammatory activity was also evaluated. The present work is a part of a project whose aim is to find new alternatives to standard treatments for these maladies, and to address the current concern of emerging resistant parasite strains. Here we report a non-clinical study focused on exploratory toxicology assays of seven IMPYs that showed the best antiparasitic and/or anti-inflammatory properties. Acute, and subacute toxicity tests were carried out. After 14-day oral treatment, liver and kidney functionality assays in combination with chemometric methods were implemented to detect hepatic and/or kidney damage. Some compounds produced off-target effects. Vehicle effects were also detected. However, no signs of hepatic or renal toxicity were observed for any IMPY. These compounds can continue non-clinical evaluations, and if possible, clinical trials as new candidates to treat trichomoniasis and amoebiasis, and inflammatory diseases. Further studies are also needed to fully elucidate a proposed dual effect that may exert these molecules against trichomoniasis and amoebiasis, which may also signify a novel mechanism of action to treat these infections.

Fluoxetine for non-arteritic anterior ischemic optic neuropathy: A double-blind, randomized clinical trial

Introduction: Fluoxetine raises the levels of BDNF (brain-derived neurotrophic factor). BDNF is known to improve neurogenesis and plasticity, so it seems to improve Anterior Ischemic Optic Neuropathy (AION). The goal of this study was to find out how Fluoxetine affects the clinical outlook of people with AION. Methods: In this double-blind, placebo-controlled, randomized clinical trial, patients with AION split into two groups: the fluoxetine group (n=50), which took 20 mg of Fluoxetine every day, and the control group (n=50), which took a placebo pill instead. Both groups were followed for six months. Before and after the trial, patients were given clinical and non-clinical evaluations. Results: 100 people took part in this study and were evaluated. Subjects in the Fluoxetine group had better visual acuity than those in the placebo group. They had lower scores on the LogMAR scale (P: 0.008 and 0.002), better MD parameters of perimetry (P: 0.003 and 0.002), and shorter VEP latencies (P (in 1st minute): 0.001 and 0.001, P (in 15th minute): 0.038 and 0.011. After the trial of Fluoxetine therapy, there were no changes in color vision, Rnfl in all dimensions, PSD parameter of perimetry, or VEP amplitudes (Ps> 0.05. Conclusion: Fluoxetine showed promise as a therapy for people with AION, and it was safe to use as a treatment option in addition to corticosteroids

Lymphocyte subsets and immunoglobulin levels in peripheral blood from cynomolgus monkeys (Macaca fascicularis) of different age groups.

Immunotoxicity assessment is an important part of non-clinical safety evaluation of biotechnology-derived pharmaceuticals. The reference ranges of evaluated parameters, which depend on the sex, age and geographical origin of animals, play a significant role in interpreting the study results. The aim of this study was to determine the reference ranges of parameters commonly used for non-clinical immunotoxicity studies in cynomolgus monkeys (Macaca fascicularis) of different ages. The percentages of CD3+, CD4+, CD8+, CD20+, CD16/56+ lymphocytes, and the serum levels of immunoglobulins A, M, G and E in clinically healthy cynomolgus monkeys of both sexes and different ages (37-104 months) were analysed. The lymphocyte subsets were assessed by flow cytometry and serum immunoglobulin levels were determined by enzyme-linked immunosorbent assay. Statistical analysis showed a weak rank correlation of CD3+, CD20+ lymphocyte levels with the ages of female and male cynomolgus monkeys, and no significant correlation with age for the combined data of both sexes. The serum levels of immunoglobulins A, M, G and E did not have a significant rank correlation with age for the combined data of both sexes, whereas IgA levels in females and IgG levels in males were weakly correlated with age. Overall, these results justify the use of animals aged three to eight years for non-clinical immunotoxicity evaluation of biotechnology-derived pharmaceuticals. It is important to avoid the formation of animal groups with extreme ages (three years or eight years) in one group during randomization.

Abstract 1260: Nonclinical evaluation of rucaparib in tumors with mutations in non-BRCA1/2 homologous recombination repair (HRR) genes

Abstract Background: Rucaparib is a PARP inhibitor approved to treat ovarian cancer (OC) and metastatic castration-resistant prostate cancer (mCRPC). Deficiencies in HRR resulting from genetic or epigenetic alterations in BRCA1/2 sensitize tumors to rucaparib through synthetic lethality. Inactivation of other HRR genes may also confer sensitivity to PARP inhibition. Here, we evaluated rucaparib efficacy in preclinical models with genomic or epigenetic alterations in an HRR gene panel. Methods: BRCA1/2 and 14 additional genes were selected for their role in DNA repair and mutation frequency in OC and mCRPC. Small interfering RNA (siRNA) knockdowns were performed in OC (OVCAR-3, OAW28, SK-OV-3) and mCRPC (DU145, PC-3) cell lines to model their inactivation and assess impact on rucaparib sensitivity (IC50) in cell viability assays. Patient-derived xenografts (PDX) harboring deleterious alterations in BRCA1/2, BARD1, BRIP1, NBN, PALB2, RAD51B, RAD51C, or RAD51D, or BRCA1/RAD51C promoter hypermethylation were treated with up to 150 mg/kg rucaparib BID for 25-42 days and tumor growth was monitored. Mutational status was confirmed by next-generation sequencing. Results: In addition to BRCA1/2 knockdown, depletion of BARD1, PALB2, or RAD51 resulted in potent rucaparib activity in OC and mCRPC cells, where the rucaparib IC50 was reduced by >70% vs control in at least 4 of the 5 cell lines tested. Rucaparib also displayed greater potency in cells with siRNA-mediated decreased expression of FANCA, NBN, RAD51C, or RAD54L, where at least 1 cell line had >50% decrease in IC50. Rucaparib treatment in 19/33 HRR-deficient PDX models resulted in significant tumor growth inhibition (TGI). These included BRCA1/2, PALB2, NBN, RAD51B, RAD51C, or RAD51D-mutated and BRCA1/RAD51C-hypermethylated models. Efficacy was strongly associated with biallelic inactivation of BRCA1/2, PALB2, RAD51C, or RAD51D with 87% vs 29% mean TGI for monoallelic BRCA1/2 alterations (P<0.0001). In addition, a single PDX model each for the exploratory genes NBN and RAD51B had 101% and 80% TGI, respectively. No significant difference was observed between PDX models with BRCA1/2 vs non-BRCA1/2 (PALB2, NBN, RAD51B, RAD51C, or RAD51D) biallelic alterations (98% vs 80% mean TGI, respectively; P=0.21). In support of these nonclinical findings, we highlight a patient with RAD51B-mutant mCRPC treated with rucaparib in the TRITON2 trial (NCT02952534) who had confirmed radiographic and PSA responses. Conclusions: Cell lines and PDX models with biallelic alterations in HRR genes other than BRCA1/2, including RAD51B, RAD51C, RAD51D, PALB2, and NBN, are comparably sensitive to rucaparib. Rucaparib is being evaluated in the LODESTAR trial (NCT04171700) in patients with tumors associated with deleterious alterations in BRCA1/2, PALB2, RAD51C, and RAD51D (cohort A) and BARD1, BRIP1, FANCA, NBN, RAD51, and RAD51B (exploratory cohort B). Citation Format: Liliane Robillard, Kevin K. Lin, Andrea Loehr, Tanya Kwan, Rachel Dusek, Andrew D. Simmons, Thomas C. Harding, Brieuc Sautois, Minh Nguyen. Nonclinical evaluation of rucaparib in tumors with mutations in non-BRCA1/2 homologous recombination repair (HRR) genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1260.

Scientific and Regulatory Policy Committee Points to Consider: Primary Digital Histopathology Evaluation and Peer Review for Good Laboratory Practice (GLP) Nonclinical Toxicology Studies.

The Society of Toxicologic Pathology's Scientific and Regulatory Policy Committee formed a working group to consider the present and future use of digital pathology in toxicologic pathology in general and specifically its use in primary evaluation and peer review in Good Laboratory Practice (GLP) environments. Digital histopathology systems can save costs by reducing travel, enhancing organizational flexibility, decreasing slide handling, improving collaboration, increasing access to historical images, and improving quality and efficiency through integration with laboratory information management systems. However, the resources to implement and operate a digital pathology system can be significant. Given the magnitude and risks involved in the decision to adopt digital histopathology, this working group used pertinent previously published survey results and its members' expertise to create a Points-to-Consider article to assist organizations with building and implementing digital pathology workflows. With the aim of providing a comprehensive perspective, the current publication summarizes aspects of digital whole-slide imaging relevant to nonclinical histopathology evaluations, and then presents points to consider applicable to both primary digital histopathology evaluation and digital peer review in GLP toxicology studies. The Supplemental Appendices provide additional tabulated resources.