Abstract
44th Annual Meeting
Poster Abstracts
Rosen Shingle Creek, Orlando, Florida
The abstracts published here were reviewed and approved by the ACT Program Committee. Every effort has been made to reproduce the abstracts as submitted. The author(s) of the abstracts are solely responsible for the data and content provided. The American College of Toxicology does not claim to validate, promote, or share in the representation of the content provided by the submitting author.
100s - General Toxicology
102 - North American Travel Grant Recipient
The Protective Effect of the Antioxidant N-Acetylcysteine on Folic Acid Induced Kidney Fibrosis
Ramji Kandel 1, Narayan Acharya1, Kamaleshwar P. Singh1, and Irfan Warraich2
1Texas Tech University, Lubbock, TX, USA
2Texas Tech University Health Science Center, Lubbock, TX USA
Kidney fibrosis is a progressive disease that can lead to kidney failure due to repetitive injury and inappropriate repair mechanisms. This study aimed to investigate the inhibitory effects of the antioxidant N-acetylcysteine (NAC) on kidney fibrosis in male C57/BL6 mice with a folic acid-induced kidney injury and fibrosis model. Mice were divided into three groups: control, folic acid only, and folic acid with NAC injections, and they received IP injections of their respective treatments. The body weight of the mice was recorded every seven days after folic acid injection. Serum and kidney samples were collected on Day 3 and Day 20. Histopathological and gene expression analyses were performed on the kidney tissue samples. The results demonstrated that the folic acid group had decreased body weight. However, co-treatment with NAC and folic acid resulted in a slight reduction in body weight and less damage to kidney tubular epithelial cells than folic acid alone, as observed in H&E staining. Furthermore, as observed in Masson trichrome staining, the NAC co-treated group showed markedly reduced extracellular matrix (ECM) deposition compared to the folic acid group. Gene expression analysis revealed statistically significant downregulation of antioxidant genes in the folic acid group, suggesting that oxidative stress plays a role in the progression of kidney fibrosis. The statistical significance level was found to be a p-value less than 0.05. Overall, the antioxidant NAC may protect kidney function and structure and could be a potential therapeutic option for kidney fibrosis.
104 - North American Travel Grant Recipient
Sex Differences in the Aspergillus Versicolor-Induced Lung-Brain Axis Transcriptome in C57BL/6J Mice
Mellessa M. Miller 1, Gus Dunbar, George D. Chittum1, Kelly L. Weaver1, Catherine B. Blackwood2, Angela R. Lemons2, Tara L. Croston2, Donald H. Beezhold3, Brett J. Green3, and Michelle L. Block1
1Department of Pharmacology and Toxicology, The Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA
2Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV, USA
3Office of the Director, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV, USA
Aspergillus versicolor (Av) is a common filamentous fungi found in damp indoor environments, where human exposure has been linked to cognitive complaints, allergy, and asthma. Recent studies have associated allergic disease with an increased risk for Alzheimer’s disease. According to the lung-brain-axis hypothesis, the pulmonary response to environmental exposures dysregulates the brain’s resident immune cells, microglia, through changes in peripheral immune cells and circulating factors, potentially leading to CNS pathology and disease. It is unknown if sex influences this progression. Male and female C57BL/6J mice were exposed to HEPA-filtered air or Av (3 × 105 spores) in nose-only chambers twice weekly for 10-weeks to explore sex differences in the midbrain, hippocampus, lung-immune and spleen-immune transcriptome; along with microglial morphology and serum cytokines. Significant sex differences were observed in the hippocampal transcriptomic fingerprint with filtered air groups, but Av exposure failed to affect gene expression in hippocampus for either sex. However, 286 genes were significantly and uniquely modified with Av in the female midbrain, indicating females’ robust, brain region specific Av responses. Females showed a greater pro-inflammatory transcriptional response to Av in lung tissue. However, Av-exposed males demonstrated greater spleen-immune transcriptome response, with significant increase in circulating serum cytokines IL-5, IP-10, IL-17 in the periphery, as well as more cortical hypertrophic microglia in the CNS. These findings support sex differences in C57BL/6J mice, Av-females exhibited significant lung-immune and midbrain responses, while Av-males demonstrated more spleen-immune and serum cytokine responses suggesting sex could influence Av exposures.
105 - International Travel Grant Recipient
Evaluation of Responses Following Exposure to Respiratory and Skin Sensitizers of an In Vitro Coculture Alveolar Model for the Prediction of Respiratory Sensitizers
Sabina Burla 1, Aline Chary2, Tommaso Serchi2, and Arno C. Gutleb2
1Invitrolize, Belvaux, Diekirch, Luxembourg
2Luxembourg Institute of Science and Technology, Belvaux, Luxembourg
An increasing trend in the development of allergies has been observed globally following exposure to chemicals. While an adverse outcome pathway describes the skin sensitization process, the immunological mechanisms underlying the development of respiratory sensitization are not fully understood. Since the in vitro models for the identification of skin sensitizers fail to differentiate skin from respiratory sensitizers, physiologically relevant test systems for the respiratory tract are required for the identification of chemical respiratory sensitizers. The aim of the present study was to evaluate the readiness of a 3D alveolar in vitro model designed for the identification of respiratory sensitizers to correctly identify respiratory sensitizers and discriminate from skin sensitizers. The model is built using human cell lines: alveolar type II epithelial (A549), endothelial (EA.hy926) and monocytes (THP-1). Respiratory-, skin- and non-sensitizing chemicals were selected, and the 3D in vitro model was exposed to the test items at the air-liquid interface (ALI). Cell viability was measured in the resazurin assay 24h post-exposure and the dose-response curves were modeled. The test system was exposed for 24h to a test item dose decreasing the cell viability of the test system by 25%, THP-1 cells were collected and expression of CD54, CD86, and TSLPr markers on the cell surface was measured by flow cytometry. Results show that the test system has the potential to distinguish respiratory- from skin- and non-sensitizers, and the TSLPr cell surface marker was confirmed as a promising marker for the prediction of respiratory sensitization potential.
106 - North American Travel Grant Recipient
Integrated Multi-Omics Revealed Deletion of Asparagine Synthetase Blocks Major Colorectal Cancer Pathways in Female CRC
Oladimeji Aladelokun 1, Caroline Johnson1, and Sajid Khan1
1Yale University, New Haven, CT, USA
The impact of sex on colorectal cancer tumor metabolism is not well understood. Female colorectal cancer (CRC) patients are characterized by increased expression of asparagine synthetase (ASNS) the gene responsible for de novo asparagine synthesis, and an elevated asparagine production that strongly correlates with inferior survival. Mutant KRAS-driven ASNS expression effects cellular processes such as dysregulated amino acid metabolism, nucleotide biosynthesis and G-protein coupled estrogen receptor (GPER) signaling. In this study, we used a combination of transcriptomics, metabolomics, and immunohistochemistry to determine the effects of ASNS knock out (KO) on the tissue metabolome. Using a CRISPR-Cas9 knockout system, ASNS-null cells showed a reduced growth rate in the absence of media-supplemented asparagine. Molecular analysis of female tumors showed that ASNS deletion caused a reduction in mitosis, cell proliferation, and activates redox signaling pathways that favorably protects female animals against tumor burden. Integrated pathway analysis revealed a disruption of amino acid uptake via solute carrier family 1 member 3 (SLC1A3) and glutamine metabolism via ASNS and glutaminase (GLS1) indicating a decreased ability to transport metabolites needed for asparagine synthesis and cell growth in the female ASNS KO mice. Kaplan Meier survival analysis shows that median survival for female ASNS KO mice was significantly extended by 11 days compared to the male animals. Together, we identified that lack of ASNS suppresses tumor progression in a pre-clinical CRC model and improved survival in female mice only. These observations are important for investigating sex-differences in CRC prevention and identification of novel targets for CRC therapy.
107 - North American Graduate Fellowship Recipient
Perfluoroalkyl Substances (PFAS) Induce Platinum Resistance in Ovarian Cancer by Increasing Mitochondrial DNA Copy Number and Altering Superoxide Production
Brittany P. Rickard 1, Marta Overchuk2, Vesna A. Chappell3, Suzanne E. Fenton3, and Imran Rizvi2
1University of North Carolina at Chapel Hill, Fuquay Varina, NC, USA
2UNC, Chapel Hill, NC, USA
3NIEHS, Research Triangle Park, NC USA
Perfluoroalkyl substances (PFAS) are widespread, persistent environmental contaminants that frequently pollute drinking water supplies worldwide. While these chemicals have been linked to a plethora of adverse outcomes, studies have shown that PFAS exposure can increase risk of infertility and ovarian cancer. Ovarian cancer is the most lethal gynecologic malignancy with a ∼65% mortality rate. One contributing factor to its high lethality is resistance to platinum-based chemotherapy, the standard of care for ovarian cancer treatment. Importantly, our recently published study showed, for the first time, that select PFAS induce platinum resistance in two ovarian cancer cell lines. Since platinum-resistant ovarian tumor populations often display increased mitochondrial networks and enhanced bioenergetic capacities, we hypothesized that PFAS induce alterations to mitochondrial biology. To determine the effect of PFAS on mitochondrial health, we evaluated changes in mitochondrial DNA (mtDNA) copy number and superoxide production using flow cytometry. Cells were exposed to PFAS for 48 hours prior to measuring mtDNA copy number using RT-PCR. In select groups where PFAS-induced platinum resistance was observed, mtDNA copy number was elevated by up to 35% compared to controls. Increased mtDNA copy number in cancer cells may enhance bioenergetic capabilities, promoting cancer progression. Additionally, superoxide production was measured to understand oxidative stress profiles post-PFAS exposure. In the OVCAR-3 cell line, superoxide production decreased compared to controls while the Caov-3 cell line produced more superoxide post-PFAS exposure. Together, these findings implicate alterations to mitochondrial functioning as a mechanism underlying PFAS-induced chemotherapy resistance.
108 - North American Graduate Fellowship Recipient
RNA-Binding Protein Tristetraprolin (TTP) Modulates Ozone-Induced Lung Injury and Inflammation in Mice
Richa Lamichhane 1, Yogesh Saini1, and Sonika Patial1
1Louisiana State University, Baton Rouge, LA, USA
Ozone (O3) is a ubiquitous air pollutant associated with increased incidence and exacerbation of pulmonary diseases. Acute O3 exposure leads to increased pulmonary inflammation and injury, primarily caused by excessive production of inflammatory cytokines and chemokines. Tristetraprolin (TTP) is an mRNA-binding protein that binds to AU-rich elements within the 3′ untranslated regions of certain mRNAs for inflammatory genes and increases their rate of decay. Here, we tested the hypothesis that TTP alleviates acute ozone-induced lung injury and inflammation. TTP knockout (TTPKO), TTP-overexpression (TTPΔARE), and wild-type (WT) adult mice were exposed to either 3ppm ozone or filtered air (FA) for three hours. Lung injury, inflammation, and airway hyperresponsiveness were assessed after 21 hours post-ozone or FA exposure. As compared to genotype-matched FA-exposed mice, ozone-exposed TTPKO, TTPΔARE, and WT mice significantly increased granulocyte infiltration (p< 0.01), lung injury (p< 0.0001), and airway hyperresponsiveness. Furthermore, as compared to ozone-exposed WT mice, TTPKO mice exhibited significantly increased immune cell infiltration in bronchoalveolar lavage fluid (BALF) (P< 0.0001). In addition, TTPKO mice exhibited exaggerated peri-arteriolar and peri-bronchiolar edema, peri-arteriolar and peri-bronchial inflammation, as well as debris within the lumen of both small and larger airways. Notably, the bronchial pattern was visibly evident in the TTPKO mice. Conversely, as compared to ozone-exposed WT mice, ozone-exposed TTPΔARE mice showed significantly decreased immune cell infiltration (p< 0.01), lung pathology, and airway hyperresponsiveness. Overall, these findings suggest that TTP may be a potential target for the development of new therapies for lung diseases associated with environmental pollutants such as ozone.
109
Potential Interactions between Narrow Therapeutic Index Medications and Other Medications in Pediatric Population
Patricia Caro Uribe 1, Marylin Acuna Hernandez2, Alfredo Portilla Pinzón2, Manuel Machado Duque3, Andrea Orozco Escobar3, and Rosangela Casanova Libreros4
1Fundación Universitaria de Ciencias de la Salud, Bogota, Distrito Capital de Bogota, Colombia
2Fundación Universitaria de Ciencias de la Salud, Bogota, Colombia
3Audifarma S.A. Colombia, Pereira, Colombia
4FUCS, Bogota, Colombia
Background: Drug interaction is the alteration in pharmacological effects due to the concomitant or prior use of another medication, food, herbal product, or other intrinsic or extrinsic factors that may generate adverse effects by decreasing the therapeutic effect or increasing the toxicity of the drugs involved. Objective: Determine the frequency of occurrence of potential drug interactions between narrow therapeutic index drugs and other medications in pediatric patients included in the general healthcare system of Colombia between January 1st to December 31st, 2021. Methods: A cross-sectional descriptive study taken the dispensing database of a pharmaceutical manager in Colombia. The data was taken from patients under 18 years old who received narrow therapeutic index drugs included in the DrugBank database between January 1st to December 31st, 2021. Results: For the time frame medications were dispensed for 777,924 patients under the age of 18, of which 24,053 received narrow therapeutic index drugs resulting in a prevalence of 3.09%. Regarding potential drug interactions of narrow therapeutic index drugs with other medications, they were present in 5% (n=1,203) of the studied population, all of which were of major clinical risk. Of these, 94% only presented one potential interaction, while the remaining 6% presented two potential interactions and the drugs that presented potential interactions in order of frequency were carbamazepine, imipramine, methotrexate, phenobarbital, phenytoin, and theophylline. Conclusions: The probability of potential drug interactions is 5 per 100 pediatric patients who use narrow therapeutic index drugs along with other types of medication.
111 - International Travel Grant Recipient
The Rescue Role of Myricetin in Testicular Oxidative Stress, Inflammation, and Apoptosis in Atrazine Model of Parkinson's Disease
Cynthia Nwamaka Ikeji 1, Isaac A. Adedara1, and Ebenezer O. Farombi1
1University of Ibadan, Ibadan, Oyo, Nigeria
Parkinson's disease (PD) is a neurological disorder that leads to motor impairment, which can cause sexual dysfunction and male infertility. Myricetin, a bioflavonoid that is abundant in grapes has various pharmacological properties, including protective effects on the reproductive system. This study investigated the impact of myricetin on testicular oxidative stress, inflammation, and apoptosis in atrazine-induced PD rat models. Thirty-two male Wistar rats (12 weeks) were randomly assigned to four groups of eight rats each. The control group received corn oil (Atrazine/myricetin Vehicle), while the MYR group received myricetin (20 mg/kg). The ATZ group received atrazine (50 mg/kg), and the ATR+MYR group was co-administered atrazine (50 mg/kg) and myricetin (20 mg/kg), all by gavage for 45 days. Employing various biochemical and immunological techniques, results revealed that myricetin administration significantly reduced the atrazine-induced increase in testicular oxidative stress (superoxide dismutase, catalase, glutathione-s-transferase, reduced glutathione, lipid peroxidation), inflammatory markers (myeloperoxidase, nitric oxide, tumor necrosis factor-α, interleukine-1β, nuclear factor-kappa B, cyclooxygenase-2, inducible nitric oxide synthase), markers of apoptosis (caspase-3, Bcl-2-associated X protein),and DNA damage (terminal deoxynucleotidyl transferase (TdT) dUTP NickEnd Labeling) in the testicular tissue, and marker of PD (alpha-synuclein) in the substantia Nigra pars compacta of the animals. Additionally, myricetin prevented the disruption of the testicular histological architecture in the atrazine-challenged rats. The findings of this study suggest that myricetin has anti-oxidative, anti-inflammatory, and anti-apoptotic effects that can ameliorate atrazine-induced testicular dysfunction in PD animal models. Therefore, myricetin may have therapeutic potential in treating male infertility and sexual dysfunction associated with Parkinson's disease.
112 - International Travel Grant Recipient
High-Fructose Diet Consumption Starting from Juvenile Age to Puberty Lead to Pro/Antioxidant Misbalance, DNA Fragmentation, and Chromatin Component Changes in the Testes of Male Rats
Ganna M. Shayakhmetova 1, Larysa B. Bondarenko1, Tetiana A. Karatsuba1, Valentina M. Kovalenko1, and Oleksandr Tkachenko2
1SI Institute of Pharmacology and Toxicology NАМS of Ukraine, Kyiv, Ukraine
2Clinic of Reproductive Medicine Lita, Kyiv, Ukraine
High-fructose diet (HFD) can induce whole range of metabolic alterations and potentially metabolic syndrome. Our previous results indicate that HFD starting from juvenile age up to puberty causes reproductive impairment in male rats; however, mechanisms involved are not fully understood. Study aim was to evaluate effects of HFD (starting from juvenile age to puberty) in rats’ testes on biochemical and molecular levels. Juvenile male Wistar rats were divided into 2 groups: control and HFD (replacement of drinking water with 10% fructose starting from postnatal day 23 up to 83). Increase in testicular level of lipid peroxidation (35%) and activity of superoxide dismutase (37%) was shown. Testicular content of reduced glutathione and serum content of ceruloplasmin (main extracellular blood antioxidant) significantly decreased. HFD consumption was accompanied by increased DNA fragmentation processes in testes with formation of 13 fractions of low molecular weight fragments. Percentage of fragmentation increased from 7.035% in control to 27.63% - in animals that consumed fructose. Eight fractions consisted of fragments with masses of 20-100 b.p., while relatively more massive fragments were represented by 5 fractions with a length of 350-500 b.p. There was significant increase in content of DNA, DNA-bound proteins, RNA, and histones in testicular cells. RNA/DNA and RNA/total nucleic acids ratios decreased, which evidenced predominance of chromatin component changes on transcriptional level. Thus, HFD starting from juvenile age up to puberty caused pro/antioxidant misbalance and changes in nucleic acids in testicular cells, which can be involved in pathogenetic mechanisms of male reproductive disorders demonstrated previously.
113 - International Travel Grant Recipient
Diphenyl Diselenide Modulate Diethylnitrosamine and Carbon Tetrachloride-Induced Nephrotoxicity in Wistar Rats
Adedoyin O. Adeoye 1, Opeyemi B. Aweda1, and Oluwatosin A. Adaramoye1
1University of Ibadan, Ibadan, Oyo, Nigeria
The pathogenesis of toxicity-mediated renal injury has been linked to reactive oxygen species. Intervention with antioxidant agents as part of therapeutic strategies is necessary. This study investigated the modulatory effects of diphenyl diselenide (PhSe)2 against diethylnitrosamine (DEN) and carbon tetrachloride (CCL4)-induced nephro-toxicity in male rats. Forty rats were assigned into five equal groups. Group 1 served as control, Group 2 received DEN (100 mg/kg) + CCL4 (0.5 mg/kg), Groups 3 and 4 received [DEN+CCL4] and treated with (PhSe)2 (5 mg/kg and 10 mg/kg), and Group 5 received [DEN+CCL4] and treated with quercetin (50 mg/kg). The DEN and CCL4 were administered intraperitoneally once weekly for three weeks after which (PhSe)2 and quercetin were administered (oral gavage) thrice weekly for another three consecutive weeks. Administration of DEN and CCL4 decreased body weight gained by 38%. The DEN and CCL4 increased serum urea, creatinine, and kidney injury molecule-1 (KIM-1) levels by 97%, 62%, and 73%. The activities of glutathione-S-transferase, catalase, superoxide-dismutase and total sulphydryl level decreased in rats administered-[DEN+CCL4]. In addition, tumor necrosis factor-α, interleukin-1β, myeloperoxidase and nitric oxide level increased by 117%, 81%, 385% and 65% respectively in [DEN+CCL4]-rats. Immunohistochemistry revealed [DEN+CCL4]-rats reduced BCL-2 associated X protein; increased caspases-3, -9 and p53 expression. Histology showed mild vascular congestion and infiltration of inflammatory cells in interstitial spaces in [DEN+CCL4]-administered rats. Treatment with diphenyl diselenide at 50 mg/kg and 100 mg/kg attenuated kidney function markers, reduced tissue inflammation and apoptosis status of [DEN+CCL4]-rats. Diphenyl diselenide abate nephrotoxicity via induction of anti-oxidative and anti-inflammatory activities.
114 - North American Travel Grant Recipient
Exposure to Inhaled Flavored E-cigarette Aerosol in Pregnant and Nonpregnant Mice
Vingie Ng 1, Morgan O. Shaffer1, Sabrina M. Scroggins1, Mei L. Lietsch1, Vernat J. Exil1, Andrea Adamcakova-Dodd1, and Peter S. Thorne1
1University of Iowa, Iowa City, IA, USA
Human and animal studies increasingly suggest that exposure to e-cigarette (e-cig) aerosol can have adverse reproductive and developmental outcomes (e.g., impaired fertility, reduced birth weight) in addition to lung inflammation. Additionally, there is a need for long-term studies, especially with newer fourth generation devices that deliver high amounts of nicotine, which is a reproductive and developmental toxicant. We investigated whether repeat dose exposure to flavored e-cig aerosol caused lung inflammation, impaired lung function, and altered gene expression of liver enzymes involved in xenobiotic and estrogen metabolism in pregnant and nonpregnant mice. C57BL/6J mice were exposed one hour daily to either Virginia Tobacco (VT), Menthol (MT)-flavored aerosol (Juul Labs, Inc.), or sham-exposed to HEPA-filtered air for 17-19 weeks in a nose-only inhalation system (InExpose, SCIREQ). Mice were mated 13 weeks into exposure, with pregnant cohorts necropsied on gestational day 18. Results show that total leukocyte counts in bronchoalveolar lavage fluid were increased in MT-exposed/nonpregnant lungs; neutrophil or lymphocyte counts or concentration of cytokines did not differ significantly among experimental groups. In contrast, VT-exposed nonpregnant mice had increased lung resistance. In addition, hepatic expression of Fasn, Scd1, Sts, Esr1, and Cyp2a5 were downregulated in VT- and MT-exposed/nonpregnant mice but upregulated in exposed/pregnant mice. Interestingly, Fasn was also upregulated in placentas from e-cig-exposed dams. Fetal/placental weights from e-cig-exposed dams were also on average significantly lower compared to sham-exposed mice. In conclusion, e-cig aerosol can elicit divergent responses depending on flavor and alter hepatic gene expression with and without pregnancy.
115 - North American Graduate Fellowship Recipient
Liver-Specific Deletion of RNA-Binding Proteins ZFP36L1 and ZFP36L2 Mitigates Carbon Tetrachloride-Induced Hepatotoxicity
Rahul Kumar 1, Sonika Patial1, and Yogesh Saini1
1Louisiana State University, Baton Rouge, LA, USA
Xenobiotic-induced hepatotoxicity remains a significant occupational hazard and mechanism-based treatments are limited. Our preliminary studies showed that deletion of mRNA destabilizing proteins, Zinc Finger Protein 36 like 1 (ZFP36L1) and Zinc Finger Protein 36 like 2 (ZFP36L2), initiates pro-inflammatory phenotype in the liver. Accordingly, we tested the hypothesis that deletion of these proteins exacerbates xenobiotic-induced hepatotoxicity. Liver-specific ZFP36L1 and ZFP36L2 double knockout (L1L2LKO; AlbCre+/Zfp36l1flox/flox/Zfp36l2flox/flox) and flox-only control (L1L2FLX; AlbCre-/Zfp36l1flox/flox/Zfp36l2flox/flox) adult mice were intraperitoneally challenged with carbon tetrachloride (CCl4, 0.8 ml CCl4 in corn oil/Kg body weight). Endpoints associated with CCl4 metabolism, glutathione (GSH) synthesis, and liver injury were assessed in unchallenged and CCl4-challenged mice at 48 h post-challenge. Liver GSH levels were significantly higher in unchallenged L1L2LKO than in L1L2FLX mice (p< 0.05). Interestingly, both mRNA and protein expressions of CYP2E1, an enzyme known to bioactivate CCl4, were significantly downregulated in unchallenged L1L2LKO compared to the L1L2FLX group. Unexpectedly, CCl4 challenge resulted in considerably less hepatic necrosis, ∼10-fold lower serum ALT and AST levels, and less perturbation in the hepatic transcriptome (359 differentially expressed genes (DEGs) versus 3,862 DEGs) in L1L2LKO mice compared to L1L2FLX mice. Notably, the mRNA levels of Gclm and Gpx1, enzymes associated with the GSH pathway, remained significantly higher in the CCl4-challenged L1L2LKO versus the CCl4-challenged L1L2FLX group. In summary, the combined deletion of ZFP36L1 and ZFP36L2 mitigated CCl4-induced hepatotoxicity by preventing the bioactivation of CCl4 and strengthening the antioxidant defense system. These findings suggest a novel pathogenic role of ZFP36L1 and ZFP36L2 in CCl4-induced hepatotoxicity.
116
Characterization of Adverse Effects on Bone Following Administration of Two Non Bile Acid Based Farnesoid X Receptor Agonists in Rats
Martin Guillot 1, Justin Schumacher2, Mélanie Felx1, Paula Katavolos3, Evan Janovitz2, William Achanzar2, Gabrielle Boyd1, Solomon Haile1, and Aurore Varela1
1Charles River Laboratories Montreal, Senneville, Quebec, Canada
2Nonclinical Safety, Bristol-Myers Squibb, New Brunswick, NJ, USA
3Bristol-Myers Squibb, New Brunswick, NJ, USA
Farnesoid X receptor (FXR) agonists are currently under development to treat various cardiac and metabolic diseases. FXR, a nuclear hormone receptor, has many functions including regulation of bile acid homeostasis and has been shown to potentially regulate bone metabolism. This study aimed to characterize FXR agonist effects on bone testing two structurally differentiated non-bile acid FXR agonists in rats. FXR agonist A (3 mg/kg/day), FXR agonist B (60 mg/kg/day) or vehicle were administered daily by oral gavage for 8 weeks to Sprague Dawley rats (n=10/sex/group). In vivo assessments (Weeks 2 and/or 4 and 8) included bone densitometry by dual-energy X-ray absorptiometry, radiography, and clinical pathology evaluations including bone-related biomarker and hormone assessments. At termination, ex vivo assessments included bone densitometry by peripheral quantitative computed tomography, biomechanical testing of the femur and lumbar vertebrae, and histopathologic evaluation of the femur, lumbar vertebrae, and sternum. Administration of FXR agonist A in males resulted in imbalanced bone turnover towards greater bone resorption associated with changes in Vitamin D with no impact or increases on serum PTH levels. These changes in bone metabolism and calcium/phosphorus homeostasis were associated with decreased in bone size/geometry, mass and density that were reflected by adverse effects on bone strength. Females presented similar but less severe effects. Administration of FXR agonist B resulted in similar but generally more severe effects leading to adverse effects on bone strength in males and females. These comparable findings for two structurally-differentiated FXR agonists suggested the observed bone toxicity was mediated by on-target pharmacology.
117
Comparison of 37.5 mg/kg and 75 mg/kg Dose of N-Methyl-N-Nitrosourea in Tg.rasH2 Mice as Positive Control in 26-Week Carcinogenicity Assays
Sumitra Sengupta 1, Jay Herman2, Gregg Goddard3, Emma Moore4, Julie Turner2, and Katie Sivak5
1Labcorp, Somerset, NJ, USA
2Labcorp Drug Development, Madison, WI, USA
3Labcorp Drug Development, Eye, United Kingdom
4Labcorp Drug Development, Huntingdon, United Kingdom
5Labcorp Drug Development, Somerset, NJ, USA
N-methyl-N-nitrosourea (MNU) is a commonly used positive control to prove the suitability of the CByB6F1-Tg(HRAS)2Jic (Tg. rasH2) mouse model in 26-week carcinogenicity assays. The dose of MNU used varies between different laboratories where 37.5 and 75 mg/kg are frequently used. We evaluated studies at three Labcorp Drug Development sites in the US and UK from 2013 to 2020 to determine if 37.5 mg/kg MNU is sufficient to induce tumorgenicity. A total of 20 studies (N = 10/dose) were analyzed to compare body weights, survivability, palpable masses, squamous cell papillomas, or lymphomas. Skin and stomach tissues are mostly evaluated histologically in laboratories using 37.5 mg/kg, while thymus and squamous cell tissues are evaluated (if identified macroscopically) for studies using 75 mg/kg. The analysis revealed that at 75 mg/kg MNU, a higher incidence of early unscheduled sacrifices occurred, with an average survival rate of approximately 34% by Week 20, compared with an average survival of approximately 70% at 37.5 mg/kg. The number of palpable masses was lower at 37.5 mg/kg compared with 75 mg/kg, but the mean onset time was comparable. Squamous cell papilloma of the skin or stomach and lymphoma were identified as a common neoplasia at both doses at similar frequencies. These results demonstrated that either dose is sufficient to provide evidence that the strain of mice used was capable to predict the carcinogenic potential of the drug of interest. Further, 37.5 mg/kg MNU in Tg.rasH2 mice carcinogenicity studies improved overall survivability.
118
Comparison of Routine Toxicology Parameters of Tg.rasH2 Mice and Non-Tg.rasH2 Mice
Guo Lei 1, Sue McPherson1, and Yan Zhang1
1WuXi AppTec, Suzhou, Jiangsu, China (People's Republic)
Tg.rasH2 mice are popular and sensitive models for rapid carcinogenicity testing of a test article, in which carcinogenic potential is determined within 26 weeks. Generally, 28-day dose range finding studies are conducted in non-Tg.rasH2 mice. Although closely related, the non Tg.rasH2 mouse it is still important to understand any potential differences between the two strains. To evaluate these a comparison of routine parameters, including body weights, food consumption, clinical chemistry parameters between the two strains was conducted. The results were analyzed using non-Tg.rasH2 mice as reference control point. For body weight and food consumption, data were collected from the 28-day DRF studies in non-Tg.rasH2 mice and the first 4-week of the 26-week carcinogenicity studies in Tg.rasH2 mice. Body weight of Tg.rasH2 mice were lower than the non-Tg.rasH2 mice for about 15% in males and 10% in females, which correlated with lower food consumption for Tg.rasH2 mice relative to non-Tg.rasH2 mice. Clinical pathology data collected at the end of the studies showed some differences between the two strains. Hematology data showed remarkably lower white blood cell count and lymphocytes in both sexes of Tg.rasH2 mice when compared to non-Tg.rasH2 mice. Differences in clinical chemistry were limited to relatively higher Total Bilirubin in male Tg.rasH2 mice, and lower alkaline phosphatase and glucose in both sexes of Tg.rasH2 mice relative to non-Tg.rasH2 mice. These differences between the strains should be considered when estimating the dosages for the 26-week carcinogenicity studies in Tg.rasH2 mice using the 28-day DRF studies in non-Tg.rasH2 mice.
119
Development of an Innovative Training Tool for Implementation of a New Technical Procedure in Nonclinical Research: First Immersive Tutorial in Virtual Reality
Gael Quesseveur 1, Michael Verset1, Nicolas Chirat1, Anthony Coillard1, Marie Latzko1, Nathalie Truchot-Limonne1, and Maxime Ros2
1Charles River Laboratories France Safety Assessment SAS, Saint-Germain-Nuelles, Rhone-Alpes, France
2Revinax SAS, Montpellier, France
The development of new therapeutic strategies for central nervous system (CNS) diseases has led to implement new administration routes (intra-thecal, intra-cerebroventricular) in non-rodent models, particularly non-human primates (NHP) because of their homology of CNS structure and function with man. Administration by such routes requires the acquisition of advanced technical skills and scientific expertise. However, the use of NHP is subject to strict regulations, particularly in Europe, which prohibits the use of NHP for staff training/teaching. In compliance with the 3Rs principles, developing new teaching methods for non-clinical research becomes critical to overcome such challenges and to reduce the number of animal use. Immersive video is an innovative teaching method based on virtual reality technology and increasingly used in clinical research. Immersive video is based on “first-person view” video (180 degrees, 3D, ultra-high definition) embedded in a virtual reality headset, which allows the development of learning by imitation, facilitated by an immersive and realistic environment and completed with additional assets (blended learning). We have developed and implemented an immersive virtual reality tutorial for teaching intrathecal administration and cerebrospinal fluid sampling techniques in Cynomolgus monkeys. Data demonstrate that immersive video facilitates the initial training and increases self-confidence of learners which results in higher operator performance, compared to conventional teaching strategies. In addition, we used the immersive virtual reality tutorial for maintaining technical skills and best practices. Therefore, immersive virtual reality tutorial will reduce animal use to develop new highly skilled technical procedures and serve as a teaching tool for non-clinical research.
120
Differential Species Sensitivity to Sulfobutylether-Beta-Cyclodextrins (SBECD)
Navatha Alugubelly 1, Nimita Dave2, and James Randazzo1
1Attentive Science, Stilwell, KS, USA
2LAPIX Therapeutics, Boston, MA, USA
Cyclodextrin (CD) compounds have been historically used as solubilizing and stabilizing agents in formulations for active pharmaceutical agents. Several CD derivatives, notably 2-hyroxyproply-beta-cyclodextrin (HPCD) and sulfobutylether-beta-cyclodextrin (SBECD), were evaluated for safety due to nephrotoxic concerns with the parent CD compounds and are now employed in several pharmaceutical drugs approved by the FDA. However, the comparative effects of SBECD in different species at a significantly higher dose weren't assessed, despite past studies showing that SBECD is safer than other CD derivatives. Due to formulation constraints, the dose level of SBECD proposed for an upcoming 28-day oral repeat dose toxicity study was 2.4g/kg/day. Therefore, we evaluated the effects of SBECD at 2.4 mg/kg/day in Beagle dogs and CD-1 mice in order to assess subsequent use at this dose level. Parameters and endpoints evaluated in this study included mortality, clinical observations, body weights, food consumption, clinical pathology parameters, organ weights, and macroscopic and microscopic observations. When compared to mice, dogs were more susceptible to SBECD. The non-adverse findings in dogs were limited to soft stool and/or diarrhea, decreased food consumption, lower urine pH, and mild mucosal edema in the colon, while no effects associated with SBECD were observed in mice. The mucosal edema and soft feces observations persisted after two weeks of the recovery period in dogs. Based on these findings, the no observed-effect-level (NOEL) of SBECD after 28 days of exposure is 2.4 g/kg/day and < 2.4 g/kg/day for mice and dogs, respectively.
122
Enhancing Seizure Liability Assessment: Integrating Target-Based Data and Addressing Knowledge Gaps
Mamta Behl 1, Alexandre Borrel2, Jonathan Hamm2, Helena Hogberg3, Oluwakemi Oyetade2, Mohan Rao1, Clifford Sachs1, and Aswani Unnikrishnan2
1Neurocrine Biosciences Inc., San Diego, CA, USA
2Inotiv, Durham, NC, USA
3NIEHS, Durham, NC, USA
Animal models are commonly employed in drug development and environmental toxicology to predict human toxicity. However, research suggests that these models have limited reliability, particularly in predicting drug safety for the central nervous system. The prediction of convulsions, specifically, is unreliable due to a significant failure rate of novel drugs in human clinical trials caused by unforeseen toxicity, revealing the inadequacy of these models. To address this issue, a collaborative government-industry initiative was conducted to identify potential biological targets associated with seizures. By combining targets from established adverse outcome pathways (AOPs) and drug discovery databases, a seizure specific AOP network was generated. In vitro assays, utilizing resources like Integrated Chemical Environment (ICE), Off-X, and other databases, were employed to identify annotated targets. Approximately 100 assays covering 21 selected targets, including neurotransmitter receptors, transporters, modulators, electrical activity, and voltage-gated calcium channels, were identified. A literature search led to the identification of 90 chemicals and drugs with potential seizure-inducing effects, along with 35 negative control compounds. ICE provided assays and targets for around 10% of seizure compounds and 30% of negative compounds, resulting in a total of 329 dose-response curves. An additional 20% of seizure-inducing compounds were identified through Off-X. This project serves as a proof-of-concept for integrating data from multiple sources based on targets to identify and categorize drugs with seizure liability. It also addresses existing knowledge gaps and offers insights into future directions.
123
Evaluation of the Extended ToxProfiler MAX Assay for Mitochondrial and Mechanistic Toxicity Assessment
Paula Van Rossum 1, Kim Elbertse1, Giel Hendriks1, Amer Jamalpoor1, Bas ter Braak1, and Liesanne Wolters1
1Toxys, Oegstgeest, Zuid-Holland, Netherlands
Human safety assessment is a crucial step in the development of new compounds in the pharmaceutical, (agro)chemical and cosmetic industries. To improve the in vitro predictions for human safety of compounds, it is important to understand the underlying mechanisms of toxicity. We previously developed ToxProfiler, a human in vitro assay that combines seven stable fluorescent reporters that can provide an extensive toxicological profile of novel drugs and chemicals. Here we describe an extended version of the assay, ToxProfiler MAX that in addition to oxidative stress, ER stress, cell cycle stress, ion stress, protein stress, autophagy and inflammation also includes assessment of mitochondrial toxicity. The extended ToxProfiler MAX assay combines the differential activation of the seven fluorescence reporters with a glucose/galactose medium switch. The assay was evaluated using 20 reference compounds with diverse and well-established toxicological mode-of-action (MoA), including mitochondrial toxicity. All 10 mitochondrial toxicants, including antimycin A and rotenone, were correctly identified in the extended ToxProfiler protocol. Interestingly, all mitochondrial toxicants also activated the ToxProfiler reporter for ER stress. For the 10 compounds that do not cause mitochondrial toxicity, oxidative stress (CDDO-me), autophagy (chloroquine) and ER stress (tunicamycin) were identified as primary toxicological MoA. The ToxProfiler MAX assay was able to discriminate between compounds that directly affect mitochondria function from those that induce other types of cellular stress (e.g., oxidative stress, autophagy or ER stress) before impacting the mitochondria. Quantitative dose-response modelling of the toxicological responses was applied for biological grouping and potency ranking of the compounds.
124
Genetic Toxicity Testing of MM-120 (lysergide D-Tartrate) Demonstrates No Evidence of Mutagenicity
Julie Tripp 1, Gennady Smagin2, and Robert Silva3
1Mind Medicine Inc, Fort Collins, CO, USA
2Mind Medicine Inc, Newark, NJ, USA
3Mind Medicine Inc, Northbridge, MA, USA
Early studies of D-lysergic acid diethylamide (LSD) indicated it caused chromosome aberrations in vitro (e.g., cultured human leukocytes; (Cohen, 1967) and in vivo (leukocytes from LSD users; [Irwin, 1967]), however, results of the many subsequent investigations have been contradictory. Therefore, we evaluated MM-120 (LSD D-tartrate) in three modern, Good Laboratory Practice compliant genetic toxicity assays including a bacterial reverse mutagenicity (Ames test), a micronuclei evaluation in cultured human lymphocytes and an in vivo micronucleus test with rat bone marrow. In each in vitro assay, MM-120 was tested in the absence and presence of metabolic activation (Aroclor-induced rat liver S-9). MM-120 did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium and one tryptophan-requiring strain (WP2 uvrA pKM101) of Escherichia coli in the Ames test at concentrations up to 5000 µg/plate in the absence or presence S-9. MM-120 did not cause an increase in the induction of micronuclei in cultured human lymphocytes. The cultures and test article were incubated in both the absence and presence of S-9 at concentrations up to 325.60 µg/mL. MM-120 did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes, or bone marrow cell toxicity, in male rats when administered orally at doses up to 12.0 mg/kg/day. Systemic exposure was confirmed with mean Day 1 Cmax for MM-120 of 144 ng/mL and mean AUC0-24h of 2180 ng·h/mL. The data demonstrate no evidence of mutagenicity and support clinical development of MM-120.
125
Intravenous Sampling and Administration in Rodents: Overcoming Challenges of Using Vascular Access Buttons
Gabriela Campoy 1, Julie Forget2, Narine Lalayeva2, Serah Tanno2, Adeline Varnet2, and Norbert Makori2
1Altasciences Preclinical Seattle LLC, Snohomish, WA, USA
2Altasciences, Everett, WA, USA
Repeated reliable access to the vascular space in rodents can be challenging in preclinical testing. Vascular access buttons (VAB) offer a solution for permanent transcutaneous catheters for blood collection and dose administration in a closed system. This technology allows for social housing of the animals therefore reducing the overall stress. In chronic toxicological studies, long-term catheter patency and integrity are crucial. As such, a retrospective review of multiple VAB studies was conducted and revealed the need for modifications of the housing environment and maintenance of the VAB to ensure successful studies, prolonged catheter patency, and quality data. During an initial study, less than 10% of VAB-implanted animals required veterinary treatments unrelated to a test article effect, nonetheless leading to important observations, like severe swelling of surgical site and histopathological findings, like vascular and surgical site inflammation, thrombi formation and/or unscheduled euthanasia. Changes in animal care included an extended acclimation period, regular site and catheter cleaning, patency checks, and adjustments in husbandry practices including use of cellulose bedding, regular nail trimming, and use of modified food hoppers and enrichment tubes. Subsequent studies conducted with these adjustments indicated a significant reduction in frequency and severity of the findings and limiting them to the acclimation phase. These modifications eliminated the surgical site inflammation and unscheduled euthanasia. Further studies are planned to continually assess the benefit of these adjustments and refine the procedures for better outcomes.
126
Introducing Philippine-Origin Cynomolgus Macaque (Macaca Fascicularis Philippensis) as a Research Model in Drug Safety Assessment
Yafei Chen 1, Tara Arndt2, George De Los Santos2, and Wendell P. Davis2
1Altasciences, Columbia, MO, USA
2Altasciences, Seattle, WA, USA
A recent special issue of Toxicologic Pathology addressed concerns that genetic and environmental variability associated with different geographical origin cynomolgus macaques (CM) could complicate the interpretation of nonclinical toxicity study results. The conclusions of this special issue support the use of different origin CMs in drug development programs as long as scientific justifications and reference data are available. However, this special issue did not include Philippine-origin CM. This current study systemically compiles genomic background, disease susceptibility, and relevant toxicological data needed to assess the feasibility of using Philippine CM in drug development. The range of genetic variation in CMs specifically within proteins involved in the metabolism and immune response has been established. Genomic background studies demonstrated the Philippine CM recapitulates the diversity and complexity of the human genome and exhibits similar or potentially lower genetic diversity for the hepatic CYP450 isoforms (CYP1A/2A/2B/2C/2D/2E1/3A4), cytokines (IL1β/IFNγ/CCL2), and MHC I (A/B/I/AG) and II (DRA/DRB) genes vs. insular Asia CMs. Subclinical gastritis is widely present in the Philippine CM with a high incidence of Helicobacter spp. infection and lymphoplasmacytic cell infiltration in the digestive tract which correlated with increased leukocyte counts. Other toxicological reference data including in-life, clinical and anatomic pathology were comparable to more extensive CM (mixed-origin) background data in the literature. These genetic heterogeneity and toxicology background data underscore the advantages of using the Philippine CM in preclinical drug development, improve scientific justifications for CM selection, and provide criteria for evaluating preclinical toxicity studies using Philippine-origin CM.
127
Investigation of Symptomatic Gastrointestinal Findings in Cynomolgus Monkeys of Different Countries of Origin
Lu Zhao 1, Fernanda Alvarez1, Geoffrey Goodrich1, and Linda Allais1
1Charles River Laboratories, Reno, NV, USA
The incidence of symptomatic gastrointestinal (GI) findings in cynomolgus monkeys of different countries of origin is not well understood. GI findings have been a concern for conducting toxicology studies, especially when monkeys of different origins are being used due to the global shortage of animal supplies. In this study, we comprehensively analyzed data from > 300 general toxicity studies conducted from 2020 to 2022 at our Testing Facility. The overall incidence of symptomatic GI findings was < 1%, primarily seen in younger animals (< 5 years old) originated from Cambodia, Vietnam, and Mauritius, and to a less extent, from China. The common pathogens resulting in clinical symptoms in monkeys ranked as Campylobacter > Entamoeba cysts > Balantidium cysts > Giardia. When these findings became symptomatic, the common clinical signs included moderate to severe diarrhea, dehydration, and low body condition. The GI findings generally had minimal to no impact on food consumption, clinical pathology parameters, cytokines, or immunophenotyping. Occasionally, crypt abscesses and other relevant histopathology findings were noted in the GI tract. Most of the symptoms were manageable and antibiotics were more effective than supportive treatments (e.g., fiber and probiotics). In severe cases (< 0.3% incidence), Campylobacter or Giardia positive animals were found in poor clinical conditions. Rarely, the GI symptoms were exacerbated by test article-related immune compromise. In conclusion, our study revealed a low incidence of symptomatic GI findings in cynomolgus monkeys of different countries of origin, which did not compromise the overall study objectives or data interpretation.
130
Proposed Best Practices for Optimizing the Number of Animals in Toxicology Studies
Narine Lalayeva 1, Norbert Makori1, and Julie Forget1
1Altasciences Preclinical Seattle LLC, Everett, WA, USA
New drug modalities, accompanied by limited availability of some of the research animal models, necessitates the continuous evaluation of study designs and technology that will enable reduction in the number of animals used for toxicology studies, an approach that aligns with the 3Rs of experimental animal welfare. In rodents, blood microsampling serves as a better option and refinement to the traditional technique of needle and syringe by leveraging the microsampling for accurate collection of low sample volume. For example, by sampling only 10 μL of whole blood, an entire cohort of study animals in the traditional needle and syringe collection was eliminated since serial samples could also be collected from the main study animals. This represented a reduction in the number of study animals by 55% and 100% for mouse and rat studies, respectively. Collection of samples from the same cohort of animals also allows correlations between pharmacodynamic findings and the actual drug exposure profile. In nonhuman primates and dogs, a review was completed of the number of animals in control and recovery cohorts in chronic study designs that had data from prior subacute studies. This enabled reduction by >25% in number of control group with no recovery cohort including in the low dose group. The Non Human Primary (NHP) design was reviewed and accepted by a regulatory agency. Each of these approaches will be discussed further to highlight the pros and cons of each in order to allow for a more informed decision when designing toxicity studies.
131
Real-Time Exposure to 3D Printer Emission Elicits Inflammatory Responses and Metabolic Perturbations in Human Airway Epithelial Cells
Lillie Barnett 1, Xiaojia He2, Jennifer Jeon2, Qian Zhang2, Saeed Alqahtani3, Jonathan Shannahan4, Marilyn Black2, and Christa Wright2
1Chemical Insights Research Institute, Underwriters Laboratories, Marietta, GA, USA
2Chemical Insights Research Institute, UL Research Institutes, Marietta, GA, USA
3Advanced Diagnostic and Therapeutics Technologies Institute, Riyadh, Saudi Arabia
4Purdue University, West Lafayette, IN, USA
Three-dimensional (3D) printer usage across homes and schools has raised health concerns regarding chemical and particle emission exposures during standard operation. However, little is known about the impact of 3D printer emission on respiratory health and underlying cellular mechanisms. In this study, we utilized an in vitro exposure chamber system to deliver real-time emissions from acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) filaments directly to human small airway epithelial cells (SAEC) cultured in air-liquid interface during 3D printer operation. Aerosol monitoring, sampling, and spectroscopic instrumentation were employed to characterize particle size distributions, concentrations, and morphological and elemental properties of 3D printer emissions. Toxicological, metabolomic, and cytokine profiles were evaluated and integrated using an xMWAS network analysis to determine the impact of filament selection on metabolism and pro-inflammatory responses. Our results revealed ABS and PLA emissions have distinct aerosol properties that led to differences in the estimated inhaled and in vitro deposited doses leading to variances in SAEC responses. Specifically, ABS emissions exposure altered amino acids and energy metabolism, whereas PLA caused fatty acid and carnitine dysregulation. Additionally, ABS emissions exposure elicited decreased glutathione levels and dysregulated redox-regulated pathways including arginine, methionine, cysteine, and vitamin B3 metabolism. Although pro-inflammatory cytokines IL-6 and IL-8 were stimulated in both exposure scenarios, IL-1β, MMP-9, and RANTES were significantly increased by ABS emissions whereas VEGF was exclusively increased by PLA emissions. Taken together, these results highlight the role filament emission properties may play in 3D printer emission-induced toxicity and in mediating diverse respiratory outcomes.
132
Reference Ranges for Immune Cell Populations in Nonhuman Primate Whole Blood Evaluated by Flow Cytometry
Amanda C. Lucchini 1, Iyanar Vetrivel2, and James Munday3
1Labcorp Early Development Laboratories Inc., Madison, WI, USA
2Labcorp Drug Development India Private Ltd, Bangalore, India
3Labcorp Early Development Laboratories Ltd, Harrogate, UK
Identification and characterization of immune cells by flow cytometry (known as immunophenotyping) is used for a wide range of modalities across all major preclinical species and is critical tool for immunosafety evaluations. However, because of the complexity of assay designs and inherent variability in methodology, industry-wide immunophenotyping reference ranges do not exist. While individual validations can provide some data, these studies have limitations. Labcorp has developed a historical database across six preclinical sites to capture standard immune cell populations in primates. Approximately 4000 whole blood collections from the predose phase of >100 studies were included. T cells (total, helper, and cytotoxic), B cells, and NK cells (collectively TBNK cells) were evaluated. For TBNK populations the mean values were generally comparable between male and female animals. Additionally, mean values were broadly comparable across sites, with the greatest variability noted in NK cells; this is particularly pertinent as the origin of the primates varied between sites and different immunophenotyping methods were used. Overall, this data provides a general reference range which may aid in interpretation of immunotoxicology data and provides more confidence in the robustness of immunophenotyping as an immunosafety evaluation.
133
Sinclair NanopigTM Standardization – Normal Reference Data
Stacy Qualls 1, Ted Baird2, Guy Bouchard1, and Derek Brocksmith1
1Sinclair Bio Resources, LLC, Auxvasse, MO, USA
2Altasciences Seattle, Seattle, WA, USA
The Sinclair NanopigTM is the outcome of 12 years of selective breeding and nutritional management of the original Sinclair miniature swine lineage. The Sinclair NanopigTM is smaller or similar in size to a Beagle within the age window used for safety assessment. This downsizing achievement provides an avenue to evaluate the safety of pharmaceutical entities and devices as swine shares several anatomical, physiological, and metabolic characteristics with humans. The objective of this study was to generate a relevant and full data set of clinical pathology, ECG, and ophthalmology at strategic timepoints for the Sinclair NanopigTM. Urine and venous blood samples were obtained from 40 to 60 equal gender Nanopigs at 3, 4, 6, 8, 10, and 12 m of age. Standard toxicology panels for hematology, clinical chemistry, coagulation, and urinalysis were generated using GLP validated equipment. ECG and ophthalmology examinations were conducted from 30 to 40 equal gender Nanopigs at 3, 4-5, 6-7, and over 9 m of age. A board-certified veterinary ophthalmologist performed the ophthalmology examinations using a portable slit-lamp biomicroscope and a binocular indirect ophthalmoscope for the posterior segment. ECG leads I, II, III, aVR, aVF, aVL were collected and the following variables were derived: heart rate, PR-interval, QT-interval, QRS-interval, ST-interval, P-wave, and T-wave durations. The rhythm was also carefully reviewed for irregularities and arrythmia. A comprehensive data set for hematology, clinical chemistry, coagulation, urinalysis, ophthalmology, and ECG was compiled specifically for the Sinclair NanopigTM to be used as a reference in safety assessment studies.
134
The Mechanisms of Degraded Microplastics-Induced Cell Toxicity
Yuya Haga 1, Sota Manabe2, Hirofumi Tsujino1, Yudai Ikuno1, Haruyasu Asahara1, Kazuma Higashisaka1, and Yasuo Tsutsumi1
1Osaka University, Suita, Osaka, Japan
Microplastics (MPs) are plastic particles with a diameter of less than 5 mm that exist in the environment. Due to increasing consumption of plastic products, it is crucial to evaluate the effect of MPs on human health. However, little is known about the toxicity of degraded MPs that mimics the chemical structure of MPs in the environment. Therefore, this study aimed to evaluate the cell toxicity and biological mechanisms of degraded MPs-induced cell toxicity using Polyethylene (PE) as a model. Degraded PE (dPE) was prepared using irradiation with vacuum ultraviolet light under air to mimic MPs in the environment. Murine macrophage cells (RAW264.7) and human monocyte cells (THP-1) were used to evaluate the toxicity of PE and dPE. First, physical properties of PE and dPE were measured by ATR-IR, and hydroxyl and carbonyl groups were observed on the surface of dPE. Moreover, MTT assay revealed that cell toxicity was increased only in dPE-treated cells. Mechanistically, we focused on lysosomal damage as small particles such as nanoparticles are known to damage lysosomes and lead to cell death. After incubation with PE and dPE, acidic lysosomes were significantly decreased in dPE-treated cells. Overall, these findings demonstrate that dPE enhances lysosomal damage and leads to cell death, providing new insight into the hazard of MPs on human health.
135
Thirteen-Week Repeated Oral Dose Toxicity Study with Four-Week Recovery Period of HemoHIM G in Sprague-Dawley Rats
Jang Hun Jeong 1, Hyun-Been Choi2, Jong Il Shin2, Duyeol Kim2, Sun-Hee Park2, Hak Sung Lee3, Su-Bin Bak3, and Jung-Jae Lee3
1Biotoxtech Co., Ltd. (GLP certified facility), Cheongju, Ch'ungch'ong-bukto, Republic of Korea
2Biotoxtech Co., Ltd., Cheongju, Republic of Korea
3Kolmar BNH CO., LTD, Seoul, Republic of Korea
HemoHIM G is a newly reconstructed herbal medicine combination of Angelica sinensis, Ligusticum chuanxiong, and Paeonia lactiflora that are highly effective in the recovery of immune-hematopoietic system among previously known herbal medicines. Although each active ingredient is known as non-toxic according to food safety data of the Republic of Korea, subchronic toxicity data on these herbal medicine combinations, HemoHim G, have not been reported to date. This study was conducted to evaluate the potential toxicity and safety of the HemoHIM G when daily orally administered to Sprague-Dawley rats of both sexes for 13 weeks. 10 male and 10 female rats per group were administered 0, 1,250, 2,500 and 5,000 mg HemoHim G formulation/kg body weight/day in water by gavage. In addition, 5 male and 5 female rats were set as 0 and 5,000 mg/kg/day groups to evaluate the reversibility of any adverse effects. There were no toxicologically significant changes in all dosing groups. Dose-dependent decreases in red blood cell mass parameters were observed, however, the decrease in RBC counts was limited to a maximum of 10 % compared to concurrent controls and was within the normal range, and the concomitant increase in erythroid cells in the spleen and bone marrow was suggestive of a compensatory response; therefore, the effects of the test article on RBCs were not toxicologically significant. Based on these results, the NOAEL (No Observable Adverse Effect Level) of the test article, HemoHIM G, was considered to be at 5,000 mg/kg/day for both sexes of Sprague-Dawley rats.
136
Tromethamine: A Suitable Counterion and/or Vehicle Component for Use in Toxicity Studies
Fatima M. Arjmand 1, Joanne Birkebak1, and Megan Wilichinsky1
1Gilead Sciences, Foster City, CA, USA
In 2015, an oral development program for “Compound A” was initiated using tromethamine (Tris) as the counterion and as the main component of the vehicle (0.5% sodium carboxymethylcellulose [medium viscosity], 1% ethanol and 98.5% 50 mM Tris buffer, pH 8 ± 0.5) used in key in vivo toxicity studies. Tris is listed as an inactive ingredient in 28 Food and Drug Administration (FDA) approved products and is the active ingredient in an approved intravenous drug (THAM) for treatment of acute acidosis. Despite the history of Tris use as both an inactive ingredient and a drug, the availability of nonclinical safety data in the public domain is limited. Literature suggests that Tris may be associated with hypoglycemia, hyponatremia, and increased urinary pH. Results of the chronic Good Laboratory Practice (GLP) repeat dose toxicity studies where Tris was a component of the vehicle, there was no evidence that these or any other parameters were impacted in mouse, rat or monkey when compared to historical control data. Additionally, there was no evidence for an effect on fertility or embryofetal development in mice and/or rabbits. In the 2-year carcinogenicity studies, both water and vehicle control groups were included. There was no statistically significant difference in any tumor type between the water and vehicle control. Thus, it is concluded that Tris is suitable for use as both a counterion and a vehicle component in routine toxicology programs.
137
Understanding Species Variation in the Occurrence of Spontaneous Background Eye Lesions in Toxicology Studies: A Comparative Study
Nirmala Chinnappareddy 1, Julie Forget1, Amanda Hunerdosse2, Simone Iwabe3, and Norbert Makori1
1Altascience, Preclinical Seattle, Everett, WA, USA
2Altasciences Preclinical Seattle, Seattle, WA, USA
3Altasciences Preclinical Scranton, Scott Township, PA, USA
Detailed ophthalmic examinations performed prior to test article administration are crucial for screening background lesions and for comparing and deriving conclusions of the potential test article induced ocular toxicity. We reviewed the acclimation or pre-test data collected at our facility for commonly observed ocular lesions in rats, mice, non-human primates (NHP), and dogs for the past one year. Rats: A total of 930 animals (465/sex) were examined of which 19.57% had background findings (cataract: 67.58%, corneal dystrophy: 30.22%, others 2.20%). Male and female animals had a comparable prevalence rate of 19.35% and 19.78%, respectively. Mice: A total of 690 mice (346 males and 344 females) were examined of which 11.01% had background lesions (cataract: 78.95%, retinopathy: 11.84%, corneal dystrophy: 5.26%, others 3.95%). Male and female animals had a comparable prevalence rate of 11.27% and 10.76% respectively. NHP: A total of 599 animals (300 males and 299 females) were examined of which 8.01% had background lesions (cataract: 56.25%, bilateral optic atrophy: 12.50%, optic disc variants: 10.42%, retinopathy: 2.08%, others: 10.42%). Males had more background findings (13.65%) compared to females (5.62%). Dogs: A total of 162 dogs (81/sex) were examined of which 9.26% had background lesions (cataract: 53.33%, corneal dystrophy: 13.33%, retinopathy: 12.5%, others 20.00%). Male and female animals had a comparable prevalence rate of 9.88% and 8.64% respectively. It is recommended either to allocate an animal with findings to a control group or to exclude it from the study depending on the severity of background lesions.
138
Use of Organotypic Gingival Cultures for Nicotine Pouch Assessment
Filippo Zanetti 1, Marion Alriquet1, Laura Ortega Torres1, Laurent Neau1, Fabio Maranzano1, Claudius Pak1, and Carole Mathis1
1Philip Morris Products S.A., Neuchatel, Neuchatel, Switzerland
Nicotine pouches (NP) are an emerging class of nicotine-containing products for oral use. Due to the absence of tobacco and combustion, NP are considered by the manufacturers to pose a lower risk to consumer health than combusted tobacco products and other oral smokeless tobacco-containing products. However, only a few studies have investigated the biological effects of NP. In this study, human organotypic gingival cultures were apically exposed to the extracts obtained from five types of NP with different flavor types and nicotine contents, a snus product with nicotine concentrations relevant to consumer use (or higher), and cigarette smoke fractions (total particulate matter combined with the gas vapor phase [TPM-GVP]) for 96 h. At concentrations representative of human use, the test products induced different degrees of morphological alterations, mostly related to increased keratinization and atrophy of the basal layers. The extract from the NP with a high menthol level induced the most pronounced alterations, followed by three other NP, the snus, and finally, the extract from the NP with the lowest nicotine content. We observed a similar product-dependent response in the inflammatory mediator profile, with the high-menthol NP extract inducing the strongest inflammatory response. TPM-GVP exposure had the strongest impact on culture morphology and inflammatory response, even at nicotine concentrations 40 times lower than those of the oral products. In conclusion, the organotypic gingival model is suitable for evaluating the biological responses to NP, snus, and TPM-GVP exposure because of the possibility to differentiate the toxicological response across different product formulations.]
139 - North American Travel Grant Recipient
HNF4alpha Is Required for CAR, PXR, and PPARα but Not AhR Function in the Liver
Manasi Kotulkar 1, Diego Paine-Cabrera1, Dakota R. Robarts1, and Udayan Apte1
1University of Kansas Medical Center, Kansas City, KS, USA
Four ligand-activated receptors including Aryl hydrocarbon Receptor (AhR), Constitutive Androstane Receptor (CAR), Pregnane X Receptor (PXR), and Peroxisome Proliferator-Activated Receptor-alpha (PPARα) function as sensors of xenobiotics in the liver. Hepatocyte Nuclear Factor 4alpha (HNF4α) is a nuclear receptor that is essential for liver function. Here, we tested the hypothesis that HNF4α is essential for the activation of these major nuclear receptors. Wild-type (WT) and hepatocyte-specific HNF4α knockout (HNF4α-KO) mice were treated with the mouse-specific activators of AhR (TCDD, 0.03 mg/kg), CAR (TCPOBOP, 2.5 mg/kg), PXR, (PCN, 100 mg/kg), and PPARα (WY-14643, 1 mg/kg). After acute activation of nuclear receptors blood and liver tissue samples were collected to study nuclear receptor activation. TCDD treatment did not affect the liver-to-body weight ratio (LW/BW) in both WT and HNF4α-KO mice. TCDD activated AhR in WT and HNF4α-KO mice. TCPOBOP significantly increased the LW/BW ratio and mRNA expression of CAR target genes in WT mice. Further, PCN significantly increased LW/BW ratio in both WT and HNF4α-KO mice; however, it induced PXR target genes only in WT mice. WY-14643 treatment increased LW/BW ratio in HNF4α-KO mice. It upregulated PPARα target genes in WT mice but not in HNF4α-KO mice. Additionally, genes positively regulated by HNF4α were significantly downregulated in WT mice after TCPOBOP, PCN and WY-14643 treatment. These data showed that hepatic CAR, PXR, and PPARα were disrupted in HNF4α-KO mice but not AhR. In conclusion, HNF4α is critical for the activation of hepatic xenosensors that are critical for the toxicological response.
140 - North American Graduate Fellowship Recipient
Cadmium Exposure Induces the Release of Extracellular Vesicles with Modified Cargo That Elicit Pro-inflammatory and Pro-angiogenetic Responses in Recipient Cells
Aikaterini Stavrou 1, Max Costa1, and Angelica Ortiz1
1NYU Grossman School of Medicine, New York, NY, USA
Cadmium (Cd), a heavy metal carcinogen, is ubiquitous in the environment resulting in daily human exposure via inhalation in highly polluted areas and via ingestion of contaminated food. This poses a great health concern due to its long half-life in humans (20-30 years). The mechanisms of Cd-induced carcinogenesis are unclear and still under investigation. Inflammation and angiogenesis are important for tumor growth. Cd is a potent inflammatory agent and has been shown to induce angiogenesis. However, the pro-inflammatory and pro-angiogenic effects of Extracellular Vesicles (EVs) derived from Cd treated cells on unexposed cells have not been explored. EVs play a vital role in cell-cell communication. EVs released by tumor cells regulate various biological processes including angiogenesis and inflammation, thus further promoting carcinogenesis. Human bronchial epithelial cells (BEAS-2B) were either treated for 24 hours with two low doses of Cd or transformed by chronic exposure to 1.6 μM CdCl2, and EVs were collected (Cd-derived EVs). These EVs were then used to investigate their ability to induce pro-inflammatory and pro-angiogenic responses in recipient cells. This study identified several pro-inflammatory genes, altered in both BEAS-2B and human umbilical vein endothelial cells (HUVEC) following Cd-derived EV treatment. Altered expression of genes involved in angiogenesis was also observed in HUVEC cells. The ability of Cd-derived EVs to induce NF-κB nuclear translocation in BEAS-2B and HUVEC cells was also demonstrated. Overall, our results revealed that Cd exposure induced the release of pro-inflammatory and pro-angiogenic EVs that infect neighboring healthy cells, altering their function further promoting carcinogenesis.
141
Absence of Functional Deficits in Rats Following Systemic Administration of an AAV Vector despite Moderate Peripheral Nerve and Dorsal Root Ganglia Findings: A Clinically Silent Peripheral Neuropathy
Seo-Kyoung Hwang 1, Cheryl Tyszkiewicz1, Jamie DaSilva1, Ramesh Kovi1, Kelly Fader1, Madhu Sirivelu2, June Liu1, Chris Somps1, Jon Cook1, and Helen Wang1
1Pfizer, Groton, CT, USA
2Pfizer, Cambridge, CT, USA
Adeno-associated virus (AAV)-based vectors are commonly used for delivering transgenes in gene therapy studies, but they are also known to cause dorsal root ganglia (DRGs) and peripheral nerve toxicities in animals. However, the functional implications of these findings and their time course remain unclear. Here, at 0, 2, 4, 6, and 8 weeks postdose of an AAV vector carrying human frataxin transgene, non-standard, functional assessments including von Frey filament, electrophysiology, and Rotarod tests were conducted longitudinally to measure allodynia, nerve conduction velocity, and coordination, respectively. Additionally, DRGs and peripheral nerves were evaluated histologically, and circulating plasma neurofilament light chain (NfL) was quantified at 1, 2, 4, and 8 weeks, respectively. At 2 and 4 weeks after dosing, minimal-to-moderate nerve fiber degeneration and neuronal degeneration were observed in the DRGs in some animals. At 8 weeks, nerve fiber degeneration was observed in DRGs, with or without neuronal degeneration, and in sciatic nerve of all AAV-dosed animals. Circulating NfL values were higher in AAV-treated animals compared with controls at weeks 4 and 8. However, there were no significant differences in the three functional endpoints between the AAV- and vehicle-dosed animals, or in a longitudinal comparison between 4, 8 weeks, and pre-dose baseline values in the AAV-dose animals. These findings show that there is no detectable functional consequence to the minimal-to-moderate neurodegeneration observed with our AAV treatment, suggesting a functional tolerance, reserve, or threshold for loss of DRG neurons and related neural plasticity after systemic administration of AAV.
200s - Regulatory Toxicology
201
Approaches to Determining Appropriate Inhalation TTC Levels for Various Patient Populations Exposed to VOCs
Erica N. Rogers 1, Samantha E. Gad1, and Shayne C. Gad1
1Gad Consulting Services, Raleigh, NC, USA
Individuals may be exposed to toxic volatile organic compounds (VOCs) in respiratory devices. In cases where a VOC lacks a regulatory limit or sufficient inhalation toxicity data, the default threshold of toxicological concern (TTC) by ISO 18562-1 may be applied. Appropriately, ISO 18562-1 calls for body weight adjustments when the inhalation TTC is used for different patient populations. However, use of one method to justify TTC limits across all populations results in restrictive limits. We therefore compare different approaches (i.e., body weight adjustment and limit of detection (LOD) methods, and ICH M7 guidance) to determine inhalation TTC levels for adults, children, infants, and neonates following exposure to the VOC, n-nonanal, which has no established regulatory limit or adequate inhalation data. Inhalation TTC limits were determined for limited (< 24 hr), prolonged (>24 hr to < 30 days) and permanent (>30 days) exposure to n-nonanal. For adults, inhalation TTC limits were set according to ISO 18652-1 standards. For pediatric and infant populations, body weight adjustments were utilized to determine inhalation TTC limits except for prolonged exposure, which were established at 10 and 4 µg/day, respectively, according to the LOD method. By contrast, limited, prolonged, and permanent inhalation TTC limits for neonates exposed to n-nonanal were set at 4.5, 1.5, and 1.5 µg/day, respectively, according to either ICH M7 guidance and/or deviations of this guidance. By presenting these different approaches, we show that multiple and a variety of methods may be required to determine the most appropriate inhalation TTC levels for diverse patient populations.
202
Developing an FDA-Qualified Drug Development Tool, the Membrane Proteome Array, for Specificity Testing of Antibodies and CAR-T Cell Therapies
Michael Phelan 1, Diana M. Norden1, Jonathan T. Sullivan1, Talia Marano1, Carmen Navia1, Rachel H. Fong1, Amrita Singh1, and Benjamin J. Doranz1
1Integral Molecular, Philadelphia, PA, USA
Detailed specificity analysis of antibody-based therapies, including CAR-T cells, is required for preclinical safety assessment and Investigational New Drug (IND) submissions to the FDA. To meet this need, we developed the Membrane Proteome Array (MPA) platform consisting of 6,000 proteins expressed in live cells. The MPA library was constructed using a bioinformatics approach that identified membrane proteins expressed in 34 normal adult human tissues recommended in FDA guidances for screening cross-reactivity of biologicals. The MPA assesses binding interactions by high-throughput flow cytometry, allowing for high sensitivity detection and rapid analysis. All targets identified on the MPA screen are validated by secondary titration analysis. Unlike tissue cross-reactivity studies, MPA assessment precisely reveals the identity of any off-target binding partners, and MPA data has been accepted by the FDA as a part of IND applications for antibody-based therapies. The MPA has been used to test the specificity of over a thousand therapeutic molecules to date. Of these molecules, approximately 25% displayed off-target binding. This included instances of off-target binding in FDA-approved antibodies, and molecules in clinical development. The FDA has established pathways to qualify Drug Development Tools through various mechanisms that now include the Innovative Science and Technology Approaches for New Drugs (ISTAND) pilot program. The MPA is the first tool to have its Letter of Intent accepted into the ISTAND program. Here, the steps undertaken to develop the MPA as a qualified assay will be described, as well as the MPA’s current status for consideration as a qualified Drug Development Tool.
203
Local Safety Assessment of Drugs Administered via the Intrathecal Route
Armaghan Emami 1, Jay H. Chang1, and R. Daniel Mellon1
1FDA, Silver Spring, MD, USA
Safety assessments to support intrathecal (IT) drug delivery require unique considerations due to the potential for permanent, life-altering toxicity if neuronal tissue is damaged. The risk assessment requires careful consideration of the potential for local toxicity which may be largely driven by drug concentration, but also rate and volume of the infusion and reduced clearance from local tissues compared to intravenous drug administration. These variables impact the potential for local toxicity and can be extremely difficult to mimic in nonclinical models. In addition, the local tissue assessment requires an understanding of not only the tissues in the local environment, but also how the drug is likely to distribute through and be cleared from the local tissues. One way to do this is to consider the “virtual space” in which the drug distributes to ensure that the toxicology studies adequately mimic the clinical setting. The U.S. FDA guidance titled Nonclinical Safety Evaluation of Reformulated Drug Products and Products Intended for Administration by an Alternative Route, provides recommendations to address specific concerns regarding the intrathecal route of administration; however, it does not provide specific details on how the studies are generally reviewed. In this poster presentation, we provide a case study to explain how local safety margins should be derived based on (1) the local concentration at the injection site, and (2) the estimated “steady state” concentrations in the intrathecal space using appropriate CSF volume scaling to ensure the adequacy of the toxicology study design to inform human risk assessment.
204
Quantitative Evaluation of the Advantages of the BMD Methodology for the 3Rs Principle
Kan Shao 1, Andrew Weissmann2, and Keith Davis2
1Indiana University, Bloomington, IN, USA
2DREAM Tech, LLC, Bloomington, IN, USA
The benchmark dose methodology has been widely accepted by regulatory agencies and industries as a default modeling method to replace the traditional NOAEL (no observed adverse effect level) approach for estimating a “safe” dose level from a dose-response dataset. Instead of performing pair-wise comparisons between the control and treatment groups, the BMD method fits a dose-response curve to the response at all tested dose groups and identifies a dose level that corresponds to an acceptable predetermined change in response. A large number of studies have investigated and demonstrated the advantages of the BMD method over the NOAEL approach, such as less dependent on experimental design and better quantification of uncertainty. However, few studies have quantitatively investigated the advantage of the BMD methodology for reducing animal use in toxicological experiments. In this study, we quantitatively evaluate how much animal use reduction can be achieved by the BMD method through BMD’s ability to better identify a “safe” dose level than the NOAEL approach so that additional testing can be avoided. In our preliminary analysis, based on 10,473 datasets collected from NTP’s Toxicological Reports, we found that NOAEL was missing in 59.3% of the datasets and NOAEL and LOAEL were both missing in 44.2% of the datasets, while a plausible BMD/BMDL was estimated for almost all the datasets, suggesting that the BMD method can prevent 25%∼50% additional animal use in half of the experiments conducted. FDA and other agencies should adopt the BMD method to align with the advocation of reducing animal use.
205
Standardized Target Carcinogenicity Assessment (TCA) Enables ICH S1B-Based Regulatory Decisions
Frances L. Hall 1, Arianna Bassan2, Paul Bradley1, Jon Chambers1, Kevin P. Cross3, Brenda Finney1, Candice Johnson3, and Glenn J. Myatt3
1Instem, Cambridge, England, United Kingdom
2Innovatune, Padova, Italy
3Instem, Columbus, OH, USA
The recently introduced ICH S1B addendum (integrated with the original S1B guideline on 4th August 2022) includes a new weight of evidence (WoE) assessment to determine whether performing the rat carcinogenicity study would add value to the assessment of human carcinogenic risk. The guideline describes six WoE factors (1) target biology, (2) secondary pharmacology, (3) histopathology chronic studies, (4) hormonal effects, (5) genotoxicity, (6) immune modulation. A target carcinogenicity assessment (TCA) produced in a consistent, transparent, and dependable manner can assess any contributions that the target biology may have to carcinogenic risk. This publication is based upon a TCA performed for a recently repurposed specific kinase inhibitor. We assembled empirical carcinogenicity data on chemicals within the same primary pharmacological class from sources including PubMed, Chembl and Drug Labels. We reviewed the well-characterized primary pharmacological pathways associated with this drug class and assessed that there are no references that mechanistically link these pathways to involvement in cancer development. Supporting data reviewed included reports on attenuation of the target kinases which indicated that inhibition is beneficial. Finally, we evaluated whether any relevant carcinogenicity risks related to the pharmacology of any major human metabolites. Crucially, the TCA is underpinned by evidence from literature and database searches collated and reviewed in a defined workflow to preserve links to reference sources. As part of the licensing application, inclusion of this TCA helped result in the approval of a drug for an underserved patient population without the need for the 2-year rodent carcinogenicity test.
206
The Surface Wipe Limit Debate: How to Set Acceptable Surface Limits for Hazardous Drugs under USP <800>
Janet Gould 1 and Mary C. Osteen1
1SafeBridge Regulatory & Life Sciences Group, New York, NY, USA
United States Pharmacopeia (USP) < 800> is a minimum standard used to prevent hazardous drug (HD; as defined by NIOSH) exposure to health care workers, and the environment in various medical facilities. These include obvious toxic oncology agents but also steroid hormones and other potent drugs. USP < 800> recommends surface wipe sampling at baseline and every six months for HDs; however, guidance is lacking on 1) sample number or area, and 2) interpretation of results. Because acceptable surface limits (ASLs) for the dermal route of exposure are not readily available, many medical facilities use an “as low as reasonably achievable” interpretation or 1 ng/cm2 limit when evaluating surface sampling results, which may require rigorous controls and procedures for each HD; yet not all HDs are equally hazardous. A health-based inhalation-route, occupational exposure limit (OEL) or an occupational exposure band (OEB), if an OEL is not available, can be used to develop an ASL. By multiplying the OEL by the air volume inhaled in an 8-hour workday (10 m3), an occupational acceptable daily exposure (OADE) is determined, which then can be adjusted for worker skin contact surface area (i.e., 100 cm2/day) and dermal absorption. By using more readily available inhalation exposure limits (OELs and OEBs) to define an ASL, each HD can be individually assessed and prioritized to meet the standard put forth by USP< 800>. In conclusion, having health based ASLs for risk assessment allows for effective controls and a prioritization of resources for overall HD program management.
207
Toxicity of Three Antisense Oligonucleotide Drugs and Eighteen of Their Impurities in Primary Human Hepatocytes
Jessica Hawes 1, Lijun Ren1, Laura Schnackenberg1, Qiang Shi1, and Kui Yang2
1NCTR, FDA, Benton, AR, USA
2CDER, FDA, St. Louis, MO, USA
Antisense oligonucleotide (ASO) drugs are synthetic polymers of RNA, DNA, or modified nucleic acids that bind to RNA or protein targets, regulating protein expression or function. Though approximately 20 ASOs have been approved worldwide, quality control and safety assessment pose significant challenges to regulatory agencies, with hepatotoxicity associated with many ASOs. Cultured human liver cells were used to assess potential hepatotoxicity of 3 marketed ASO therapeutics (mipomersen, inotersen, and nusinersen) and 18 of associated commonly-found ASO impurities, such as exclusion of 1 nucleotide (n-1). Pooled primary human hepatocytes (p-PHHs) were maintained in sandwich culture and treated for 3 days with ASOs at concentrations normalized to human peak blood concentrations (Cmax). Cellular ATP, albumin secretion, and urea production were measured to reflect toxicity. At 100-fold Cmax, mipomersen and inotersen, but not nusinersen, caused significant inhibition of albumin; however, none of the parent compounds showed noticeable effects on ATP and urea production. Most of the impurities tested caused remarkable inhibition of albumin, with half leading to decreases of >90%, and several also triggered >90% ATP inhibition. The p-PHHs appeared to be a useful in vitro model in ranking the relative hepatoxicity risk of ASO impurities to parent ASOs, wherein some impurities were comparable while others were remarkably more toxic than the parent drug themselves. These data provide novel insights into hepatotoxicity risks of ASO impurities, highlighting the importance of impurity quality control in ASO manufacturing and safety assessments.
208
Toxicological Risk Assessment and Exposure Limit Derivation for Human Transferrin as a Serum-Free Media Residual in Cell-Based Drug Products
Isaac Mohar 1 and Tom Lewandowski1
1Gradient, Seattle, WA, USA
The production of human cell-based drug products (e.g., hematopoietic stem cells) often involves the use of serum-free media. As a result, residual media components may be present in the final drug product. Transferrin is an 80-kDa glycoprotein that is essential for iron homeostasis and a common component of serum-free media. However, a toxicology-based acceptance limit for residual transferrin in drug products has not been established. The objectives of this assessment were to evaluate the safety of human transferrin as a potential residual in cell-based drug products and, if possible, propose a toxicology-based exposure limit (EL) for single administration drug products. The potential risks were evaluated based on the results of a comprehensive literature review of regulatory guidance, in vitro data, pharmacology data (six studies), and clinical and nonclinical toxicology data (two and six studies, respectively). The normal serum reference range of transferrin is 200 to 400 mg/dL in healthy adults, which was estimated to be 400 to 800 times greater than concentrations used in tissue culture applications. Nonclinical and clinical studies support no observed adverse effect levels of 300 mg/kg-bw/day for 60 days and 26 mg/kg-bw/dose for nine doses administered every other day, respectively. Based on the available data, three possible ELs of 0.83, 1.30, and 1.33 mg/kg-bw were derived. Of these, an EL of 1.33 mg/kg-bw, based on a 1% increase in the reference human transferrin blood concentration and supported by pharmacokinetic modeling, was proposed as a toxicology-based EL for human transferrin in cell-based drug products.
209
Using a Nonanimal Tiered-Testing Protocol to Predict GHS Categorization of Eye Irritants and Nonirritants
George DeGeorge 1, Kayla Costan1, Chris Kalimtzis1, Puneet Vij1, Timothy Shaak1, and Rachael Koch1
1MB Research Laboratories, Spinnerstown, PA, USA
Non-animal test guidelines for identifying ocular hazards using individual in vitro or ex vivo methods have limited accuracy (as low as 69.2%) in predicting GHS Ocular Category (1, 2, NC) irritants. To address this issue, we have optimized an integrated testing strategy (ITS) that combines the Eye Irritation Test (EIT) outlined in OECD TG 492 with the Bovine Corneal Opacity and Permeability (BCOP) Test described in OECD TG 437. By leveraging the complementary nature of the EIT and BCOP test, we can effectively differentiate between eye irritants and corrosives and accurately identify GHS Acute Eye Hazard Category 2 chemicals, which induce reversible eye irritation. Our approach involves utilizing the BCOP test to rule out GHS Category 1 and the EIT to exclude GHS No Category. Analyzing the results obtained from these tests enables the determination of the remaining designation, which is Category 2. In accordance with GHS guidelines, Category 2 is defaulted to Category 2A due to the inability to differentiate between Category 2A and 2B. Through blinded testing of our dataset including 51 chemicals, we achieved a 92% correct identification rate for Category 2A/B chemicals as Category 2. The novel EIT+BCOP-based ITS exhibits an overall accuracy of 90% in predicting transitions from Category 1 to 2 or from No Category to any other category. This represents a substantial improvement compared to the maximal accuracy range of 55.3% to 85.4% observed with any single test alone.
300s - Safety Evaluation Nonpharmaceuticals
301
Cell-Mediated Immunity Exacerbates Amorphous Silica Nanoparticles-Induced Hepatic Damage
Kazuma Higashisaka1 and Yuya Haga1
1Osaka University, Suita, Osaka, Japan
Nanotechnology has advanced rapidly, and many products containing nanoparticles are now essential for our daily lives. Thus, opportunities for the exposure to nanoparticles have been increasing with the expanding use of these materials. However, information regarding their adverse effects occurring due to acquired immune responses which is activated after continuous exposure to nanoparticles remains limited. Here, to assess the effects of amorphous silica nanoparticles on acquired immunity, we analyzed changes in acute hepatotoxicity after pretreatment with amorphous silica nanoparticles (50 nm in diameter; nSP50). BALB/c mice and C.B-17 SCID mice were intradermally pretreated with PBS or nSP50 once a week for a total of 4 times. After that, acute hepatotoxicity was induced by injecting an excessive amount of nSP50. Pretreatment with nSP50 biochemically and pathologically exacerbated nSP50-induced hepatic damage in immunocompetent mice, while pretreatment with nSP50 did not exacerbate hepatic damage in immunodeficient mice. Moreover, we clarified that CD8+ cell–mediated immune response played a crucial role in the exacerbation of the hepatic damage induced by multiple exposure of nSP50, and that the blockade of IFN-γ decreased plasma levels of ALT and AST, hepatotoxicity markers, levels in nSP50-pretreated mice. Collectively, we showed that multiple exposure of nSP50 exacerbate the hepatic damage initiated by a single dose and that this exacerbation is mediated through acquired immunity. Mechanistically, CD8+ T lymphocytes and IFN-γ play critical roles in this process. Our current findings suggest that nSP50-induced acquired immunity leads to exacerbation of hepatic damage through the activation of cytotoxic T lymphocytes.
302
Toxicological Evaluations of 2-Alkylcyclobutanones (2-ACBs) in the Liver and Thyroid: 4-Week Toxicity Study and Cytochrome P450 (CYP) Inhibition Assay in SD Rats
Min-Kyung Cho 1, Hee-Seon Park1, Kang-Hyun Han1, Young-Bum Kim1, and Min Seong Jang1
1Korea Institute of Toxicology, Yuseong-gu, Taejon-jikhalsi, Republic of Korea
2-Alkylcyclobutanones (2-ACBs) are known as chemical markers of irradiated lipid-containing foods. 2-ACBs are important because they are produced solely through irradiation and not subjected to any other food processing method. Despite the importance of evaluating the toxicity of 2-ACBs, the toxic potential of 2-ACBs has not been fully understood. In this study, we aimed to investigate the toxicity of 2-ACBs, 2-tDeCB, 2-tDCB and 2-dDCB, through a 4-week oral administration at doses of 0, 100, 300, and 1000 mg/kg in SD rats. The results of the 4-week toxicity study showed that the administration of 2-ACBs resulted in toxic effects on hematology, serum chemistry, organ weights, and histopathology in both male and female SD rats. Histopathological examinations revealed that oral administration to 2-tDeCB, 2-tDCB, and 2-dDCB induced hepatocellular hypertrophy in the liver at a dose of 1000 mg/kg, and follicular cell hypertrophy in the thyroid at a dose of >300 mg/kg. Furthermore, a cytochrome P450 inhibition assay was conducted on 2-ACBs, which demonstrated CYP inhibition effects by 2-dDCB and 2-tDCB. These findings suggest that 2-ACBs have the capacity to inhibit the specific CYP enzymes, thus potentially exacerbating toxicity. Overall, our comprehensive data reveals that the potential toxicity of 2-ACBs in liver and thyroid, highlighting the necessity for further study to understand the toxicological significance of 2-ACBs. Acknowledgement: This research was financially supported by the Korea Institute of Toxicology of the Republic of Korea (No. 1711195883 and 1711195885)
305
New Approaches for Acute Inhalation Studies Only Considering Animal Survival
Gustav Bruer 1, Paula Janssen2, Nils Krüger3, Klaus Weber4, Nils Warfving5, Jürgen Nolde6, Tobias Schuster3, Otto Creutzenberg2, Benno Wessely7, and Michael Stintz7
1Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Hannover, Niedersachsen, Germany
2Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Hannover, Niedersachsen, Germany
3Evonik Operations GmbH, Hanau-Wolfgang, Germany
4AnaPath Services GmbH, Oberbuchsiten, Switzerland
5AnaPath Services GmbH, Liestal, Switzerland
6Grace Europe Holding GmbH, Worms, Germany
7Technische Universität Dresden, Dresden, Germany
In case of lethality, the OECD technical guideline 436 only requires the count of dead animals and a rough macroscopical examination of the outer surfaces of the organs in the abdominal and thoracal cavity. Low-density particles, such as hydrophobic Synthetic Amorphous Silica (SAS) show no systemic toxicity, however they can cause lethality at concentrations in the range of 100-1000 mg/m³ in an acute inhalation test. Seven of these particle types were selected at concentrations in the range of 500 mg/m3 for acute inhalation studies, each conducted with three male and female rats for 4 hours. By adding additional parameters like an enlargement of the aerosol characteristics and a thorough histopathological examination of the respiratory tract, the expanded experimental design was used to assure the scientific validity of older results. For one of seven substances animals died spontaneously or were sacrificed due to moribund conditions during or shortly after cessation of exposure ahead of schedule. Prior to death, the animals showed signs of anemia and a decreased respiratory rate. The histopathology screening confirmed the cause of morbidity/death in all animals by asphyxia and mechanical blockage of the upper respiratory tract. Partial blockages were also shown for other of the 7 substances. This impaired respiration up to total airway obstruction, which can be misdiagnosed as a toxic effect, suggests that acute high dose inhalation studies are not suitable for certain substances.
306
Practical Application of the GARDskin Dose-Response Assay to Derive a No Expected Sensitization Induction Level (NESIL) Value for Confirmatory Human Patch Studies to Determine Safe Use Level for Novel Fragrance Ingredients
Tim Lindberg 1, Satoshi Sasaki2, Christopher Choi3, Andy Forreryd4, and Ulrika Mattson4
1Senzagen AB, Lund, Skane Lan, Sweden
2Takasago International Corp, Hiratsuka City, Japan
3Takasago International Corp, Rockleigh, NJ, USA
4Senzagen AB, Lund, Sweden
Skin sensitization is one of the required endpoints for the development and registration of novel fragrance ingredients. Traditionally, assessment has been performed combining in vitro and in vivo assays, but recent developments has shifted to the use of New Approach Methodologies (NAMs), without need for in vivo methods. However, none of the proposed NAMs are currently validated for continuous potency predictions, which is required for quantitative risk assessments of novel fragrance ingredients. The GARD®skin assay (OECD TG 442E) is a genomics-based assay for hazard identification of sensitizers. To meet the need for quantitative potency information, GARD®skin Dose-Response has been developed based on the validated protocols of GARD®skin and generates a dose-response curve to identify the lowest concentration of a test compound required to elicit a positive classification (cDV0-value). These values correlate significantly to LLNA EC3 and human NESIL values. This study presents the use of the GARDskin Dose-Response assay to determine safe human user levels for one novel fragrance ingredient. The experimentally predicted NESIL value was determined to 37800µg/cm2. Using a weight-of-evidence approach mainly guided by the quantitative data from the GARDskin Dose-Response, confirmatory Human Repeated Insult Patch Testing (HRIPT) studies were conducted and at the tested concentrations, no sensitization reactions were observed. In conclusion, this study expands the toxicologist´s toolbox and illustrates the potential to use the GARDskin Dose-Response assay to derive NESIL values that are protective of human health, without having to rely on the Dermal Sensitization Threshold (DST) approach or reverting to traditional animal testing approaches.
307
Toxicologic Evaluation of Oubli Fruit Sweet Protein: A Novel Sweetener Alternative
Mark R. Bauter 1, Barry Lynch2, Brittany Bamonte3, Tina Wang4, and Jason Ryder4
1Product Safety Labs, Dayton, NJ, USA
2Intertek, Mississauga, ON, Canada
3Product Safety Labs, Dayton, OH, USA
4Oobli Inc., Davis, CA, USA
Oubli fruit sweet protein (OFSP) contains a naturally sweet protein brazzein present in the fruit of the West African oubli fruit (Pentadiplandra brazzeana Baillon). Like other sweet proteins of similar origin, OFSP is 500-2000 times sweeter than commonly used sweeteners such as sucrose, and has been consumed by humans and other primates in its area of origin for a long time. Applications utilizing OFSP provide an alternative to low-calorie sweeteners and offers a new approach to sweetness for consumers actively working to reduce sugar intake to improve their health. GLP studies were performed to evaluate the safety profile, including the mutagenic, clastogenic, and toxicologic potential of the protein. In an investigation for its potential to induce gene mutations according to an in vitro plate incorporation and pre-incubation test (Ames) no concentration- or test article- related increases in revertant colony numbers in any of the five tester strains was observed. OFSP did not cause gene mutations by base pair or frameshift changes in the genome of the strains and was found to be non-mutagenic. In an in vitro micronucleus assay, OFSP did not induce structural and/or numerical chromosomal damage in human lymphocytes. In a 14-day and 90-Day feeding study targeting dietary intakes of 0 (standard chow), 250, 500, 1000 mg/kg/day in Sprague-Dawley male and female rats, there were no adverse, clinical, body weight, food consumption, functional observations, motor activity effects, as well as no clinical pathology, macroscopic, or microscopic changes attributable to test substance administration.
308 - North American Graduate Fellowship Recipient
Maternal Electronic Cigarette (E-Cig) Use Induces Oxidative Stress, Neuroinflammation, and Causes Mitochondrial Dysfunction in Neonatal Brain
Sabrina Rahman Archie 1, Ali Ehsan Sifat1, David Mara1, Yeseul Ahn1, Khondker Ayesha Akter1, Yong Zhang1, and Thomas Abbruscato1
1Texas Tech University Health Sciences Center, Amarillo, TX, USA
Despite the prevalence of the perception that electronic cigarettes (e-Cig) are a safe alternative to tobacco smoke, growing concern related to its potential toxic impact warrants investigation focusing on special populations like maternal and pediatric groups. In this study, we have evaluated the consequences of maternal e-Cig use on neonatal neuro-inflammation, oxidative stress, and mitochondrial function in primary neuron, postnatal day (PD) 7 and 90. Pregnant CD1 mice were exposed to e-Cig vapor (2.4% nicotine) or oxygenated air (control) from gestational day 5 (E5) till PD7, and primary neurons were isolated from pups at E16/17. Cellular total reactive oxygen species (ROS) and mitochondrial superoxide were measured in primary neurons using CM-H2DCFDA and Mitosox red, respectively. Mitochondrial function was assessed by Seahorse XF Cell Mitostress analysis. Level of pro-inflammatory cytokines was measured in primary neurons, PD7 and PD90 by RT-PCR and immunobead assay. Expression of anti-oxidative markers (SOD-2, HO-1, NRF2, NQO1) and proinflammatory modulator (NFκB) were evaluated by Western blot in primary neurons, PD7 and PD90. Significantly higher level of total ROS (P < 0.05) and mitochondrial superoxide (P < 0.01) was observed in prenatally e-cig exposed primary neurons. We also observed significantly reduced expression of anti-oxidative markers, increased expression of proinflammatory modulator and cytokines in primary neurons and postnatal brains (P < 0.05). Our findings suggest that prenatal e-Cig exposure induces postnatal neuroinflammation by increasing cytokines levels, oxidative stress and disrupting mitochondrial function which may alter fetal brain immune function that makes such offspring more vulnerable to brain insults.
309 - International Travel Grant Recipient
A 21-Day Toxicity Screening of o-Toluic Acid, a Versatile Organic Intermediate, in Wistar Rats
Chiagoziem A. Otuechere 1, Titilayo E. Ayoade1, and Adedotun F. Adesina1
1Redeemer's University, Akoda Ede, Osun, Nigeria
o -toluic acid (TOL), an isomer of p-toluic acid and m-toluic acid, is widely used industrially as a component of food spices, pesticides, animal feed supplements, and drugs. However, no comprehensive toxicity screening has been conducted in rats. This study was designed to evaluate the toxicity profile of TOL, especially in the heart and kidney. o -toluic acid was administered via gavage to male Wistar rats at the dose of 100 mg/kg (TOL 1) and 200 mg/kg (TOL 2) for 21 days. Plasma clinical indices, body and organ weights, cardio-renal redox status, and histopathology were assessed. The administration of TOL significantly (p< 0.05) increased the heart organ weights, albumin and sodium levels. However, TOL caused insignificant (p>0.05) changes in aspartate aminotransferase, gamma-glutamyl transferase, bilirubin, cholesterol, triglyceride, creatinine, and C - reactive protein levels relative to the controls. In the kidney, TOL significantly (p< 0.05) increased glutathione S-transferase and catalase activities but depleted superoxide dismutase and nitric oxide levels. In the heart, TOL significantly depleted catalase activity but had no effect on the nitric oxide, myeloperoxidase, superoxide dismutase, glutathione S-transferase and lipid peroxidation levels. Histopathological examination of the organs from TOL- treated rats showed minimal aberrations from the control group. It appears that TOL could be generally considered as safe, due to its minimal distortion of the sero-clinical and redox profiles of rats, especially at the investigated doses.
400s - Toxicology Methods
401
A Mechanistic Investigation of Electroretinogram (ERG) Suppression in the Rabbit: Role of an Excipient or a Species-Related Sensitivity?
William J. Brock 1, Andrew J. Kocab2, Jeffrey A. Jamison3, Marianna Bacellar-Galdino3, and David N. Zacks2
1Brock Scientific Consulting, LLC, Hilton Head Island, SC, USA
2ONL Therapeutics, Ann Arbor, MI, USA
3Experimentica, Fort Worth, TX, USA
Suppression of the dark-adapted ERG only was observed in the Dutch-belted (DB) rabbit but not minipigs (MP) following intravitreal (IVT) injection of a small peptide (MW < 5kDa) containing poloxamer 407 (PX407). ERG suppression of the b-wave with no effect on the a-wave is unusual, and mechanistic studies were carried out to investigate this finding. DB or New Zealand White (NZW) rabbits (n=2-3) were given bilateral injections (50 or 100 µL/eye) of vehicles used in nonclinical studies with ocular effects evaluated with optical coherence tomography (OCT), direct and indirect ophthalmoscopy (OE) and ERG examinations for up to 16 weeks. Findings were compared to IVT injection of saline or a PX407-free vehicle. Globes were collected for histopathology and immunohistochemical (IHC) staining. Following injection to DB or NZW rabbits, a vitreal haze (opacity) was observed with the vehicles but not saline and confirmed by OCT imaging. Approximate 50% reduction (100 µV) in b-wave response at 0.1 cd.s/m2 light stimulation was observed at 2-weeks post-injection. At 8 to 16 weeks, a ≥3-fold maximum b-wave suppression was observed at 1000 cd.s/m2 light stimulation. Slight b-wave suppression (< 1.2-fold) occurred at 8 and 16 weeks with PX407-free vehicle. No retinal degeneration was observed on OE or OCT examination or histopathology. Normal IHC rhodopsin staining of photoreceptor rods and IBA-1 normal staining of the optic nerves were observed. These studies suggest that the ERG suppression was likely due poloxamer 407 in the formulation. However, relative to MP, a rabbit-specific sensitivity to the ERG suppression cannot be ruled out.
402
A Population Pharmacokinetic Model with Tissue Compartments for Oxytetracycline in Sheep
Zhoumeng Lin 1 and Malek Hajjawi1
1University of Florida, Gainesville, FL, USA
The objective of this project is to build a population pharmacokinetic (PopPK) model for oxytetracycline in sheep by including tissue compartments and then apply it to predict withdrawal intervals (WDIs) in sheep after extralabel administration. Pharmacokinetic data for oxytetracycline in sheep were collected from the Food Animal Residue Avoidance Databank (FARAD) database. A structural PopPK model with one, two, or three compartments was evaluated with Monolix. Individual tissue compartments, including liver, kidney, and muscle, were incorporated into the model. WDIs were predicted using the model for representative dose regimens (20 and 40 mg/kg single intramuscular administration) using Monte Carlo simulations. The optimal model was a two-compartment model with three additional tissue compartments (liver, kidney, and muscle). The PopPK parameter estimates were as follows: ka_pop = 1.5 h-1, F_pop = 0.99, CL_pop = 5.65 L/h, Q2_pop = 1.2 L/h, Q3_pop = 2.14 L/h, Q4_pop = 19.45 L/h, Q5_pop = 0.23 L/h, V1_pop = 90 L, V2_pop = 60.78 L, V3_pop = 4.74 L, V4_pop = 29.78 L, and V5_pop = 1.6 L. The model-predicted WDI results for liver, kidney, and muscle were: 6, 2, and 11 days for the 20 mg/kg dose; and 8, 3, and 14 days for the 40 mg/kg dose. In summary, incorporating tissue compartments into a traditional PopPK model of oxytetracycline in sheep offers a useful tool to predict drug tissue residues and WDIs in food-producing animals. This plasma and tissue composite PopPK modeling approach can be applied to other drugs in other food animal species.
403
A Retrospective Analysis of Internal In Vitro Phototoxicity Data
Liwen Gao 1, Mengqi Zhang1, Xiaohui Zhu1, Ke Xu1, Ning Xu1, and Millie Chen1
1WuXi AppTec (Suzhou) Co., Ltd, Suzhou, Jiangsu, China (People's Republic)
The 3T3 Neutral Red Uptake Phototoxicity (NRU-PT) assay is an established in vitro assay used to evaluate the potential phototoxicity of a test article. Since recommended by EMA and ICH guidance, the 3T3 NRU-PT assay has been used quite extensively in the pharmaceutical industry. However, there are some limitations of this assay including high false positive rate and compound solubility issue. To better understand the assay, we retrospectively analyzed the data generated in our laboratory. Data were collated for 100 compounds from 2017 to 2022. Of these, 52 compounds produced a photo irritation factor (PIF) value < 2 (or mean photo effect, MPE value of < 0.1) and were considered negative (52%), 8 compounds produced a PIF value of 2 to 5 (or MPE value of 0.1 to 0.15) and were considered equivocal (8%), whilst 40 compounds produced a PIF value ≥5 (or MPE value ≥0.15) and were considered positive (40%). Thus, the overall positive/equivocal ratio in the 3T3 NRU-PT assay is approaching to 50%, which is consistent with the value reported in the literatures. Among these studies, only 28% of final calls were made based on PIF values, most conclusions (72%) were made based on the MPE values, since the IC50 values were not determined due to solubility issue (43%) or low toxic effect (29%). Based on these results, it appears that this assay is substantially oversensitive for predicting photosafety hazard (in contrast, high negative predictivity) and MPE is a good supplementary parameter if no IC50 values can be derived.
404
Acute Liver Toxicity Associated with Clinical Administration of the MAT2A Inhibitor AG-270 Can Be Predicted Preclinically and Is Chemotype-Specific
Claire L. Neilan 1, Paul A. Barsanti1, Melissa Fleury1, and Jay P. Jain1
1IDEAYA Biosciences, South San Francisco, CA, USA
Acute liver toxicity, manifested as elevations in AST, ALT, bilirubin and ALKP, occurred following administration of the MAT2A inhibitor AG-270 in a Phase I trial of patients harboring MTAP-deleted tumors (NCT# 03435250). This toxicity was not observed in earlier studies in preclinical species. The aim of the current study was to 1) elucidate the mechanism behind the liver toxicity, and 2) determine if the liver toxicity was AG-270 chemotype specific. To this end we interrogated structurally distinct MAT2A inhibitors, including AG-270 and IDEAYA clinical candidate IDE397 (NCT# 04794699), for liver toxicity potential in a suite of assays including an imaging assay in primary human hepatocytes and in various hepatic enzymes and transporters. In primary human hepatocytes, AG-270 and structurally related MAT2A inhibitors caused an increase in reactive oxygen species, increased nuclear size and intensity, and a slight decrease in mitochondrial membrane potential, which were not observed in IDE397 or other structurally related MAT2A inhibitors. AG-270 inhibited the transporters BSEP and MRP4; inhibition of MRP4 in addition to BSEP is reported to be a risk factor for cholestatic injury. This data suggests that AG-270-associated liver toxicity may be due to oxidative stress leading to increased membrane permeability and cell death, with potential contribution from inhibition of two key hepatic transporters. Moreover, this toxicity could be predicted using relevant in vitro assays utilizing human cells/proteins. Importantly, our findings suggest that AG-270-associated liver toxicity is chemotype-specific.
405
Applying ICH E14/S7B: The Cardiovascular Effects of Moxifloxacin in Conscious, Telemetered Cynomolgus Monkeys
Ty Speece 1, Noel Horton2, Jacob Rivers2, Phil Downing2, and Joel Baublits3
1Inotiv, Osage City, KS, USA
2Inotiv, Evansville, IN, USA
3Cardiovascular Analytics, Newbury Park, CA USA
The ICH E14/S7B regulatory guidance documents provide recommendations for developing drugs with low proarrhythmic potential, specifically to provide a framework for incorporating best practice approaches in nonclinical models with clinical data, as part of an integrated risk assessment. The purpose of this study was to investigate the effects of moxifloxacin (QT reference drug) and apply best practices recommendations to evaluate the sensitivity and validity of the model to be an effective preclinical predictor of QT prolongation in humans. Methods: Four telemetered cynomolgus monkeys were monitored for 48 h following administration of vehicle and 3 dose levels of moxifloxacin using a Latin square dose design. Pharmacokinetic profiles at the high dose were evaluated, at one timepoint on each PD day, and separately as a PK leg performed on the last treatment day. ER analysis was conducted to determine the relationship of moxifloxacin plasma concentration to QTc prolongation. Power analysis (measurement of study quality) was conducted. Results: Sufficient sensitivity to detect a clinically relevant increase in the QTc interval (statistical comparisons by timepoint) was demonstrated using an appropriate positive control and statistical power analysis, where minimum detectable difference and least significant difference (changes from the control group) were 11.4 ms (4.0%) and 8.0 ms (3.1%), respectively. The ER curve showed a positive slope (0.0048 ms/ng/ml) consistent with previously published nonclinical and clinical literature. Discussion: ER method is a useful translational tool that should be regarded as a valuable supplement to comparisons by timepoint.
406
Building Confidence to Wave a 2-Year Rodent Bioassay through an Expert-Driven Weight-of-Evidence Approach
Loannis Xanthis 1, Antonio A. Oliveira1, Susanne Stalford1, and Alex Cayley1
1Lhasa Limited, Leeds, England, UK
Within the usual drug development process, a battery of safety studies is conducted to profile the toxicological properties of the active ingredient, including an understanding of the human carcinogenic potential in most cases. The adoption of ICH S1B(R1) guidance allows for the use of a weight-of-evidence (WoE) approach to waive the 2-year rat carcinogenicity study, if it can be shown that it will add no additional value beyond the existing data. WoE assessments pose certain challenges, including how to organise the body of evidence and how to perform review in a systematic manner to produce interpretable and robust conclusions. An in silico system has been developed to support these assessments, which is centred around adverse outcome pathways (AOPs) to capture mechanistic data and contextualise assays. Rules have been established to reason between evidence and generate classifications for each of the six factors that need to be considered for S1 WoE assessments. Expert review can then be performed across the organised evidence-base and conclusions can be documented from the assay to the factor level. In addition to reviewing data for the query compound, relevant analogues can be examined allowing to build on the WoE where data gaps exist. This is facilitated by using existing data which has been populated in the system for over 17000 chemicals from over 50 relevant assays. Overall, in silico systems can facilitate complex WoE assessment, thus enabling multiple stakeholders to reach consensus and confidently waive unnecessary in vivo testing.
407
Characterization of a Model of Neurotoxicity by Histology
Sébastien Brèche, Sr.1, David Babin1, Elise Esneault1, Christelle Froger-Colléaux1, Guillaume Peyon1, and Florian Simon1
1Porsolt, Le Genest Saint Isle, Pays de la Loire, France
N-methyl-D-aspartate receptor (NMDAr) antagonists are effective therapeutics for treating CNS disorders such as pain, epilepsy, neurodegenerative disorders, drug-resistant depression and other psychiatric disorders. However, these substances are also known for inducing neurotoxicity, called ‘Olney’s lesions’ following an acute exposure. This raises the need to evaluate the safety profile of New Chemical Entities (NCE) with the same mechanism of action. The aim of this study was to evaluate the effects of a dose-range of MK-801 to develop a model of neurotoxicity. Female Sprague Dawley rats (3 per group) of 10 weeks old were administered subcutaneously with physiological saline or MK-801 at 2, 5 and 10 mg/kg. Following administration, the rats were observed twice daily for behavioral changes and body weight and rectal temperature were recorded daily. Seventy-two hours after administration, the rats were transcardially perfused with 4% paraformaldehyde (PFA) fixative before brain sampling. Seven transverse sections were cut to target the main brain areas. After paraffin-embedding, slices of 5 µm were stained using hematoxylin and eosin to determine morphologic changes. MK-801, dose-dependently, impaired the behavior and induced body weight loss. Neuronal vacuolation and necrosis, graded from 0 (absent or same as control) to 5 (prominent presence) were dose-dependently increased with MK-801 and identified mainly in cortical neurons. The presence of astrogliosis and microglial activation were also observed at high dose. This experiment highlights the value of evaluating neurotoxicity of a NCE at an early stage of development to ensure their use and safety.
408
Characterization of Novel Selective DAT Inhibitors Using Multivariate qEEG and Sleep/Wake Metrics
Monica R. Metea 1, Ernesto Palmero-Soler2, Kimberly M. Holter3, Amy H. Newman4, and Robert W. Gould3
1Preclinical Electrophysiology Consulting, LLC, Mattapoisett, MA, USA
2Preclinical Electrophysiology Consulting, LLC, Mattapoisett, MA, USA
3Wake Forest University School of Medicine, Winston Salem, NC, USA
4National Institute on Drug Abuse Intramural Research Program, National Institutes of Health, Baltimore, MD, USA
Deficits in dopamine neurotransmission have been shown to contribute to cognitive decline in aging and other CNS disorders. Elevating endogenous dopamine levels using dopamine transporter (DAT) inhibitors represents a promising therapeutic strategy. However, elevated extracellular dopamine concentrations via DAT inhibition is generally associated with addictive risk. Therefore, recent efforts have centered on compounds that differentially alter DAT conformation yet have limited misuse potential. Given the sensitivity of quantitative electroencephalography (qEEG) to drug-induced oscillatory and synaptic changes, rapid in vivo qEEG assessments may be advantageous to streamline drug discovery processes prior to lengthy addictive risk assessments. In this study, we examined the effects of two known (cocaine, modafinil) and two novel DAT inhibitors (Compounds JJC8-008 and JJC8-091) on brain function and sleep/wake duration using qEEG to identify predictive biomarkers related to abuse liability. Male and female Sprague-Dawley rats were implanted with wireless EEG devices and administered the above compounds. EEG spectral distributions were computed for each vigilance state (wake, REM, NREM) across the 24-hour recording. Statistical analyses were performed for each compound and a principal component analysis (PCA) -based algorithm was employed to reduce data dimensionality and identify components representing the maximum variance over the combined multivariate dataset. All compounds led to dose-related changes in wakefulness and qEEG metrics, while principal components enabled misuse potential class separation based on the multivariate dataset. In summary, we present a process using multivariate sleep/wake and qEEG metrics for rapid characterization in an attempt to distinguish DAT inhibitors with and without abuse risk.
409
Chasing Biomarkers of Seizure Activity and Neuropharmacology in Toxicology: Opportunities and Limitations
Simon Authier 1, Cristina Ballesteros1, Nehla Bennamoune1, Kim Bujold1, Christian Li1, and Mylene Pouliot1
1Charles River Laboratories, Laval, Quebec, Canada
Drug-induced seizures are a common concern in preclinical development given the life-threatening consequences. EEG traces were obtained in cynomolgus monkeys and Beagle dogs from general toxicology studies using telemetry with implanted electrodes from the 10-20 system (Cz-Oz, C3-O1 and C4-O2). The use of implanted telemetry devices and appropriate recovery (i.e., 21 days or more) did not significantly alter general toxicology endpoints including body weights, clinical signs, clinical pathology and anatomic pathology. Automated seizure detection tools identified spike trains but could not detect more complex biomarkers of increased susceptibility to seizure such as increased synchrony, isolated sharp waves and isolated spikes or isolated spike-and-waves patterns. Automated EEG analysis alone is insufficient to evaluate traces for biomarkers of seizure activity but can serve as a first line tool to identify areas of interest. EEG traces obtained in a Cz-Oz derivation presented the lowest artifact level which was optimal for automated seizure detection and this derivation was systematically used for the primary analysis including, ictal activity detection, qEEG and/or polysomnography. Tremors or myoclonus in normal animals were generally not associated with abnormal EEG activity. Based on video-EEG, the incidence of tremors in normal dogs and cynomolgus monkeys was 5.3% and 5.6%, respectively. The incidence of physiological myoclonus in normal healthy dogs and cynomolgus monkeys was 0.7% and 0.9%, respectively. Salivation was observed in 9.9% and 2.6% of normal dogs and cynomolgus monkeys, respectively. Overall, the data presented here in supports the inclusion of EEG monitoring using telemetry in dog and non-human primate toxicology studies.
410
Comparison of Cardiovascular and Respiratory Parameters in Three Strains of Research Pigs: Sinclair Nanopig™, Göttingen Minipig®, and Yucatan™ Miniature Swine
Miri K. Pannu 1, Guy F. Bouchard2, Derek F. Brocksmith2, Emily M. Griffith1, Sandra D. Love1, Seth J. Mueller1, and Rebecca M. Smith1
1Altasciences Preclinical Columbia, Auxvasse, MO, USA
2Sinclair Bio Resources, Auxvasse, MO, USA
Minipigs are growing in popularity for pre-clinical safety assessment studies, including safety pharmacology. The Sinclair Nanopig™ is a new, smaller version of the original Sinclair minipig. This strain was developed by selective breeding and diet management. Sexual maturity is reached at approximately 4.5 months (6 to 8 kg). The cardiovascular and respiratory parameters from Sinclair Nanopigs™ were compared to Göttingen® and Yucatan™ minipigs (total of 62 pigs). The average weight of all pigs was 19±6 kg (10 to 28 kg), with an average age of 6.5 mon. Minipigs were instrumented with DSI L11R implants placed preperitoneal, using the jugular vein for ECG and the femoral artery for BP. Ponemah® software was used for data capture and analysis. Average baseline values obtained from 13 Sinclair Nanopigs™ were SBP=121±9 mmHg, DBP=87±15 mmHg, HR=88±18 bpm, PR=100±43 ms, QRS=60±20 ms, QT=296±27 ms, QTcH=282±13 ms, BT=37.7±0.7 °C, respiratory rate=22±4 Rf, tidal volume=185±90 ml, and minute volume=4084±1854 ml/min (mean±SD). On average, values were within 11% of the average values obtained from Göttingen® and Yucatan™ minipigs except for blood pressure which was approximately 22% lower than the other strains. To confirm the Sinclair Nanopig™ sensitivity to a cardiovascular compound, dofetilide was administered to a group of five pigs. Dofetilide produced the expected prolongation of QTcH; 54 ms at 0.1 mg/kg and 71 ms at 0.3 mg/kg with an MDD=11.5 ms. In conclusion, the cardiovascular and respiratory parameters from the new Sinclair Nanopig™ were comparable to those of the Göttingen® and Yucatan™ minipigs.
411
Creation of a Swine Model of Oral Angioedema
Kyle R. Klepner 1, Megan Haney1, Steve Wargacki2, Rajesh Kainthan2, and Holly Ponichtera2
1Altasciences Preclinical Columbia, LLC, Columbia, MO, USA
2Aquestive Therapeutics, Warren, NJ, USA
Angioedema, a sudden swelling often caused by allergic reactions, commonly affects the mouth. This can be life threatening, especially if breathing is obstructed. Angioedema in the oral cavity may affect the absorption of medication. Though swelling has been induced in the skin of miniature swine (MS) using histamine injections, no large animal model of angioedema in the oral cavity exists. Therefore, an animal model was created to investigate the impact of facial angioedema on sublingual drug absorption. It was hypothesized that histamine would induce swelling and erythema in the lips and tongue. 6 male anesthetized Yucatan MS were injected in the inner lip and tongue with varying levels of histamine dihydrochloride (125 ug/ml – 10 mg/ml). Calipers were used to measure tissue swelling and Draize scores were performed to monitor each injection site. The 10 mg/ml animal displayed the desired reaction, and 4 additional animals were dosed at 10 mg/ml for reproducibility. All animals dosed at 10 mg/ml displayed lip and tongue swelling within the first 30 minutes (average change from baseline = 3.82 ml (lips) and 3.32 ml (tongue)). Lip thickness mildly increased over 90 minutes, whereas tongue swelling decreased. Draize scores showed increased erythema and edema starting at 10 minutes. Animals were given diphenhydramine during recovery to reduce swelling. In conclusion, histamine dihydrochloride is a suitable agent for inducing angioedema in the tongue and lips of MS. Additionally, MS are a valuable large animal model for assessing treatments of angioedema and the impact of angioedema on drug absorption.
412
Determining the Genotoxic Mode of Action of N-Nitrosamines Using DNA Repair-Deficient Cell Lines
Dan Roberts 1, Marit E. Hoogenboom-Geijer2, Dan Roberts3, Inger Brandsma2, Nynke Moelijker2, and Giel Hendriks2
1Toxys, Canandaigua, NY, USA
2Toxys, B.V., Oegstgeest, Netherlands
3Toxys, Inc., New York, NY, USA
N-Nitrosamines (NAs) are considered probable human carcinogens and were recently detected as impurities in pharmaceuticals leading to a concern for human health. NAs require metabolic activation and induce alkylating DNA damage that may be mutagenic. There are many, differently sized NAs and not all NAs are mutagenic. Understanding which NAs are genotoxic and their mode of action (MoA) will improve understanding of the mutagenic potential of these drug impurities as it relates to their structure. DNA Repair-Profiler can be used for MoA assessment of genotoxic substances and consists of a collection of mammalian DNA repair-deficient cell lines, that when used together, provide insight into the type of induced DNA lesions by identifying the impact of DNA repair on cell viability, genome stability, and mutation induction. As part of the EMA-funded MutaMind project, focused on building quantitative structure activity relationship (QSAR) models, we studied different types of NAs using DNA Repair-Profiler to predict carcinogenic potential for select NAs. NAs predominantly induce different forms of alkylating damage. Hence, we studied the relevance of base excision repair and nucleotide excision repair in removal of NA-induced DNA damage following exposure to 10 different small and bulky NAs. In each cell line, genotoxicity profiles and the DNA repair mechanisms that were important for the removal of the induced DNA damage were determined and incorporated into the QSAR models to relate structure to potential mutagenic and subsequent carcinogenic activity.
413
Development of a Direct Cisterna Magna Injection Method for Juvenile Rats Study
Raluca Emanuela Kubaszky 1, Cristina Ballesteros2, Francine Beaudry2, Simon Authier2, Christian Li2, and Said Maghezzi2
1Otsuka Pharmaceutical Companies, Blainville, Quebec, Canada
2Charles River Laboratories, Laval, PQ, Canada
Direct injection into the cisterna magna is a minimally invasive yet efficient method that provides direct access to the CSF for delivery of molecules at a controlled rate. To support the significance of this method in the pediatric population because of their differences compared to adults in respect of size, growth and maturation of organs and functions, sensitivity and responsiveness to intervention, this study was conducted to assess the impact of the procedure in juvenile animals. Herein, the safety, reproducibility and effectiveness of the method were determined in fully anesthetized postnatal Day (PND) 10 rats. Using a stereotaxic apparatus and a syringe with 31G (8mm) needle, 10µL aCSF was administered to the cervical intervertebral (Atlas-axis), with the accuracy of the position confirmed by presence of CSF during withdrawal prior to injection. The formulation was administered over 30 seconds and the needle was kept in place for an additional 30 seconds, then slowly exteriorized over 30 seconds. At 2 weeks postdose, the rats were euthanized. Based on the data obtained, our cisterna magna injection method produced no mortalities or clinical signs, and no effects on body weights or organ weights. Tissue sections of the spinal cord (cervical, thoracic, and lumbar), injection site, and dorsal root ganglia (DRG; lumbar region) did not show any significant macroscopic findings. In conclusion, direct injection to the cisterna magna was successfully implemented and is a feasible method for delivering test items in juvenile rats at PND 10.
414
Dog Telemetry Assay Sensitivity to Detect QTc Prolongation: Retrospective Statistical Power Analysis and Moxifloxacin Effects by Timepoint and Concentration-QTc Relationship Analysis
Sonia Goineau 1, Guillaume Froget1, Sébastien Brèche1, Fréderic Tantot1, and Philippe Guillaume1
1Porsolt, Le Genest Saint Isle, Pays de la Loire, France
The ICH Guideline E14/S7B Q&A gives information on the Best Practices to be followed to demonstrate the sensitivity of the nonclinical in vivo QT assay, with the aim of reducing the number of clinical studies. In order to determine the sensitivity of the assay, the Minimum Detectable Difference (MDD) was estimated for QTcR (individual regression correction) by conducting a retrospective statistical power analysis, using historical data from telemetry studies with the same design (either 4 or 6 instrumented dogs, 24-hour recording, randomized design). The effects of moxifloxacin (10, 30 and 100 mg/kg) were evaluated by a time-point analysis using the same randomized study design (4 dogs). The same animals received moxifloxacin again in additional sessions, to establish a full pharmacokinetic profile. QTc and exposure data were combined to conduct a concentration-QTc relationship analysis. The MDD for QTcR has been shown to be 6.5 ms [4-9.6] (16 studies, 4 dogs) and 4.7 ms [3.7-5.7] (6 studies, 6 dogs), with 80% power and a 5% confidence level. Using the timepoint analysis, moxifloxacin lengthened ΔΔQTcR by +8, +27 and +52 ms at 10, 30 and 100 mg/kg, respectively. Using the concentration-QTcR analysis, the predicted ΔΔQtcR increase at respective Cmax values was 6.8, 25.6 and 38.7 ms. The predictive exposure that resulted in 5 or 10 ms ΔΔQTcR increase (1141 or 2398 ng/mL free moxifloxacin) were approximately 2.5-fold larger than human thorough QT data study. These data therefore confirm the sensitivity and validity of experimental conditions used in the dog assay.
416
Generation and Characterization of a FcγR-Humanized HFCGR2–3 Mice Line
Cristina Bertinetti-Lapatki 1, Kerstin Hahn1, Martina Stirn1, Felix Weber1, Elisabeth Husar1, Michael Gertz1, Francois Christen1, Megana Prasad1, Roland Schmucki1, and Antonio Iglesias1
1Hoffmann - La Roche, Basel, Basel-Landschaft, Switzerland
Pre-clinical in vivo studies constitute a valuable tool to better understand the exposure-response relationships governing therapeutic antibodies’ efficacy and toxicity, contributing to increase the confidence in the translational relevance to humans. However, the effector functions of therapeutic antibodies are mediated by the interaction of their Fc domains with FcγRs. And although animal models can be informative, the FcγRs interspecies differences in the structural diversity and cellular expression patterns limit their usage for the assessment and prediction of the efficacy and safety consequences of engaging these receptors in humans. Here we describe the characterization of a humanized mouse model generated by the replacement of the murine FcgR2b with the huFCGR2B gene in the genome of our previously engineered C57BL/6 mice, already expressing human FcγRIIIa, FcγRIIIb, FcγRIIa and FcγRIIc. Given the major role played by liver sinusoidal endothelial cells (LSEC) in mediating the clearance of circulating small antigen-antibody complexes (IC) via the uptake of ICs by FcγRIIb, a dedicated characterization of the expression pattern of this receptor in the liver was conducted. IHC and single nucleus sequencing revealed high expression of huFCGR2B in LSECS. Platelets and mononuclear cells analyzed by FACS showed that the FcγRs in the transgenic mouse mirror the expression and cellular distribution pattern of human immune cells. In particular, this model can be used to support preclinical PK profiling and safety risk assessment of antibodies designed to clear soluble targets by selectively increasing FcγRIIb binding while reducing FcγRIIa/IIIa binding to prevent platelet activation, ADCC and first infusion reactions.
417
Heart Rate Corrected JTp and TpTe as Proarrhythmia Biomarkers in Safety Pharmacology Nonhuman Primate Studies: Outcome from a HESI Consortium Prospective Study
Brian Roche 1, Andrea Greiter-Wilke2, Yevgeniya Koshman3, Derek Leishman4, Jill Nichols5, Theresa Bartko5, Eric I. Rossman6, Jean-Pierre Valentin7, Jose Vicente8, and Todd Wisialowski9
1Charles River Laboratories, Kansas City, KS, USA
2Roche Pharma Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center, Basel, Switzerland
3AbbVie, Chicago, IL, USA
4Lilly Corporate Center, Eli Lilly & Co, Indianapolis, IN, USA
5Labcorp, Madison, WI, USA
6GlaxoSmithKline, Collegeville, PA, USA
7UCB, Brussels, Belgium
8Food and Drug Administration, Silver Spring, MD, USA
9Pfizer, Inc., Groton, CT, USA
QTc is an established biomarker for drug-induced Torsade de Pointes (TdP) but with concerns for a false positive signal. Clinically, JTpc and TpTec have emerged as ECG sub-intervals to differentiate predominant hERG vs. mixed ion channel blocking drugs that prolong QTc. Drugs that prolong QTc with increased TpTe, but without JTpc effects, are likely mixed ion channel blockers with low TdP risk. Drug effects on QTc, JTpc and TpTec were characterized with cynomolgus monkeys using telemetry in a Lead II configuration. Drugs and vehicle were administered orally to group sizes of 4 to 8 animals, in 4 laboratories. In monkeys, dofetilide (0.03-0.3 mg/kg, PO) was associated with exposure dependent QTc and JTpc increases but no significant TpTec effect. Quinidine (2 to 50 mg/kg, PO) increased QTc and JTpc but did not change TpTec. Mexiletine (1-15 mg/kg, PO) and verapamil (50 mg/kg, PO) did not induce any significant effect on QTc, JTpc or TpTec. Clinically, predominant hERG blockers (dofetilide and quinidine) prolong QTc, JTpc and TpTec and are associated with an increased risk for TdP. Results from the current study demonstrate that ECG changes after dofetilide and quinidine administration to telemetered monkeys differ from the clinical response, lacking the expected effects on TpTec. Potential explanations for the lack of translation include physio-pharmacology species differences or ECG recording and analysis methodology variations. Mixed ion channel blockers verapamil and mexiletine administered to monkeys showed no significant QTc, JTpc or TpTec prolongation as expected based on the similar clinical response for these agents.
418
Historical Review of In-Life Data from Preclinical Studies Utilizing AAVs for Gene Therapy
Julie Forget 1, Kelsey Brooks1, and Norbert Makori1
1Altasciences, Seattle, WA, USA
The field of gene therapy has made considerable progress over the past several years, specifically regarding Adeno-associated virus (AAV) vectors which have emerged as promising tools for in vivo gene therapy. Prior to clinical trials, safety assessment studies of AAV vector-based therapeutics often utilize nonhuman primates (NHP) as the primary test species, which requires prescreening for naturally occurring neutralizing antibodies (nAb) against AAV prior to dosing. Additionally, pre-treatment with corticosteroids prior to AAV administration is often required to mitigate adverse immune response. Given the unique challenges of executing successful preclinical AAV studies, we reviewed data from >20 studies conducted in the last 3 years to identify (1) the minimal number of NHPs needed for nAb prescreening; (2) successful corticosteroid premedication regimes; (3) common in-life findings. Only 38% (430/1119) of NHPs screened for nAb against AAV8 had low or negative viral titers indicating suitability for use on study, although nAb against other AAVs serotypes were less common (∼80% suitable). Pretreatment with 2 mg/kg of dexamethasone approximately 1-2 hours prior to AAV administration was adequate to mediate adverse immune-related responses. Increases in complement fragment C3a concentrations were most often noted 6h postdose on Day 1, at an average of 4.3-fold over baseline. No AAV related effects were noted on body weights, and most abnormal clinical observations post AAV administration were not significantly difference than concurrent controls. In conclusion, this data review can serve as a guide to inform future AAV study designs and identified areas for reduction and refinement of animal use.
419
Human 3D Hepatic Spheroids: A Micro-Physiological System to Study Cholestatic Drug-Induced Liver Injury
Armin Wolf 1, Maria Vittoria Colombo1, Friederike Wenz1, Lola Fäs1, Wolfgang Moritz1, Jonathan P. Jackson2, and Bruno Filippi1
1InSphero, Schlieren, Zurich, Switzerland
2Pfizer, Groton, CT, USA
Bile acids (BAs) have a significant impact on cellular metabolism but can be cytotoxic when their balance is disrupted. Certain drugs interfering with BA homeostasis can cause cholestasis. Evaluating the cholestatic potential of drug candidates is crucial during pre-clinical development. Hepatic spheroids, consisting of primary hepatocytes and non-parenchymal cells, with essential liver features (canaliculi structures, BA synthesis and secretion), were utilized as a screening-tool for this purpose. Spheroids were co-treated with 17 FDA-approved compounds known to induce or not induce cholestasis and increasing concentrations of a physiological BA mixture. To evaluate cytotoxicity, LDH release and cellular ATP content were measured. The presence of BA did not affect LDH levels, but slightly decreased ATP at the highest BA concentration. Increased cytotoxicity of compound during co-treatment with BA was considered indicative of cholestatic potential. The results were compared to clinical observations of cholestasis to define the specificity and sensitivity of the assay. Co-treatment with 100µM BA resulted in 43% sensitivity and 70% specificity for ATP, whilst LDH displayed 43% and 100%, respectively. Co-treatment with 200µM BA increased specificity for ATP and LDH (57% and 71%, respectively), but decreased specificity in both assays (50% and 90%, respectively). Co-treatment with 300µM BA yielded 100% sensitivity and 10% specificity for ATP assay, whilst LDH presented 100% sensitivity and 50% specificity. Overall, LDH readout demonstrated higher sensitivity than ATP in detecting BA-driven sensitization of cholestatic compounds. These findings highlight the relevance of hepatic spheroids as a micro-physiological system for assessing cholestatic potential of new drug candidates.
420
Imaging-Based Detection of Acute Systemic Tissue Injury by the Black Box Drug Celecoxib Reveals Age-Based Differences in Susceptibility to Drug-Induced Tissue Damage
Steven E. Johnson 1, Justin Jeffery2, Andrew Mazar3, and Ming Zhao1
1Northwestern University/Durametrix LLC, Chicago, IL, USA
2University of Wisconsin, Madison, WI, USA
3Durametrix LLC, formerly affiliated with Northwestern University, Milwaukee, WI, USA
Objective: In drug development, late-stage and even approved pharmaceutical candidates can still carry risk of adverse effects due to unforeseen drug-induced tissue damage. The ability to assess such systemic tissue injury early will have a significant impact on pharmaceutical development and drug safety. The goal of this study was to investigate the feasibility of imaging with 99mTc-duramycin, which detects membrane reorganization as a surrogate marker for apoptosis/necrosis, for assessing systemic tissue injury using celecoxib as a canonical black box model. Method: Four groups of Sprague Dawley rats were enrolled, including young nontreated control (8-10wk), nontreated older control (>30wk), treated young rats (8-10wk, celecoxib 200 mg/kg, P.O.), and treated older rats (>30wk, celecoxib 200 mg/kg, P.O.) (n=8). In vivo SPECT imaging was performed 72h post-treatment with 99mTc-duramycin. Radioactivity uptake in tissues was compared, using statistical analyses, between control and treated groups to determine tissue susceptibility to drug toxicity. Results: Significantly elevated 99mTc-duramycin uptake was detected in multiple organs/tissues in treated animals consistent with known toxicities. These changes were more apparent and diverse in older over younger subjects. SPECT studies indicated changes in 99mTc-duramycin uptake due to drug toxicity were detectable and quantifiable in an age-dependent manner. Histopathology is in progress to validate these findings. Conclusion: This imaging technology provides an opportunity for studying differences in drug-induced tissue damage in a dynamic, whole-body, minimally invasive, and age-dependent manner and has potential to preemptively identify safety issues early and to generate a real impact on pharmaceutical development, drug discovery, and drug safety.
421
Intraparenchymal Dosing in Beagle Dogs using Convection Enhanced Delivery Guided by Real-Time MRI
Steve Bulera 1, Said Maghezzi2, Morgan Forest2, Nehla Bennamoune2, Clotilde Lecrux2, Kim Bujold2, Archival Santos Galicia2, Christian Li2, Chiara Orciani2, and Simon Authier2
1Charles River Laboratories, Reno, NV, USA
2Charles River Laboratories, Laval, Quebec, Canada
Convection-enhanced delivery (CED) is used in patients for intraparenchymal brain dosing, spanning a range of neurological indications. Non-clinical toxicology is required to closely mirror clinic practices. To achieve CED, neurosurgical procedures incorporate real-time MRI to target and quantify local delivery to desired brain regions by employing a step-infusion paradigm coupled with a contrast agent (i.e., gadolinium). This procedure was established in non-human primates (NHPs) and novel neurological therapeutics were successfully delivered to brain regions selected based on therapeutic indication. A Beagle dog model was developed as an additional non-rodent non-clinical alternative for toxicology and biodistribution studies. In dogs, up to six different parenchymal sites per animal have been dosed at rates of 120 to 300 µL per hour with dose volumes ranging between 30 to 50 µL per site. Real-time MRI was used to accurately target relevant brain regions while also monitoring dose distribution over time. As observed in NHPs, Beagle dogs recovered well from the neurosurgery and presented with minimal procedure-related clinical signs including mild tremors, cutaneous changes, and decreased activity which were expected after surgery and resolved within approximately 4-days after the procedure. From an ethical standpoint, Beagle dogs represent an advantageous alternative to non-human primates. This work demonstrates an additional non-clinical model for CED using real-time MRI, with a workflow that utilizes clinically relevant methods and devices, ultimately addressing the need for alternative non-rodent non-clinical models for assessing toxicology of neurological therapeutics.
422
Investigating the Effect of Positive Chronotropic Compounds on Cardiac Troponin I Release from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
Jomana Hatahet 1, Jessica Treadway2, Yevgeniya E. Koshman2, Amanda S. Wildey2, Brandan M. Bird2, Mark T. Zafiratos2, Aimee L. Bielinski2, and Michael C. Foley2
1AbbVie, Lake Bluff, IL, USA
2AbbVie, North Chicago, IL, USA
Cardiac troponin I (cTnI) is a key regulatory protein of cardiomyocytes contractile machinery. The release of cTnI from myocytes has been attributed to necrotic injury. However, it has been recently shown that cTnI can be released into the bloodstream after physical exercise in the apparent absence of myocardial damage. This indicates that cTnI release might not always be an indicator of myocardial necrosis. Currently, troponin release in pre-clinical safety studies is often seen as a show-stopping red flag for investigational compounds. Therefore, it is important to differentiate between benign and necrotic troponin release in the field of safety pharmacology to better distinguish between cardiotoxic and nontoxic compounds. In this study, we investigated whether the increase in heart rate in telemetry instrumented conscious dogs as well as an increase in beat rate in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) would lead to elevated levels of cTnI. In conscious dogs, an increase in heart rate was observed following atropine treatment. However, cTnI levels measured in the plasma were not elevated. Treatment of healthy hiPSC-CMs with positive chronotropic compounds caused significant increases in beat rate that was sustained over 72 hours. In addition, cTnI levels in the media were significantly increased. However, this increase was not accompanied with changes in cell viability. Our results suggest that increases in beat rate caused a benign release of cTnI in the absence of myocyte necrosis. Our future studies will focus on investigating the difference in mechanisms of cTnI release between benign and cardiotoxic effects.
423
MCL1 Inhibitor-Mediated hiPSC-CM Cell Death and Troponin I Release Is Transiently Delayed by β-Agonist-Mediated Phosphorylation of Troponin I (Ser23/24)
Jessica L. Treadway 1 and Jomana Hatahet1
1AbbVie Inc., North Chicago, IL, USA
Cardiac Troponin I release has long been considered a key clinical indicator of myocardial necrosis and damage. In vitro, Troponin I is released into the media in response to cardiotoxic compound-induced cell death of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Myeloid cell leukemia (MCL1) promotes cell survival by inhibiting apoptotic cell death. As such, MCL1 inhibitors are currently being developed by several pharmaceutical companies for hematological cancers such as AML and non-Hodgkin lymphoma. However, we demonstrate here that small molecule MCL1 inhibitors caused Troponin I release and cell death in a concentration and Caspase-dependent manner in hiPSC-CMs, even after as little as 1 hour of treatment. In this study, we found that MCL1 inhibitor-mediated Troponin release and cell death was abrogated when incubated in combination with the β-agonist, Isoproterenol, despite an increase in beat rate. Isoproterenol-mediated PKA activation induces Troponin I phosphorylation at Ser-23/24, thereby blocking Calpain-mediated Troponin cleavage. In addition to inhibiting Troponin I cleavage, this phosphorylation event also temporarily promotes cell survival, delays caspase activation, and reduces Troponin I release. Furthermore, we show that inhibition of Caspase 3/7 not only prevents cell death, but also prevents Troponin I cleavage, suggesting the two events are interdependent.
424
Nonclinical Safety Assessment of Vectorized ADAR-Mediated Programmable RNA Editing
Thomas J. Long 1, Aditya Radhakrishnan1, Paul Meyers1, Andrew C. Dietz1, and Ron Hause1
1Shape Therapeutics, Seattle, WA, USA
RNA-guided Adenosine Deaminase Acting on RNA (ADAR)-mediated A-to-I editing has emerged as a powerful tool to correct disease-causing mutations and modulate gene expression and protein function. Compared with DNA editing techniques, RNA editing offers unique advantages in certain therapeutic contexts, particularly due to the lack of permanent off-target modifications. The RNAfix® platform uses AAV vectors to stably deliver guide RNAs (gRNA) designed to recruit ADAR and facilitate targeted mRNA base editing. By engineering gRNA sequence and structure, exquisite selectivity for the intended mRNA target can be achieved, maximizing pharmacological activity while minimizing off-target editing. Here, we present robust methods to profile the specificity of candidate gRNAs across therapeutically-relevant cellular model systems. Deep RNA sequencing allows us to characterize alterations across the cellular transcriptome and quantify the local and global editing signature of a gRNA. We have developed a sensitive and reproducible computational pipeline for identifying A-to-I editing events from RNA-seq data. Our analytical pipeline has been designed to minimize spurious off-target calls resulting from natural variation in ADAR editing across biological replicates. Notably, we rarely observe distal off-target editing sites, with editing primarily localized to the on-target adenosine and its immediate vicinity. This underscores the tailored activity of ADAR at the intended edit site facilitated by the gRNA. Global gRNA editing signatures identified through these methods can be used to assess off-target risk of therapeutic candidates.
425
Nonclinical Safety of Long-Acting Anti-SARS-CoV-2 Monoclonal Antibodies to Support Authorization
Patricia C. Ryan 1, David Henry2, Adam Hey2, Matthew Jacobsen2, Maryam Rafie-Kolpin3, Bodrul Noor2, Mats Någård1, David W. Boulton1, and Taylor Cohen1
1AstraZeneca, Gaithersburg, MD, USA
2AstraZeneca, Cambridge, England, United Kingdom
3AstraZeneca, Waltham, MA, USA
AZD7442, comprised of tixagevimab and cilgavimab, and AZD5156, comprised of cilgavimab and a novel monoclonal antibody (mAb) AZD3152, bind to distinct non-overlapping epitopes on the SARS-CoV-2 spike protein to neutralize viral binding and cellular fusion. These mAbs are being developed for prevention and treatment of COVID-19. The Fc region of each of these mAbs has been engineered to extend the half-life and to reduce effector function, and the cynomolgus monkey was selected for toxicology studies based on the similar binding affinity to cynomolgus monkey FcRn compared to human FcRn. The regulatory toxicology packages to support development and authorization included GLP toxicology studies in NHP and tissue cross-reactivity studies (TCR). A single IV dose of AZD7442 at 600 mg/kg or a single IM injection of 150 mg/kg was well tolerated in cynomolgus monkeys with no adverse findings following a 2-week or an 8-week observation period (NOAEL 600 mg/kg IV; 150 mg/kg IM). Similarly, AZD5156 was well tolerated when administered once weekly for 3 weeks by IM injection or IV infusion at 300 mg/kg followed by an 8-week treatment-free period, with no adverse findings observed (NOAEL 300 mg/kg IM or IV). No binding to any tissues was observed in the TCR studies against panels of human tissues. Toxicokinetics confirmed similar systemic exposure following IM and IV dosing with bioavailability of approximately 100% and average half-life ranging from 17 to 21 days. The results of these nonclinical studies supported the clinical development and authorization of both the individual mAbs and these mAb combinations.
426
Nonclinical Safety of Tofersen, an Antisense Oligonucleotide for the Treatment of SOD1-ALS
Christopher Hurst 1, Martin Lamb2, Tom Magee2, Tom Zanardi3, Scott P. Henry3, and Jeff Engelhardt3
1Biogen, West Roxbury, MA, USA
2Biogen, Cambridge, MA, USA
3Ionis, Carlsbad, CA, USA
ALS is a neurodegenerative disease that causes loss of upper and lower motor neurons within the cortex, brainstem, and spinal cord. It has historically been classified as familial or sporadic with SOD1 ALS representing ∼2% of the ALS population. Data suggest that toxic gain of function of mutant SOD1 protein is the trigger that initiates the cascade of events resulting in motor neuron death. Tofersen is an intrathecally (IT) administered antisense oligonucleotide designed to bind and degrade SOD1 mRNA to reduce synthesis of SOD1 protein. Toxicology studies were conducted in mice, rats, rabbits, and monkeys. In a single IT dose rat study, decreases in arousal, gait, mobility, respiration, and sensorimotor observations were observed. In mice, SC administration of tofersen was well tolerated after administration for 26 weeks, with 150 mg/kg as the NOAEL. In a 9 month IT monkey study, adverse clinical observations were noted in 1 female monkey administered 35 mg. After the second dose, the female exhibited transient muscle cramping, prolonged recovery from anesthesia, and intermittent tremors. An EEG analysis confirmed this was not a seizure, and there were no correlates in clinical and anatomic pathology. The NOAEL was 12 mg. In vitro and in vivo studies indicated no evidence of genotoxicity. There were no effects on mating and fertility, embryofetal development, or perinatal and postnatal reproduction in mice or rabbits. Results of the nonclinical safety program for tofersen support the clinical program and provide appropriate safety data for the use of tofersen in the treatment of SOD1-ALS.
427
Nonclinical Safety Profile of an Antibody-Oligonucleotide Conjugate, AOC 1001, for the Treatment of Myotonic Dystrophy Type 1
Laura Leung 1, Aaron Anderson1, Eileen Blasi1, Rob Burke1, Philip Kovach1, Arthur A. Levin1, and Husam S. Younis1
1Avidity Biosciences, Inc., San Diego, CA, USA
Antibody oligonucleotide conjugates (AOCs) combine the tissue specificity of monoclonal antibodies with the precision and potency of oligonucleotides to enable the targeted delivery of oligonucleotides to previously untreatable tissues and cell types. AOC 1001 is comprised of a siRNA conjugated to an antibody targeting human transferrin receptor 1 (TfR1), designed for functional delivery to muscle cells, where it can reduce the levels of myotonic dystrophy protein kinase (DMPK) mRNA implicated in myotonic dystrophy type 1 (DM1) pathogenesis. DM1 is a rare dominantly inherited progressive neuromuscular disease caused by toxic gain-of-function mutation in the DMPK gene. The safety profile of AOC 1001 or its components (antibody or siRNA) were evaluated in cynomolgus monkeys for up to 9 months of repeat dosing (IV, Q6W). AOC 1001 was pharmacologically active in monkeys producing DMPK mRNA reduction (> 80%) in multiple muscle tissues, including the heart. There were no adverse AOC 1001-related effects based on all examined endpoints (clinical pathology, pathology, safety pharmacology). The no-observed-adverse-effect level (NOAEL) was the highest dose tested of 537 mg/kg (45 mg/kg siRNA). Non-adverse minimal to mild findings include hematology changes consistent with the transient modulation of TfR1. Histopathology findings were limited to basophilic granularity in the liver and vacuolation in the mesenteric lymph node and are known effects due to siRNA oligonucleotide accumulation in the endolysosome. Importantly, no safety concerns were associated with DMPK mRNA reduction including on cardiovascular parameters. This study enabled the selection of safe starting doses to test AOC 1001 in individuals with DM1.
428
Phenolsulfonephthalein, a Phenol Red Herring?
Douglas J. Ball 1 and William P. Beierschmitt1
1D&B ChemTox, LLC, Lakewood Ranch, FL, USA
Phenolsulfonephthalein (CAS# 143-74-8), also known as phenol red, is a widely used pH color indicator used in cell biology media. Its color exhibits a gradual transition from yellow (λmax = 443 nm) to red (λmax = 570 nm) over a pH range of 6.8 to 8.2. A small amount of phenol red added to growth media (∼15 mg/mL) will have a pink-red color under normal conditions, and as cells eliminate waste and/or die (increasing the acidity of the media) the color will change to orange and then to yellow. Albeit out of scope for a biological drug substance, phenol red was identified as a process-related impurity at levels greater than the ICH Q3A qualification limit; therefore, a risk assessment was conducted to determine whether exposure to trace levels of phenol red represents any patient risk during clinical development. One concern was if the carcinogenic risk associated with a structural analog, phenolphthalein, could be the same for phenol red. As there was limited toxicological data available for phenol red, surrogate and read-across data were used to conduct a thorough risk assessment. Based on the available data, it was determined that the presence of phenol red as a specified drug substance impurity poses negligible patient risk in planned and ongoing clinical trials.
429
Preclinical Development of a Novel Gene Therapy for the Treatment of Peripheral Neuropathic Pain through Zinc Finger Repressor-Mediated Repression of the SCN9A Gene
Toufan Parman 1, Mohammad Samie2, Yonghua Pan1, Brian Jones2, Khaled Hettini1, Andres G. Villegas1, Kenneth Kennard2, David Clark3, Rebekah I. Keesler3, and Kathleen Meyer2
1Sangamo Therapeutics, Brisbane, CA, USA
2Sangamo Therapeutics, Inc., Richmond, CA, USA
3Charles River Laboratories, Reno, NV, USA
Peripheral neuropathy (PN) is a debilitating and painful condition affecting millions with a significant unmet need due to the ineffectiveness of present therapies. Loss of function mutations in human Nav1.7 voltage gated sodium channel, encoded by the SCN9A gene, have been shown to cause insensitivity to pain. We have developed a novel therapeutic for treatment of PN by epigenetically repressing the SCN9A gene using highly specific engineered zinc finger repressors (ZF-Rs) targeting mouse (Scn9a) or human/nonhuman primate (NHP) SCN9A genes in nociceptive neurons within the dorsal root ganglia (DRG). Mouse and human/NHP ZF-Rs were tested for efficacy in the gold-standard Spared Nerve Injury (SNI) mouse model of chronic PN, and safety in a 1-month dose range-finding (DRF) toxicology study in NHPs, respectively. ZF-Rs were administered intrathecally using the clinical recombinant adeno-associated viral vector (AAV) as the delivery system. In the SNI model, ZF-Rs led to significant repression of the Scn9a gene in DRG neurons at bulk and single cell levels, restored mechanical- and cold-induced pain responses to normal levels, and were well-tolerated (no neuroinflammation or neuronal loss). In the DRF study, ZF-Rs resulted in a well-tolerated 40-60% reduction of SCN9A gene expression in the DRG neurons with no dose-limiting toxicity. Anti-AAV neutralizing antibodies were detected in serum at all doses, and cerebrospinal fluid primarily at high doses, neither of which impacted pharmacology or safety parameters. Taken together our results describe successful design and selection of a novel AAV-ZF-R therapeutic for potential treatment of PN, and support advancement to IND-enabling studies.
430
Preclinical Electrophysiologic Nerve Conduction Assessments of Peripheral Neuropathies (PN) Secondary to Administration of AAV-Based Gene Therapies: Focus On Early Detection and Severity Ranking of Sensory PN in Primates
Mark Jahnke 1, Monica R. Metea1, and Ernesto P. Solar1
1Preclinical Electrophysiology Consulting, Mattapoisett, MA, USA
Nerve conduction tests assessing the peripheral neuropathy (PN) risk of novel therapeutic compounds are frequently included in preclinical drug development programs of small molecule drugs or adeno-associated virus (AAV)-based gene therapy (GT) products. However, limited regulatory guidelines are in place for assessing the presence and for ranking the severity of preclinical PN findings. Here we present criteria for designing sensory-focused serial nerve conduction studies in primates including selection of nerves and recording timepoints, as well as a framework for interpretation of changes in nerve action potential (AP) metrics using combined ‘within subject’, group statistics, and normative database comparisons. A rigorous and clinically translatable approach to ranking the severity of PN findings (including alignment with histological and behavioral endpoints) is presented, with examples from several long term (6 to 50 months) case studies where minor to severe PN axonopathy were present in sensory nerves from multiple DRG levels secondary to AAV-GT-therapy. The case studies presented here highlight the value of a data-driven systematic approach to assessing preclinical PN risk based on electrophysiological assays with established sensitivity and known interuser variability, and support the efforts towards standardization of these assays.
431
Preclinical Safety Assessment for Specificity of Biotherapeutics Using the Membrane Proteome Array
Diana Norden 1, Rachel Fong1, Jonathan Sullivan1, Carmen Navia1, Rona Wilf1, and Benjamin Doranz1
1Integral Molecular, Philadelphia, PA, USA
Investigational new drug (IND) applications for biotherapeutics require detailed cross-reactivity assessment for safety profiling to uncover potential off-target effects. Specificity is especially vital for cytotoxic biotherapeutics, including bispecifics and CAR-T cell therapies, where even minimal cross-reactivity could result in serious or even life-threatening consequences. Tissue cross-reactivity (TCR) studies have conventionally been employed to screen for off-target binding; however, their predictive value regarding in vivo safety and toxicity is limited. To address this challenge, we developed the Membrane Proteome Array (MPA) platform, which offers a comprehensive approach to assess specificity across the entire human membrane proteome to identify potential off-target binding liabilities. The MPA assesses binding interactions across 6,000 proteins expressed in live cells using high-throughput flow cytometry, allowing for high-sensitivity detection and rapid analysis. Recently, we used the MPA to select a lead antibody candidate targeting the oncotherapeutic target Claudin 6 (CLDN6). Therapeutic antibodies targeting CLDN6 must be highly specific, excluding closely related CLDN family members widely expressed on healthy tissues, as other CLDN6 antibodies in clinical development have demonstrated considerable cross-reactivity and safety issues in clinical trials. Through MPA screening, we successfully identified a lead candidate devoid of cross-reactivity against other CLDN family members or any other membrane protein across the human genome. We will also present case studies where the MPA has been used to rapidly evaluate the specificity of novel CAR-T cell therapies for cancer and autoimmunity.
432
Qualification of a Proprietary High-Throughput Immunoassay for Determining Symmetric Dimethylarginine (SDMA) Concentrations in Rhesus Macaque Serum
Valerie M. Wong 1, Kendall E. Clark2, Paul A. Vue2, Chelsea D. Landon3, Francis J. Sun3, Lee-Ronn Paluch4, Alyson R. Guy4, Colleen E. Thurman5, Rebecca K. Tierce6, and JodiAnne T. Wood2
1IDEXX BioAnalytics, IDEXX Laboratories, Sacramento, CA, USA
2IDEXX BioAnalytics, IDEXX Laboratories, Westbrook, ME, USA
3Duke University Medical Center, Durham, NC, USA
4New York University, New York, NY, USA
5Boston University, Boston, MA, USA
6Columbia University, New York, NY, USA
Symmetric dimethylarginine (SDMA) is a serum biomarker for kidney failure. In a puromycin aminonucleoside-induced rat model for kidney disease, serum SDMA concentration was useful for early detection of acute glomerular toxicities. The utility of SDMA in detecting kidney toxicity in non-human primates (NHP) has not been established. The “gold standard” method for measuring serum SDMA concentrations is liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). However, this method is labor-intensive and time-consuming. Our laboratory has previously qualified a proprietary, high-throughput SDMA immunoassay using a routine chemistry analyzer (Beckman Coulter AU680) against the LC-MS/MS assay for rat use. The objective of this study was to determine if the high-throughput immunoassay would be appropriate for NHP use. Within a toxicologically relevant range (7.4 to 108 µg/dL), the immunoassay showed high intra-assay (coefficient of variation (CV) < 10%) and inter-assay (CV < 5%) precision. When compared against LC-MS/MS, the immunoassay demonstrated accuracy, as indicated by good correlation (r2 = 0.99) and minimal bias (y-intercept = 0.62, 95% confidence interval (CI): -1.3, 2.5; slope = 0.99, 95% CI: 0.95, 1.03). Bland-Altman analysis showed negligible difference (mean ± standard deviation (SD): -4.08% ± 9.43%) between the immunoassay and LC-MS/MS. The immunoassay had a limit of detection (LOD) of 2.96 µg/dL and a limit of quantitation (LOQ) of 4.6 µg/dL. These results demonstrate the appropriateness of the high-throughput immunoassay for NHP use. Investigative work for determining serum SDMA concentrations in rhesus macaques with normal and compromised kidney functions are currently underway.
433
Quantitative Systems Toxicology (QST) Modeling Using a New Virtual Population in BIOLOGXsym Offers Mechanistic Insights into Bile Acid-Mediated Biologics-Induced Liver Injury (BILI) upon Cimaglermin Alfa (GGF2) Administration
Brett A. Howell 1, James J. Beaudoin1, Lawrence A. Vernetti2, D. Lansing Taylor2, Albert H. Gough2, Lara Clemens1, Christina Battista1, Scott Q. Siler1, Lisl K. M. Shoda1, and Kyunghee Yang1
1DILIsym Services Division, Simulations Plus, Inc., Research Triangle Park, NC, USA
2University of Pittsburgh Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA, USA
Biopharmaceuticals are increasingly used to treat various medical conditions, but BILI events can end the clinical development of otherwise promising therapies, such as the treatment of heart failure patients with the growth factor protein GGF2. Transient increases in the BILI biomarkers plasma alanine aminotransferase (ALT) and total bilirubin suspended phase I trials of GGF2, but the mechanism underlying these biomarker elevations was not understood. Combining assay outputs from a human biomimetic liver acinus microphysiology system (LAMPS) with BIOLOGXsym, a QST modeling platform for macromolecules, representing relevant liver biochemistry and mechanistic effects of biologics on liver pathophysiology, has recently provided mechanistic understanding of GGF2-induced hepatotoxicity, and this approach was further investigated in the present work. Transcriptional data and GGF2-mediated reduction of bile acid (BA) secretion in LAMPS were used to derive mechanistic toxicity parameters in BIOLOGXsym. Proof-of-concept simulations of GGF2 suggested that downregulation of basolateral BA transport was the main contributor to plasma ALT elevations in a representative virtual subject in BIOLOGXsym. In the current work, an extended BA homeostasis representation, and a virtual healthy population with interindividual variability in mechanistic pathways were implemented within BIOLOGXsym. While new simulations with a representative subject in BIOLOGXsym continue to support a key role for basolateral BA transport downregulation in explaining GGF2-induced ALT elevations, a more dominant role for the downregulation of biliary BA excretion, and subsequent hepatocellular BA accumulation, is predicted at the population level. In conclusion, population-based simulations in BIOLOGXsym can provide new mechanistic insights into BILI. Supported by NIH R44TR003535.
434
Refining Inter-individual Variability Adjustment Factors for Occupational Exposure Limits
Kelli F. Malott 1, Janet C. Gould2, Erica L. Dahl2, Michael J. Olson3, and Robert G. Sussman2
1SafeBridge Regulatory & Life Sciences Group, Los Angeles, CA, USA
2SafeBridge Life Sciences & Regulatory Group, New York City, NY, USA
3SafeBridge Life Sciences & Regulatory Group, Raleigh, NC, USA
Using a default adjustment factor (AF) of 10 for inter-individual variability is a common practice when setting occupational exposure limits (OELs) to protect workers in pharmaceutical manufacturing environments. Human pharmacokinetic (PK) data are generally available for active pharmaceutical ingredients (APIs), and a chemical-specific adjustment factor (CSAF) can be derived from the area-under-the-curve (AUC) or maximum blood concentration (Cmax) by taking the mean AUC/Cmax plus two standard deviations and dividing by the mean AUC/Cmax. Previous work by our group concluded that a default factor of 5 may be sufficient to estimate inter-individual variability of PK in healthy workers, and that the default factor of 10 may be unnecessarily conservative. In this analysis, we compared the CSAFs calculated for 140 unique APIs including small molecules and mAbs using the reported arithmetic or geometric means and standard deviations of the appropriate PK parameter. We compared the CSAFs derived using arithmetic vs. geometric means, small molecule vs. monoclonal antibodies (mAbs), males vs. females, and healthy vs. patient populations. We found that there was little difference and the mean CSAF value generally rounded to 5, even with the comparison of these subpopulation variations. These findings further strengthen the use of a default AF of 5 for inter-individual variability when calculating OELs for APIs. While appropriate for healthy adult workers, a default adjustment factor of 10 should be used for other calculated exposure limits.
435
Retrospective Review of Histopathology Findings in Sprague Dawley Rats in Powder Inhalation Toxicology Studies
Predrag Novakovic 1, Stephen Groom1, Lev Kolodzyeski1, William Lee1, Kevin McInally1, and Joseph Younan1
1ITR Laboratories Canada Inc., Baie D'Urfe, Quebec, Canada
Global increases in the use of inhaled drugs in the form of fine powders demands a better understanding of the effects of this delivery method on the respiratory system of laboratory animals used in preclinical toxicology studies. The objectives were to review the historical incidence and severity of histopathological findings in the respiratory tract of Sprague Dawley (SD) rats exposed to powder carriers and establish the recommendations for the typical background findings observed in powder inhalation toxicology studies. This retrospective review was performed on archived studies conducted at ITR between 2008 and 2022 comprising a total of 97 powder inhalation studies with 1962 control animals. Exposure to air, or the typical vehicles employed in respirable test item powder blends, such as lactose, leucine, magnesium stearate and trehalose, resulted in background histopathological findings consisting mainly of minimal hyperplasia of the mucous cells of respiratory epithelium at various levels of the respiratory tract, increased eosinophilic globules of the olfactory epithelium, epithelial alteration of the larynx, loss of cilia with flattening of respiratory epithelium at the carina and increased numbers of alveolar macrophages in lungs. The results of this review suggest that typical powder carriers used in test item powder blends are associated with an increase in the incidence (e.g., atrophy of the carina was present in 22% powder control animals compared to 13% air control animals), but not the severity of common background histopathological findings in the respiratory tract of SD rats, as compared to findings observed in animals exposed to air.
436
Enhanced Analysis of Immune Cell Responses in the Mouse Spleen
Yunling Song 1, Andrea Matter1, Andrew Dunn1, Min Wang1, Santo Cruz1, Leila Eslamizar1, Charles Wood1, and Elizabeth Clark1
1Boehringer Ingelheim, Ridgefield, CT, USA
Evaluation of immune safety is a critical part of preclinical biopharmaceutical development. Currently, however, there is limited information on tissue-based biomarkers and methods to assess spatial dynamics of immune cell populations. In this study, we describe the anatomic distribution of major immune cell types in the mouse spleen, concordance of cell percentages between single sections of splenic tissue and referent bulk tissue immunophenotyping data, and spatial patterns using HALO image analysis to establish baseline data for major splenic immune cell types in naïve BALB/cByJ mice. Immunohistochemistry (IHC) was performed in formalin-fixed paraffin-embedded (FFPE) splenic tissue for CD3 (all T cells), CD4 (T Helper cells), CD8 (Cytotoxic T cells), FOXP3 (Regulatory T cells), CD19 (B cells), CD163 (red pulp macrophages), and CD169 (marginal zone/metallophilic macrophages). Additionally, we conducted double IHC for select marker pairs and performed proximity and nearest neighbor analyses. Image analysis showed the distribution of cells: CD3+, 26.5%; CD4+, 14.4%; CD8+, 11.8%; FOXP3+, 3.7%; CD19+, 42.5%; CD163+, 9.6%; and CD169+, 5.1%. Our results on immune cell proportions are similar to internal reference data generated by flow cytometry: CD3+, 28.8%; FOXP3+, 5.8%; CD19+, 43.2%; and CD169+, 3.0%. and to published results. Spatial distance was measured between two selected cell types, for example, the average proximity distance between Foxp-3+ cells to CD169+ cells was 29.2 µm. The percentage of CD169+ cells within 10 µm of Foxp3+ cells was 6.86%. These results provide important baseline information for assessing changes in immune cell types and investigating their spatial dynamics.
437
The MultilinkTM Linker Is a Promising Approach to Improve Efficacy and Safety of ADCs
Josée Hue-Perron 1, Noémie Luong2, Lisa Ivanschitz2, Nathalie Bellocq3, Léo Marx3, and Ulf Andersson2
1Debiopharm International SA, Lausanne, Vaud, Switzerland
2Debiopharm International SA, Lausanne, Vaud, Switzerland
3Debiopharm Research & Manufacturing SA, Martigny, Valais, Switzerland
Antibody drug conjugates (ADCs) represent an attractive method to deliver cytotoxic payloads into tumor cells while reducing systemic toxicities. To improve efficacy, increasing DAR (drug–antibody ratio) is a good approach, although lipophilicity of such constructions can lead to unselective uptake. Non-specific linker cleavage is also associated with off-target toxicity. We have developed the MultilinkTM technology, which allows rapid and specific intracellular cleavage of a hydrophilic linker, resulting in efficient payload release from the ADCs. We investigated the toxicology profiles in mouse of two ADCs targeting CD37. Both ADCs carry the cytotoxic payload DM1; one with a non-cleavable thioether linker (DAR3.5) and the other with our MultilinkTM linker (DAR8). Both ADCs demonstrate a toxicity profile expected with DM1, such as hematologic abnormalities, infusion reactions and hepatic lesions. Despite higher DAR, there were fewer toxicities with the Multilink TM ADC, and a trend to lower severity of histopathological lesions. Notably, there were no hemorrhage or degeneration in the sciatic nerve when compared to the thioether-conjugated-ADC, which suggests reduced risk of peripheral neuropathy. When Multilink TM was conjugated with DM1 alone, it was not associated with any signs of toxicity. When evaluated in xenograft efficacy models, the Multilink TM ADC demonstrated significantly better tumor regression. In conclusion, following normalization for dose and difference in DAR in the efficacy and toxicology studies, it was demonstrated that the Multilink TM -conjugated ADC displayed higher therapeutic index (TI) compared to the thioether-conjugated-ADC. This demonstrates the importance of optimizing linker chemistry to improve efficacy and safety, resulting in increased TI.
438
Toxicology Profile of PN-943, an Oral α4β7 Integrin Inhibitor, Supports Further Clinical Development in Ulcerative Colitis Patients
Jingsong Zhao 1, Jill Kohler1, James Tovera1, Shu Li1, Grace Ledet1, and Mohammad Masjedizadeh1
1Protagonist Therapeutics, Inc., Newark, CA, USA
PN-943, a specific α4β7 integrin inhibitor, is an oral, gastrointestinal-restricted, peptide-based drug candidate intended for moderate to severe ulcerative colitis (UC). In GLP repeat-dose toxicology studies, PN-943 did not elicit any treatment-related adverse effects up to 6-month in rats and 9-month in cynomolgus monkeys. All in vitro and in vivo genetic toxicology studies were negative. No PN-943 treatment related effects were observed in the safety pharmacology studies for cardiovascular, respiratory, and central nervous systems. Margins-of-safety (MOS) were thus calculated to be 10.4x and 57.6x from the steady-state area-under-curve (AUC) values at no-observed-adverse-effect-levels (NOAELs) obtained from the 6-month rat and 9-month monkey toxicity studies, respectively. The observed steady-state human exposure of PN-943 following multiple oral doses of 150 mg twice daily (BID) (300 mg daily) in healthy volunteers was used for MOS comparisons. Furthermore, the highest dose tested in a 4-week rat limit dose toxicity study, 1000 mg/kg/day, was considered as no-observed-effect-level (NOEL), resulting in MOS of 37.2x over 150 mg BID (300 mg daily) in humans. In definitive embryo-fetal development (EFD) studies, both maternal and developmental NOEL was 1000 mg/kg/day in rats and 75 mg/kg/day in rabbits. In male and female rat fertility and early embryonic development (FEED) studies, paternal/maternal and male/female FEED NOELs were both 1000 mg/kg/day. Dose range-finding toxicity studies were also completed in both mice and rats to enable the dose selections for rodent 2-year carcinogenicity studies. Taken together, the up-to-date PN-943 toxicology profile supports its further clinical development as a potential therapeutic for UC patients.
439
Translational and In Silico Assessment of Liver Injury for 4-Hydroxy-2,6-Dichlorodiphenylamine
Brenda Finney 1, Arianna Bassan3 Kevin Cross2, Candice Johnson2, and Glenn Myatt2
1Instem, Hereford, England, United Kingdom
2Instem, Columbus, OH, USA
3Innovatune, Padua, Italy
The ability to predict clinical outcomes from pre-clinical data is of interest as this could improve the safety profile of new drugs. Liver toxicity is one reason why drugs may fail. Previous work led to development of in silico tools for assessment of drug induced liver injury (DILI) such as statistically-based and alert-based human relevant models. These computational tools combined with review of public clinical data using Instem’s Centrus (harmonized data sharing and review) platform allow for a more robust means of predicting clinical outcomes. In this study, in silico models were used to predict the propensity of 4-hydroxy-2,6-dichlorodiphenylamine (target) to induce liver injury through an assessment of bile duct disorders, cholestasis, liver damage and abnormal liver enzymes. Currently, available databases do not contain experimental studies on the target specifically therefore, analogs of the target structure were searched to gain insights potential toxicity. Diclofenac was identified as a mechanistically similar analog following a comparison of the in silico profiles based on potential bioactivation alerts and peptide reactivity. The human relevance of the hypothesis was further investigated using Centrus to retrieve clinical findings related to diclofenac and additional analogs with identification of many clinical findings of liver injury (bile duct structure/liver structure). However, given the strength of similarity between diclofenac and the target, it is likely that 4-hydroxy-2,6-dichlorodiphenylamine undergoes metabolic transformation to a reactive species that could result in clinically observed liver injury. The combination of in silico tools and access to clinical data provides a means for conducting translational assessments.
440
Why Do Some Mutagenic N-Nitrosamines Not Require Metabolic Activation?
Rachael E. Tennant 1, David J. Ponting1, Andrew Thresher1, and Anne-Laure D. Werner1
1Lhasa Limited, Leeds, England, United Kingdom
The detection of highly potent carcinogenic N-nitrosamine impurities in pharmaceuticals has resulted in industry-wide efforts to better understand their carcinogenic potential. The OECD-471 bacterial reverse mutation (Ames) assay is the gold-standard primary screen to assess drug impurities for potential mutagenic risk and forms a core component of the safety assessment data required by regulatory agencies for acceptance of new drug substances. It is therefore important to understand how metabolic conditions in the Ames test may affect its ability to identify potentially carcinogenic N-nitrosamines. Although it is widely understood that the primary mechanism of mutagenicity for N-nitrosamines usually requires an initial metabolic activation step to form a DNA-reactive diazonium ion, investigation of historical Ames assay data indicates that some sub-classes of N-nitrosamine compounds, primarily those bearing various oxy groups alpha- and/or beta- to the amine, may undergo spontaneous decomposition to the reactive species or an alternative mechanism of mutagenic activity.1 Analysis of a large curated Ames data set, consisting of over 300 public and proprietary compounds, indicates that a significant proportion of N-nitrosamines demonstrate mutagenic activity both in the presence and absence of an exogenous source of metabolic enzymes. Molecular features have been identified alongside potential mechanistic rationale for sub-classes that are likely to result in mutagenicity via a pathway other than the traditional activation route. This knowledge can be used for better interpretation of Ames test results for N-nitrosamines during chemical safety assessments.
500s - Safety Evaluation Pharmaceuticals
502
A 12-Day Intracerebroventricular Infusion via a Catheter and an Implantable Infusion Pump Research and Development Study
Jay K. Herman 1, Laura Boone2, Jennifer Chapman3, Todd Quackenboss2, Michael Taschwer2, Kyle Wegner2 Mollie McArthur2, Angela Favreau2, Dwight Skinner4, Eric Adams5, and Michelle Boyd4
1Labcorp Drug Development, Madison, WI, USA
2Labcorp Drug Development, Madison, WI, USA
3Labcorp Drug Development, Chantilly, VA, USA
4Cerebral Therapeutics, Aurora, CO, USA
5Neurosite Consulting, Ada, MI, USA
The purpose of this study was to evaluate the catheterization of the intracerebroventricular (ICV) space of beagle dogs to allow dosing via continuous infusion using the fully implanted Cerebral Therapeutics K9-ICVRxTM Infusion System. Dogs were assigned to two groups and doses were administered continuously at a rate of either 2.2 mL/day (vehicle: 1 male) or 2.0 mL/day Test Article (TA: 1 male/1 female) for 12 days. No clinical signs, changes in body weight, food consumption or neurobehavioral observations were observed with the exception of surgical-related seromas on the lateral abdomen, at the site of pump implantation, which resolved spontaneously during the dosing phase. A TA-related mildly decreased absolute lymphocyte count was observed in animals administered TA but without microscopic correlates. No other TA-related effects were identified in coagulation, clinical chemistry, CSF, or CSF cytology. All microscopic findings were considered procedure-related findings in the brain. These included one or more of the following: presence of a catheter track, necrosis, compression, axonal degeneration, microgliosis, astrocytosis, hemorrhage, and/or perivascular mononuclear cell infiltrate. Procedure-related immunolabeling for Iba-1 and GFAP was observed and characterized by increased microglial cells and astrocytes, respectively, associated with the catheter track and/or associated with axonal degeneration adjacent to the ventricles and occasionally extending along white matter tracts. These microscopic findings were not associated with neurobehavioral or clinical findings in the dogs; therefore, the implantation procedure and 12-day infusion via the ICVRx Infusion System was considered to be viable and supportive of longer-term ICV delivery to dogs at similar doses.
503
A Canadian Case Study in Chemical Risk Assessment Using a Refined Zebrafish Model to Reduce the Number of Animals to Generate Toxicity Data While Meeting Regulatory Data Requirements under the New Substances Notification Regulations
Cindy LA Woodland 1, Lee D. Ellis2, Joëlle Pinsonnault Cooper3, and Deborah E. Ratzlaff3
1Government of Canada, Bainsville, Ontario, Canada
2National Research Council of Canada, Halifax, Nova Scotia, Canada
3Government of Canada, Ottawa, Ontario, Canada
Health Canada currently uses animal toxicity testing data to inform chemical risk assessments under the Canadian Environmental Protection Act (CEPA) and the New Substances Notification Regulations (NSNR). These data are often required to be provided by industry to determine if a new substance is potentially harmful to human health and the environment. In 2021, Bill S-5 was introduced in the Senate of the Government of Canada to amend CEPA to promote activities to replace, reduce and refine the use of vertebrate animals. On January 14, 2022, the Government of Canada published a ‘Notice of Intent (NOI)’ to promote reduced reliance on animal testing and the alignment of regulatory requirements under NSNR with advancements in science. In support of these objectives, the Government of Canada has developed a refined zebrafish embryo/larval model intended for use in quantitative chemical risk assessment. This model is currently being cross-validated internationally and is moving towards regulatory acceptance in Canada. A case study in risk assessment using this refined zebrafish model as a replacement for the rodent model was conducted using morphological, behavioural, transcriptomic and toxicokinetic data generated for Raloxifene, a test substance used in the development of the model. Comparisons between risk assessments conducted using the zebrafish test data and historical rodent data were made to evaluate the robustness of the zebrafish model for predictive toxicity. Preliminary results suggest promise for use of the evolving refined zebrafish model as a potential alternative to the rodent model for use in chemical risk assessments.
505
Assess the Effects of Dopamine Receptor Agonists on the Estradiol-Induced Prolactin Surge in Ovariectomized Wistar Han Rats
Atish Patel 1, Travis L. Clakins2, Justin Vidal2, Pragati Coder2, Scott Carrier1, Giri Gokulrangan1, Srinivas Chakilam1, Sandeep Akare1, Stefanos Aivazidis1, and Marci L. Harter1
1Cerevel Therapeutics, Cambridge, MA, USA
2Charles River Laboratories, Ashland, OH, USA
This study shows the effects of dopamine receptor agonists, pergolide and rotigotine on estradiol (E2)-induced prolactin surge in ovariectomized (OVX) Wistar Han rats which can be used to explain the rat specific endocrine tumor response observed with dopamine agonists. All rats were OVX and implanted with a silastic capsule containing E2-benzoate, except for the OVX control group, to produce a synchronized cohort of animals with E2 levels comparable to intact animals. The study included five groups: OVX-Control, OVX-E2-Control, pergolide (oral gavage) at 0.25 mg/kg/day, rotigotine (subcutaneous) at 0.5 mg/kg/day, and 5 mg/kg/day. Mortality, clinical signs, body weights, body weight gains, and plasma prolactin levels (Days 4 and 7) were evaluated. The OVX-E2 group showed an afternoon surge (1500 and 1700 h, up to 100x compared to control) in prolactin on both days 4 and 7, with an expected decline in prolactin at 1900 h (75x compared to control) post lights on. The OVX-control group showed low prolactin levels as expected for females with no endogenous or exogeneous source of estradiol. The pergolide group showed a significant decrease in E2-induced prolactin surge on days 4 and 7 (up to 98% decrease compared to OVX-E2-Control) while rotigotine low and rotigotine high dose groups showed a dose-dependent decrease in E2-induced prolactin surge (up to 75% and 96% compared to OXV-E2-Control). Thus, these data demonstrate that the OVX rat model with a silastic capsule loaded with E2 is suitable for evaluating the effects of dopamine receptor agonists, to alter (reduce) the E2-induced prolactin surge.
506
Cell Therapy Safety Study Considerations Relevant to the Immunodeficient NCG-X Mouse Model
Cristina Ballesteros 1, Simon Authier1 Steven Bronson2, Steve Festin2, Stephanie Harris1, Zeinab Noroozian1, Camila Para1, Jenny Rowe2, Michael Ruppert1, and Christian Li1
1Charles River Laboratories, Laval, Quebec, Canada
2Charles River, Wilmington, NC, USA
The NCG triple-immunodeficient mouse model, which lacks functional T, B, and NK cells, is used to develop therapies for tumor, transplant, and immune-related diseases. Its SCID-like phenotype facilitates the engraftment of human hematopoietic stem cells (hHSC). However, human cell engraftment typically requires irradiation or chemoablation of the animal. This procedure can cause background changes in toxicology endpoints and can often negatively impact animal sensitivity, bodyweight, behavior, and rates of survival. To circumvent these issues, the NCG-X mouse model was developed by deriving the NCG model with a mutation in the Kit gene increasing the immunodeficient state of the mouse. This additional gene editing allows for human cell engraftment without prior immuno-conditioning. We assessed the engraftment rates in NCG-X and compared the results to the NCG model with irradiation and busulfan conditioning. A consistent trend towards increased engraftment was observed in the NCG-X when compared to NCG with immuno-conditioning with human levels reaching above 50% human cells in most animals. Expected body weight gains were observed with the NCG and NCG-X strains and minimal mortalities were observed over a 20-week period which is typical for toxicology investigations of gene edited stem cell therapies. Thus, gene editing for c-KIT in the NCG-X mice enhanced human cell engraftment resulting in an improved murine model which can be considered in the development of cell therapies and the associated toxicology assessments.
507
Daily Six-Hour Restraint Decreases Sprague Dawley Body Weight Gain in Subchronic Toxicity Studies
Emily Resseguie1
1Labcorp Early Development Laboratories, Inc, Franklin Park, NJ, USA
Safety assessment of pharmaceuticals and chemicals with potential for repeated human exposure involves determining toxicity in studies that align with country-specific regulatory frameworks. A minimum 13-week study is required to support Phase II or III clinical trials, and a 13-week study following Organisation for Economic Co-operation and Development (OECD) guidelines is required for chemicals depending on the tonnage band (OECD 408 for oral ingestion risk or OECD 413 for inhalation risk). Exposure duration for inhaled therapeutics depends on clinical safety margins, while rodent exposure in compliance with the OECD 413 guideline is 6 hours/day for 5 days/week. For orally consumed pharmaceuticals and chemicals, rodent administration is daily and only requires a few minutes handling per animal. A comparison of 13-week studies with different dose routes using Crl:CD®(SD) rats was conducted to assess the impact of restraint on body weight. Data were averaged from the control groups of recent studies for daily oral gavage and 6 hours/5-7 days/week inhalation dosing scenarios (9 and 6 studies, respectively). Body weights of animals following 13 weeks of inhalation dosing were significantly lower than the body weight of animals in oral gavage studies (-17% for males and -13% for females). Food consumption did not change over time. Organ weights did not reveal macro-level changes associated with lower body weight gain. Higher respiratory rates during nose-only exposure (previous research) may contribute to increased metabolic demand that is uncompensated for by feeding. Thus, consideration to potential impact of metabolic differences on toxicity is important for study interpretation.
509
Enhancing Decision Making for Genotoxicity Assessments Using Transparent In Silico Systems
Adrian Fowkes 1, Rachael Tennant1, Graham Heaven1, and Robert Foster1
1Lhasa Limited, Leeds, England, United Kingdom
QSAR systems for predicting genotoxicity are widely used for a variety of chemical safety assessments, from prioritisation to deriving regulatory conclusions. However, the performance of models for different genotoxicity endpoints varies. For bacterial mutagenicity, models provide sufficient coverage and accuracy to allow them to be used in a regulatory setting, whilst for other genotoxicity endpoints such as chromosome damage, models are less robust hindering their applicability to support certain decisions. To further develop in silico systems with improved predictions across genotoxicity endpoints, relevant sets of expert-derived structural alerts within Derek Nexus were reviewed alongside expert-curated datasets. Review of the existing knowledge and data supported improvements to mutagenicity alerts, creation of 34 refined chromosome damage alerts and the generation of training sets for transparent statistical models. These complementary models and datasets can then be combined into in silico workflows, to enhance chemical safety assessments such as satisfying the EFSA residue definition guidance. Example case studies showed that instances where pesticide metabolites did not fire an expert alert for chromosome damage, increased confidence in the absence of toxicity was provided by accompanying negative predictions from the statistical system where relevant nearest neighbours could be examined. This overall approach allows for new genotoxicity knowledge to be easily incorporated into in silico systems as new data becomes available and support experts with decision making in lieu of data through leveraging transparency, even when endpoints are more challenging to model.
510
Establishing Modified Cerebrospinal Fluid (CSF) Collection in Cynomolgus Monkeys and Rodents: Overcoming Limitations of Conventional Approaches
Bo-Sung Baek 1, Sang-Hee Lee1, Jung-Ho Noh1, Tae-Ho Hwang1, Eun-Jung Park2, and Hong-Soo Lee1
1Keyprime Research Co., Ltd., Cheongju-si, Ch'ungch'ong-bukto, Republic of Korea
2Kyung Hee University, Seoul, Republic of Korea
Cerebrospinal fluid (CSF) collection plays a pivotal role in advancing our understanding of the central nervous system's physiology and pathophysiology in neuroscience research to offer compelling prospects for enhancing diagnostic capabilities, understanding disease mechanisms, and developing targeted therapies. In rodents, while a single-time sampling approach through the lumbar puncture, ventricular puncture, and puncture of the cisterna magna is typically employed due to technical limitations, using a butterfly needle has proven effective in reducing the risk of blood contamination during CSF collection (Atulya, 2021). However, this method is hindered by challenges associated with repeated sampling and standardizing the technique. In order to address these shortcomings, a novel and modified CSF collection method has been developed, integrating the utilization of a butterfly needle and stereotaxic apparatus into the cisterna magna. This innovative approach has not only been successfully implemented in rodents but has also been extended to CSF collection in cynomolgus monkeys, showcasing remarkable efficacy, high success rates, and the ability to facilitate repeated samplings without involving the implantation of catheters or cannulas through a surgical process; no signs of contamination nor inflammation have been observed even after 18 times repeatedly collecting CSF over three months. By adopting this modified method, researchers can overcome the limitations associated with conventional CSF collection methods, significantly enhancing the reliability, accuracy, and applicability of CSF analysis which holds great potential advancements in understanding neurological disorders, identifying therapeutic targets, and eventually improving patient outcomes. Keywords CSF collection, cynomolgus monkeys, rodents, Cisterna Magna, butterfly needle
512
MRI Guided Delivery to the Putamen Using The Northern ARC: A Custom Frame Allowing Occipital Approaches in Large Animal Models
Christen D. Simon 1, Andy Carlson1, Eva Wembacher2, and Mark Johnson1
1Northern Biomedical Research, Norton Shores, MI, USA
2Brainlab AG, Munich, Germany
Delivery of gene therapies directly to the central nervous system allows the blood brain barrier to be bypassed and decreases the risk for systemic exposure and toxicity. Intraparenchymal (IP) delivery directly to the brain using Convection Enhanced Delivery (CED) is ideal for maximizing exposure of the target of interest to the therapeutic agent. Northern Biomedical Research (NBR) has developed a custom Magnetic Resonance (MR) compatible system (the Northern ARC) to allow bilateral infusion device placement and for simultaneous, real-time MRI guided CED. The objective of this study was to assess feasibility and effectiveness of the Northern ARC for infusions into the putamen using occipital trajectories. Three cynomolgus macaques were anesthetized and placed in a semi-prone position in the Northern ARC frame. The putamen was targeted using gadolinium-filled arrays attached to the frame. The cannulae (Clearpoint Neuro Smarflow Cannula) were inserted to the proximal boundary of the target and advanced stepwise to optimize target coverage. Infusions commenced at 1 – 3 µL per minute. T1 scans were performed prior infusion and continuously during the infusions for distribution monitoring. The bilateral infusions were performed simultaneously. Injection volumes of 85 – 125 µL per putamen, distributed over 4 injection sites, resulted in a coverage of 63 ± 3% of the putamen (maximum: 67%, minimum 58%). All catheters were placed on target. In conclusion, targeting the putamen for catheter placement by an occipital approach for simultaneous, bilateral CED infusions is feasible and effective in large animal species using the NBR Northern ARC Infusion System.
513 - International Travel Grant Recipient
Protocatechuic Acid (PCA) Protects against 7,12-Dimethylbenza[A]Anthracene (DMBA)-Induced Mammary Gland Carcinogenesis in Wistar Rats via Antioxidative, Anti-inflammatory, Apoptotic, and Anti-proliferative Pathways
Chijioke N. Onah 1, Adeleye A. Edema1, Adedoyin O. Adefisan1, Anuoluwa A. Oloyede1, and Oluwatosin Adaramoye1
1University of Ibadan, Ibadan, Oyo, Nigeria
Breast cancer is the commonest cancer killer in women globally. Natural products, due to their ability to interfere in several molecular signaling processes, have shown promise as novel therapeutics against complex ailments such as cancer. We investigated the effects of protocatechuic acid (PCA) on 7,12 dimethylbenz[a]anthracene (DMBA)-induced mammary gland carcinogenesis in Wistar rats. Thirty-five female Wistar rats were divided into five groups (n=7): Group 1 served as control, group 2 received DMBA (80mg/kg), groups 3 and 4 received DMBA and PCA at 50 and 100 mg/kg, respectively, and group 5 received DMBA and vincristine at 0.5 mg/kg. DMBA (intraperitoneal) was dissolved in corn oil, PCA (oral), and vincristine (intraperitoneal) were given thrice and once per week, respectively for 15 weeks. DMBA administration significantly (p< 0.05) reduced rats’ body weight gain by 18%. Also, DMBA increased oxidative stress markers; nitric oxide (NO), lipid peroxidation (LPO), and myeloperoxidase (MPO) by 25%, 480%, and 35%, respectively. Furthermore, DMBA decreased mammary levels and activities of GSH, GST, and GPx by 16%, 68%, and 30%, respectively. TNF-α, Bax, and Bcl-2 levels increased by 4.7, 3.5, and 1.9 fold, respectively in DMBA-treated rats, measured by ELISA. In DMBA rats, histology showed mammary glandular epithelial cells with hyperchromatic nuclei and infiltrating inflammatory cells, while immunohistochemistry revealed the expression of estrogen receptors, epidermal growth factor receptors, and E-cadherin. PCA treatment restored mammary gland cyto-architecture through anti-oxidative, anti-inflammatory, and apoptotic pathways, and inhibited carcinogenesis by reversing DMBA-induced oxidative stress and inflammation. Therefore, PCA may be a powerful anticancer agent.
514
Nonclinical Toxicology Study Designs for Advanced Therapies: Practical Approaches to Address Resource Limitations
Jesse M. Lueth 1 and Marcie Wood2
1Toxstrategies, LLC, Deforest, WI, USA
2Toxstrategies, LLC, Katy, TX, USA
Nonclinical programs for advanced therapeutics often have unique considerations, including the use of nonstandard toxicology study designs and procedures. For example, cell or gene therapy studies may include nonstandard sample collections to assess biodistribution, or on- or off-target effects. Advanced dose routes often face equipment limitations or require advanced training and experienced staff. Lastly, the COVID pandemic has strained the supply of nonhuman primates, a species commonly used for testing advanced therapies. A need exists to determine common nonclinical study approaches to address ever-evolving challenges, while still maintaining study integrity and addressing the ultimate goal of characterizing the safety of candidate therapies. Herein, we explore various scenarios and, for each, approaches to overcoming limitations on staffing, equipment, and animal resources to execute successful nonclinical toxicology studies. These approaches may be applied for future studies where appropriate. 1. A nonhuman primate AAV gene therapy study requiring prescreening of neutralizing antibodies (nAB), in which staggering multiple arrivals addressed concerns of appropriate animal numbers due to low success of baseline nAB results during candidate screening. 2. A mouse cell therapy study with tissue biodistribution collections where flexibility in the timing of necropsy procedures due to staffing limitations was addressed in reporting by reorganization of the data for increased understanding using additional assignment strategies. 3. An intrathecal port surgery animal study with multiple staggered dosing initiation events to address equipment and staffing limitations.
515
Prevalence of Adeno Associated Virus Serotypes in Nonhuman Primates
Rajeev K. Dhawan 1, Connor Balzer1, LaChelle Campbell1, Brittany Egan1, and Donald Nicholson1
1Charles River Laboratories, Wilmington, MA, USA
Adeno-associated viruses (AAVs) are preferred vectors for gene therapy studies but can be limited by the presence of neutralizing antibodies (NAbs), from prior exposure to the virus, in patients or research animals. Therefore, cell based AAV NAb prescreening of subjects prior to enrollment in studies has become commonplace. Researchers routinely screen 8X-10X the number of NHPs enrolled in studies to find enough AAV seronegative animals. Using data collected by our lab over the past 2 years, we sought to identify variables that better predict the likelihood of AAV seropositivity to minimize the number of NHPs for prescreening. To this end, data for four AAV serotypes including AAV2, AAV8, AAV9 and AAVrh74 from nearly 2500 cynomolgus macaque sera was collated and analyzed. Results showed that seroprevalence ranged between 50% and 80% with AAV2 being the most prevalent and AAV9 the least. Both sexes, males and females had similar prevalence for all serotypes. NHPs in the 2-3 year age group had no significant variation despite suggestions that older animals are more likely to be positive for AAV NAb. Small variations in seroprevalence were found in NHPs from different countries of origin and even different colonies from the same country, e.g., between two Asian countries, one had ∼15% higher prevalence of AAV9 NAbs but ∼10% lower prevalence of AAV2 NAbs. In summary, when screening NHPs to select candidates for studies, it’s important to consult the NHP supplier for suggestions about optimal screening numbers and timing based on specific seroprevalence observed in their colonies.
516
Repeated Intracerebroventricular Administration in Nonhuman Primates for the Long-Term Nonclinical Safety and Efficacy Studies
Tetsuya Yoshikawa 1, Yuki Minayoshi1, Makoto Tanoue1, Yuma Ariki1, Haruna Nagae1, Ryutaro Nishikata1, Yuri Nakamura1, and Tomonobu Yamada1
1SNBL, Ltd, Kagoshima, Kagoshima, Japan
Intracerebroventricular (ICV) administration is a valuable route for delivering the central nervous system (CNS) therapeutic drugs into the brain and used for therapy of CNS infections and neoplastic diseases. In this study, repeated ICV dosing method in cynomolgus monkey was developed for the long-term evaluation. Before surgery, MRI scanning was performed to determine the coordination for the implantation of an indwelling needle into the lateral ventricle. Fourteen cynomolgus monkeys (7 males and 7 females, 2 years old, 1.7 to 2.5 kg) were sedated by ketamine and underwent surgery under inhalation isoflurane anesthesia. The cranium was perforated using a drill, the needle was inserted into the lateral ventricle though the hole in the cranium, and cerebrospinal fluid (CSF) was checked for outflow. The indwelling needle was fixed in place, and the muscle and skin incisions were sutured. After the 3-week post-surgery recovery period, artificial CSF was repeatedly administered intracerebroventricularly via the indwelling needle once every 2 weeks for 28 weeks (1 mL/body, 100 μL/min, 14 doses in total). Clinical signs and food consumption were monitored daily, body weight was measured weekly, and hematology and blood chemistry were examined on Weeks 4, 16, and 28. No abnormal clinical signs were observed in any animal after ICV dosing, and there were no apparent changes in body weights, food consumption, hematology, or blood chemistry. In conclusion, our method of repeated ICV administration in cynomolgus monkeys is a successful procedure for use in long-term nonclinical safety and efficacy evaluations of novel CNS therapeutics.
517
Species Selection for Biologics: Exploring Increased Use of Minipigs and Dogs for Nonclinical Safety Evaluation
Nawshaba Nawreen 1, David Clarke2, Eric Wen Su2, Michael Pritt2, Matthew Abernathy2, and Meredith Steeves2
1Eli Lilly, IND - Indianapolis, IN, USA
2Eli Lilly, Indianapolis, IN, USA
Appropriate species selection is a key decision to enable nonclinical safety evaluation in drug development. Historically for biologics, particularly monoclonal antibodies (mAbs), and antibody constructs, non-human primates (NHPs) have been the species of choice. Ethical considerations with 3Rs goals and a global shortage of NHPs drive an increased need to consider alternative non-rodent species for safety evaluation of biologics. In this study we performed a meta-analysis of published work to investigate the usage of alternate non-rodent species, the minipig and dog, for safety evaluation of biologics. We built Linguamatics Interactive Information Extraction (i2e) queries to mine FDA documents of approved biologics for preclinical use of minipigs and dogs. We then evaluated literature evidence to characterize the suitability of using NHPs, minipigs, or dogs for preclinical safety assessment and for human risk prediction. A minimal number of programs were identified that used minipigs or dogs for evaluation of mAbs. Key species differences to consider during non-rodent species selection under certain situations include pharmacological response, immunogenicity/hypersensitivity reactions, and anti-drug antibody (ADA)-mediated differences in drug clearance and exposure. Other factors influencing alternate species choices are higher test article requirements, synthesis time and lack of well-validated methodology (reagents and assays) for evaluating specialized endpoints such as immunomodulation/immunotoxicity. The work signifies important aspects to take into consideration when evaluating alternate non-rodent species during safety assessment of biologics in drug development. Our work highlights the importance of building the database of alternate non-rodent species to enable their usage for safety assessment of biologics.
518
Virtual Control Groups (VCGs) in Preclinical Testing: A Promising Tool for Reducing Animal Usage in Safety Assessment
Laura Lotfi 1, Sihong Ma2, and Steven Bulera3
1Charles River Laboratories, Boston, MA, USA
2Charles River Laboratories, Wilmington, MA, USA
3Charles River Laboratories, Reno, NV, USA
Virtual Control Groups (VCGs) have emerged as a promising tool for reducing animal usage by replacing selected control group animals with virtual counterparts in preclinical studies. The characterization of selecting historical study data for defined study parameters is being studied and the data science models built are undergoing validation. These key aspects must be understood to qualify and compare the VCGs with concurrent control group datasets using statistical metrics and algorithms to demonstrate equivalency or better outcomes. VCGs have the potential to revolutionize preclinical studies by reducing and partially replacing for selected control group animals. VCGs database development uses an advanced analytics model to select and provide best control matched virtual animal to a targeted study design. The development of VCGs is still in its early stages, but significant progress has been made. We will present some of these selection criteria analysis (i.e., Species, Strain, Sex, etc.) and their impact by endpoints (i.e., Body weight, Clinical pathology parameters) for rodent and non rodent models. These results are expected to be used to qualify the biological and statistical relevance of VCGs database and drive a regulatory guidance document for wider adoption of VCGs in the safety assessment industry. In conclusion, VCGs offer a promising tool for reducing animal usage in preclinical studies, while also providing a better understanding on critical parameters variation in preclinical testing. Continued efforts to develop and qualify VCGs will be crucial for the advancement of animal welfare and the conduct of preclinical studies.
519
What Are Our Options for CNS Delivery of AAV Therapies: Systemic, Intrathecal, Intracerebroventricular, Convection-Enhanced Delivery, or MRI-Guided Focused Ultrasound?
Alan Hoberman 1, Said Maghezzi2, Morgan Foret2, Karen Wong2, Camila Para, Srishti Vats2, Simon Authier2, Christian Li, Zeinab Noroozian2, and Michael Ruppert2
1Charles River Laboratories, Horsham, PA, USA
2Charles River, Laval, Quebec, Canada
The use of non-human primates (NHPs) in gene therapy studies is necessary to map the efficacy, limitations, and potential of adeno-associated virus (AAV) delivery to the central nervous system for the clinic. Drug transport to the brain has proven difficult due to the protection by the blood-brain barrier (BBB). Multiple strategies have been established to overcome this challenge. Previously, we delivered AAVs to the brain of NHPs using intrathecal, intracerebroventricular, and convection-enhanced delivery. However, drug administration via brain surgery is a traumatic procedure that damages tissue and can often lead to neuronal degeneration at the sites of injection. Thus, we have most recently considered MRI-guided focused ultrasound (MRIgFUS) which transiently permeabilizes the BBB in a minimally invasive manner to grant access of parenchymal brain tissue to systemically injected AAV. Based on the toxicological, pathological, histological, and clinical observations of each approach, we have prepared a comparative portfolio of the advantages, potential for translation, applications, and risks of multiple strategies of AAV delivery to the brain. The biodistribution of the transgene in peripheral tissue is also measured to determine risk assessment more accurately. Applications and clinical availability of each modality are discussed to provide comprehensive guidelines for selecting the most efficient approach for gene therapy in NHPs. The significance of selecting the proper route of administration is reflected in the reduction of risks, such as off-target expression of the transgene, and in the enhancement of therapy efficacy, which improves the potential for translation of preclinical studies.