Rodent Toxicity Studies Research Articles

The effect of aging on the repeated-dose liver micronucleus assay using N-nitrosodipropylamine, quinoline, and carbendazim

The repeated dose liver micronucleus (RDLMN) assay has been sufficiently validated in terms of the numbers and types of chemicals studied. However, it remains unclear whether aging affects assay results. The OECD Test Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents) indicates that dosing should begin as soon as feasible after weaning and in any event before 9 weeks of age. Therefore, it is particularly important to determine whether there are age-related differences between 6 and 8 weeks of age at the start of dosing when considering the possibility of integrating this assay into a 4-week repeated dose general toxicity study. We evaluated the impact of the rats’ age on the RDLMN assay with three chemicals: N-nitrosodipropylamine, quinoline, and carbendazim. There were no significant age-related differences for the first two chemicals, whereas a markedly higher frequency of micronucleated hepatocytes (MNHEPs) was observed in younger rats for carbendazim. However, regardless of the age of animals, micronucleus induction was detected in all three chemicals. Combined with the previous reports on clofibrate and diethylnitrosamine, we concluded that animals of any age from 6 to 8 weeks could be used in the RDLMN assay.

Abstract PO3-26-11: Enhancing endoplasmic reticulum stress for treating endocrine therapy resistant breast cancers driven by mutant estrogen receptors

Abstract Background: Estrogen receptor alpha (ERα) mutations are common (30-40%) in metastatic endocrine therapy-resistant breast cancers (ETR-BC), enable resistance to endocrine therapies and are the molecular drivers of ETR-BC. We had previously shown that an oligobenzamide, ERX-41, could enhance endoplasmic reticulum stress in ETR-BCs driven by mutant (MT) ERα, resulting in cancer cell death in vitro and and in vivo. To enable clinical translation of ERX-41, we performed lead optimization, followed by preclinical and IND-enabling studies. Methods: Over 2000 oligo-benzamides were designed, synthesized and tested in multiple BC models including those that express WT-ERα (MCF7, and ZR75) and BC models with acquired resistance (MCF7-Tam, and MCF7-LTLT) and engineered models that express MT-ERα (MCF7-ERα-D538G, MCF7-ERα-Y537S, ZR75-ERα-D538G, ZR75-ERα-Y537S). For our lead compound, mechanistic validation studies were performed using CRISPR LIPA mutants, RT-qPCR and Western blotting. Explants, organoids, cell line-(CDX) and patient-derived (PDX) xenografts were used to test the ex vivo and in vivo effectiveness of our lead compound as a monotherapy and in combination with abemaciclib. Results: Testing of >2000 synthesized oligo-benzamides identified a lead compound, ERX-315, that had broad and potent activity (IC50 between 20-100 nM) against both WT and MT (mutant) ERα-driven BC cells in in vitro assays. CRISPR KO of LIPA (which encodes lysosomal acid lipase (LAL) in BC cells abrogated cytotoxic response to ERX-315, validating LIPA as the molecular target of ERX-315. Ultrastructural and molecular studies confirm that ERX-315 induces significant endoplasmic reticulum stress, leading to a shutdown of de novo protein synthesis and apoptotic cell death in BC. Importantly, ERX-315 does not induce endoplasmic reticulum stress or cell death in normal cells and is non-toxic in animal models. We have shown that this capacity of ERX-315 to induce endoplasmic reticulum stress is unique among drugs targeting ERα, including selective ERα modulators and degraders, such as GDC-0180, AZD-9496 and fulvestrant. ERX-315 has potent anti-proliferative activity against MT-ERα-driven BC, as seen in genetically modified cell lines both grown as monolayer or spheroids in vitro, patient derived explants (PDEs) ex vivo and cell line derived (CDX) and patient-derived (PDX) xenografts in vivo. The combination of ERX-315 and CDK4/6 inhibitor abemaciclib was synergistic in decreasing the proliferation of both endocrine therapy-sensitive and endocrine therapy-resistant BC cells, in vitro, in xenograft models in vivo, and in primary patient-derived explants ex vivo. We are currently optimizing both the synthesis of ERX-315 using GMP process for kilogram scale production and validated methods for pharmacokinetic studies. We have developed formulations for both intravenous and oral administration with favorable pharmacokinetic parameters and proven utility of the formulated ERX-315 against CDX and PDX tumors in vivo. Toxicity studies in dogs and rodents have demonstrated >8-fold therapeutic to toxicity window. A phase I clinical trial is planned to be open to enrollment by Q1 2024. Conclusions: We have identified a lead compound ERX-315, which represents a novel class of agent that induce catastrophic levels of ER stress resulting in cancer cell death and that can effectively work against multiple forms of ETR-BC, including those driven by MT-ERα. Preclinical studies, GMP manufacturing, formulation and IND-enabling studies are being completed in time for the commencement of the phase I clinical trial by Q1 2024. Citation Format: Suryavathi Viswanadhapalli, Karla Parra, Tae-Kyung Lee, Rahul Gopalam, Kara Kassees, Tanner Reese, Gaurav Sharma, Xihui Liu, Xue Yang, ChiaYuan Chen, Carlos Roggero, Liping Chen, Sautam Bhattacharya, Uday Pratap, Russell Hayward, Sharron Gargosky, Jung-Mo Ahn, Ganesh V. Raj, Ratna Vadlamudi. Enhancing endoplasmic reticulum stress for treating endocrine therapy resistant breast cancers driven by mutant estrogen receptors [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-11.

The association between histopathologic effects and liver weight changes induced in mice and rats by chemical exposures: an analysis of the data from Toxicity Reference Database (ToxRefDB).

Absolute (ALW) and relative (RLW) liver weight changes are sensitive endpoints in repeat-dose rodent toxicity studies, and their changes are often used for quantitative assessment of health effects induced by hepatotoxic chemicals using the benchmark dose-response modeling (BMD) approach. To find biologically relevant liver weight changes to chemical exposures, we evaluated all data available for liver weight changes and associated liver histopathologic findings from the Toxicity Reference Database (ToxRefDB). Our analysis of 389 subchronic mouse and rat studies for 273 chemicals found significant differences in treatment-related ALW and RLW changes between dose groups with and without liver histopathologic changes. In addition, we demonstrate that chemical treatment-induced ALW and RLW changes can predict the presence of histopathologic findings and inform the selection of biologically relevant liver weight changes for BMD modeling and derivation of toxicity values.

The road to virtual control groups and the importance of proper body-weight selection.

Virtual control groups (VCGs) created from historical control data (HCD) can reduce the number of concurrent control group animals needed in regulatory toxicity studies by up to 25%. This study investigates the performance of VCGs on statistical outcomes of body weight development between treatment and control groups in legacy studies. The objective is to reproduce the statistical outcomes of 28-day sub-chronic studies (legacy studies) after replacing the concurrent control group with virtual ones. In rodent toxicity studies initial body weight is used as surrogate for the age of animals. For the assessment of VCG-sampling methods, three different approaches were explored: (i) sam­pling VCGs from the entire HCD, ignoring initial body weight information of the legacy study, (ii) sampling from HCD by matching the legacy study’s initial body weights, and (iii) sampling from HCD with assigned statistical weights derived from legacy study initial body weight information. The ability to reproduce statistical outcomes using virtual controls was determined by the congruence between the legacy study and the HCD weight distribution: regardless of the chosen approach, the ability to reproduce statistical outcomes was high for VCGs when the legacy study’s initial body weight distribution was similar to the HCD’s. When the initial body weight range of the legacy study was at the extreme ends of the HCD’s distribution, the weighted sampling approach was superior. This article demonstrates the importance of proper HCD matching by the legacy study’s initial body weight and discusses conditions to accurately reproduce body weight development.

Acute Effects of Brevetoxin-3 Administered via Oral Gavage to Mice.

Brevetoxins (BTXs) constitute a family of lipid-soluble toxic cyclic polyethers mainly produced by Karenia brevis, which is the main vector for a foodborne syndrome known as neurotoxic shellfish poisoning (NSP) in humans. To prevent health risks associated with the consumption of contaminated shellfish in France, the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) recommended assessing the effects of BTXs via an acute oral toxicity study in rodents. Here, we investigated the effect of a single oral administration in both male and female mice with several doses of BTX-3 (100 to 1,500 µg kg-1 bw) during a 48 h observation period in order to provide toxicity data to be used as a starting point for establishing an acute oral reference dose (ARfD). We monitored biological parameters and observed symptomatology, revealing different effects of this toxin depending on the sex. Females were more sensitive than males to the impact of BTX-3 at the lowest doses on weight loss. For both males and females, BTX-3 induced a rapid, transient and dose-dependent decrease in body temperature, and a transient dose-dependent reduced muscle activity. Males were more sensitive to BTX-3 than females with more frequent observations of failures in the grip test, convulsive jaw movements, and tremors. BTX-3's impacts on symptomatology were rapid, appearing during the 2 h after administration, and were transient, disappearing 24 h after administration. The highest dose of BTX-3 administered in this study, 1,500 µg kg-1 bw, was more toxic to males, leading to the euthanasia of three out of five males only 4 h after administration. BTX-3 had no effect on water intake, and affected neither the plasma chemistry parameters nor the organs' weight. We identified potential points of departure that could be used to establish an ARfD (decrease in body weight, body temperature, and muscle activity).

Safety evaluation of the food enzyme triacylglycerol lipase from the genetically modified Aspergillus luchuensis strain FL108SC.

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is produced with the genetically modified Aspergillus luchuensis strain FL108SC by Advanced Enzyme Technologies Ltd. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in an immobilised form for the production of modified fats and oils by interesterification. Genotoxicity tests did not raise safety concerns. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rodents. The Panel identified a no observed adverse effect level (NOAEL) at the highest dose of 774 mg TOS/kg body weight per day. As the food enzyme is used in an immobilised form and as any residual amounts of the food enzyme-total organic solids (TOS) are removed during the food manufacturing process, dietary exposure was not calculated. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Evaluating endocrine disrupting chemicals: A perspective on the novel assessments in CLARITY-BPA.

The Consortium Linking Academic and Regulatory Insights on Bisphenol A Toxicity (CLARITY-BPA) was a collaborative research effort to better link academic research with governmental guideline studies. This review explores the secondary goal of CLARITY-BPA: to identify endpoints or technologies from CLARITY-BPA and prior/concurrent literature from these laboratories that may enhance the capacity of rodent toxicity studies to detect endocrine disrupting chemicals (EDCs). A systematic literature search was conducted with search terms for BPA and the CLARITY-BPA participants. Relevant studies employed a laboratory rodent model and reported results on 1 of the 10 organs/organ systems evaluated in CLARITY-BPA (brain and behavior, cardiac, immune, mammary gland, ovary, penile function, prostate gland and urethra, testis and epididymis, thyroid hormone and metabolism, and uterus). Study design and findings were summarized, and a risk-of-bias assessment was conducted. Several endpoints and methods were identified as potentially helpful to detect effects of EDCs. For example, molecular and quantitative morphological approaches were sensitive in detecting alterations in early postnatal development of the brain, ovary, and mammary glands. Hormone challenge studies mimicking human aging reported increased susceptibility of the prostate to disease following developmental BPA exposure. Statistical analyses for nonmonotonic dose responses, and computational approaches assessing multiple treatment-related outcomes concurrently in linked hormone-sensitive organ systems, reported effects at low BPA doses. This review provided an opportunity to evaluate the unique insights provided by nontraditional assessments in CLARITY-BPA to identify technologies and endpoints to enhance detection of EDCs in future studies.

Abstract 3448: Discovery of HYP-2A,a 2nd- generation KRASG12C inhibitor exhibits potent in vivo efficacy in drug resistant tumor models

Abstract Although activating mutations in KRAS are well recognized as a hallmark of cancer, KRAS was considered an undruggable target for over 30 years after its discovery, due to the intrinsic characteristics of KRAS proteins. The pocket of KRAS is small and has a considerably smooth and shallow surface, resulting in difficulty for small molecule to bind to the protein. Recent accelerated approval of Sotorasib (AMG 510) by FDA marked the first marketed KRASG12C inhibitor for the treatment of 2nd line KRASG12C mutation-positive NSCLC patients. Although great clinical benefits were delivered by this breakthrough medicine, intrinsic or acquired drug resistances were developed in a large portion of patients treated. HYP-2A is a new chemical entity (NCE) being developed by Sichuan Huiyu Pharmaceuticals for the treatment of KRASG12C inhibitor resistant tumors. It is an orally bioavailable, potent KRASG12C inhibitor that shows very high anti-proliferation activities in NCI-H358 cell line and various Sotorasib and Adagrasib (MRTX 849) resistant tumor cell lines in vitro and demonstrates strong in vivo efficacy in the corresponding xenograft mouse models. It inhibits NCI-H358, H358 AMGR, Mia PaCa-2, PaCa-2 AMGR, KYSE-410, and SW1573 cell lines with IC50 values range from low double-digit to single-digit nanomolar. When dosing at 10 mpk via PO, HYP-2A demonstrates significant efficacy (TGI > 75%) in SW1573 Xenograph mouse model, whereas neither Sotorasib nor Adagrasib shows any obvious efficacy at 30 mpk with TGIs less than 20% in the same experiment. Similarly, when dosing at 10 mpk via PO, HYP-2A exhibits strong efficacy (TGI > 90%) in KYSE-410 Xenograph mouse model, while neither Sotorasib nor Adagrasib shows any significant efficacy (TGI < 50%) at 30 mpk via PO dosing in the same experiment. In an acute toxicity study in rats, the maximum tolerated dose (MTD) of HYP-2A is found higher than 30 mpk. In a two-week toxicity studies in rodents, rats are found well tolerated when treated with HYP-2A via PO at 3-10mpk (QD) for 14 consecutive days. The MTD of HYP-2A is determined higher than 10 mpk. In this presentation, we will highlight the research program that leads to the discovery of HYP-2A, which is currently under IND-enabling studies. Citation Format: Shoujun Chen, Xiaoming Qiang, Haibo Wang, Yong Xiong, Ke Liu, Dengming Liao, Nan Zhong, Mingdeng Liu, Xuemei Mu, Rudan Huang, Min Li, Dengwei Gui, Meilin Huang, Yuting Chen, Yuanfu Pan, Wengang Yao, Yingte Song, Zhongbo Wang, Wenbin Wang, Jun Liu, Xingchi Yu, Zhen Liang, Yike Yuan, Zhao Ding. Discovery of HYP-2A,a 2nd- generation KRASG12C inhibitor exhibits potent in vivo efficacy in drug resistant tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3448.

Discovery of Novel Human Constitutive Androstane Receptor Agonists with the Imidazo[1,2-a]pyridine Structure.

The nuclear constitutive androstane receptor (CAR, NR1I3) plays significant roles in many hepatic functions, such as fatty acid oxidation, biotransformation, liver regeneration, as well as clearance of steroid hormones, cholesterol, and bilirubin. CAR has been proposed as a hypothetical target receptor for metabolic or liver disease therapy. Currently known prototype high-affinity human CAR agonists such as CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) have limited selectivity, activating the pregnane X receptor (PXR) receptor, a related receptor of the NR1I subfamily. We have discovered several derivatives of 3-(1H-1,2,3-triazol-4-yl)imidazo[1,2-a]pyridine that directly activate human CAR in nanomolar concentrations. While compound 39 regulates CAR target genes in humanized CAR mice as well as human hepatocytes, it does not activate other nuclear receptors and is nontoxic in cellular and genotoxic assays as well as in rodent toxicity studies. Our findings concerning potent human CAR agonists with in vivo activity reinforce the role of CAR as a possible therapeutic target.

Transcriptional profiling of the developing rat ovary following intrauterine exposure to the endocrine disruptors diethylstilbestrol and ketoconazole

Exposure to endocrine-disrupting chemicals (EDCs) during development may cause reproductive disorders in women. Although female reproductive endpoints are assessed in rodent toxicity studies, a concern is that typical endpoints are not sensitive enough to detect chemicals of concern to human health. If so, measured endpoints must be improved or new biomarkers of effects included. Herein, we have characterized the dynamic transcriptional landscape of developing rat ovaries exposed to two well-known EDCs, diethylstilbestrol (DES) and ketoconazole (KTZ), by 3’ RNA sequencing. Rats were orally exposed from day 7 of gestation until birth, and from postnatal day 1 until days 6, 14 or 22. Three exposure doses for each chemical were used: 3, 6 and 12 µg/kg bw/day of DES; 3, 6, 12 mg/kg bw/day of KTZ. The transcriptome changed dynamically during perinatal development in control ovaries, with 1137 differentially expressed genes (DEGs) partitioned into 3 broad expression patterns. A cross-species deconvolution strategy based on a mouse ovary developmental cell atlas was used to map any changes to ovarian cellularity across the perinatal period to allow for characterization of actual changes to gene transcript levels. A total of 184 DEGs were observed across dose groups and developmental stages in DES-exposed ovaries, and 111 DEGs in KTZ-exposed ovaries across dose groups and developmental stages. Based on our analyses, we have identified new candidate biomarkers for female reproductive toxicity induced by EDC, including Kcne2, Calb2 and Insl3.

An In Vitro Model to Investigate the Potential of Solid Dispersions to Form Pharmacobezoars.

The formation of pharmacobezoars from suspensions of spray-dried amorphous solid dispersions (SD-ASDs) of new chemical entities (NCEs) and hydroxypropyl methylcellulose acetate succinate (HPMC-AS) represents a non-compound related adverse effect in preclinical oral toxicity studies in rodents. Whereas the contribution of the insolubility of the carrier polymer to this process taking place in the acidic environment of the rodent stomach is conclusive, unawareness of the extent of in vivo pharmacobezoar formation is adverse. In order to evaluate the risk of pharmacobezoar formation before in vivo administration, we subsequently introduce an in vitro model to assess the agglomeration potential of solid dispersions. To verify that the pharmacobezoar formation potential can be assessed based on the observed agglomeration potential, we conducted a sequence of experiments with two HPMC-AS-based SD-ASD formulations. In vitro, we found their different in vivo pharmacobezoar formation potential reflected by a significantly increased agglomerated mass of formulation 1 per day compared to formulation 2. In order to find an approach to reduce the agglomeration potential of solid dispersion from suspensions, we further applied the model to investigate the impact of the viscosity of the vehicle used to prepare suspensions on agglomerate formation.

Abstract LB144: Antagonism of SF-1 as a potential targeted therapy for malignant Leydig cell tumors

Abstract Leydig cell tumors (LCT), originating from androgen-producing interstitial Leydig cells of the testis, represent about 3% of all testicular cancers. LCT belong to the family of sex cord stromal tumors (SCST), a collection of tumors formed in the supporting tissue within the ovaries or testes. Although 90% of LCT are considered benign and can be cured by orchiectomy, in adult patients about 10% of LCT are malignant and metastasis is common. Non-resectable metastatic disease is poorly responsive to radiation and chemotherapy and patients are advised to seek clinical trials. Steroidogenic Factor 1 (SF-1, or NR5A1) is a transcription factor that is essential for the development of the adrenal gland and gonads. SF-1 mutations result in disorders of sexual development, ovarian failure, and adrenal insufficiency. SF-1 is necessary for development of fetal and adult Leydig cells and is strongly expressed in LCT. Significant data support the role of SF-1 in adrenocortical cancer (ACC). To address the need for a targeted therapy in ACC and other SF-1-dependent malignancies, Orphagen has identified potent small molecule antagonists to SF-1*. Here, using R2C, a rat Leydig tumor cell line, we demonstrate that small molecule antagonists of SF-1 inhibit Leydig tumor cell proliferation in vitro and in vivo. OR-449, an orally available inhibitor of SF-1 transcriptional activity (SF-1 Luc IC50 = 16 nM) and OR-907S, a probe SF-1 antagonist (SF-1 Luc IC50 = 22 nM), exhibit striking anti-proliferative activity in R2C cell cultures, inhibiting DNA synthesis by >90% at 1 μM with estimated IC50’s of 0.068 μM and 0.074 μM, respectively. OR-907R, the ~100-fold less active stereoisomer of OR-907S (SF-1 Luc IC50 = 2 μM) was significantly less active in the R2C proliferation assay (estimated IC50 >10 μM). Moreover, in cell lines lacking SF-1 expression, such as HEK293, SF-1 antagonists have no anti-proliferative activity up to 20 μM, suggesting that the anti-proliferative effect on R2C is SF-1-mediated and not due to cytotoxicity. Furthermore, OR-449 completely blocks R2C xenograft tumor growth in immunocompromised mice at an oral dose of 30 mg/kg/day. OR-449 also dose-dependently regulates expression of SF-1 responsive genes, a mRNA signature first identified in R2C culture by comparison of the activity of OR-907S and OR-907R at 1 μM. OR-449 has excellent pharmacokinetic properties and is well-tolerated in repeat dosing toxicity studies in rodents and non-rodents*. These results highlight SF-1 antagonism as a novel targeted therapeutic approach with potential utility in the treatment of LCT and other SCST. OR-449 is currently in IND-enabling studies in order to enter the clinic by the end of 2022. * P. Crowe, et al. A novel steroidogenic factor-1 antagonist, OR-449, as a targeted therapy for adrenocortical cancer. ENDO 2021: J Endocr Soc, Vol5, Supplement_1, A1010 Citation Format: Haiyan Tao, Emily Eastwood, Raymond G. Fox, Neil Raheja, Paul D. Crowe, Scott M. Thacher. Antagonism of SF-1 as a potential targeted therapy for malignant Leydig cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB144.

Pharmacological effects and safety of Andrographis paniculata (Burm.f.) Nees.

Andrographis paniculata (Burm.f.) Nees (AP) is widely used in most Asian and some Western countries. However, its main effects and underlying pharmacological mechanism have not been thoroughly characterized, and its safety has not been sufficiently investigated. The present study aimed to predict and visualize the potential targets and pathways, clarify the main pharmacological effects, and investigate the toxicological properties of AP extract (APE). First, ingenuity pathway analysis (IPA) was performed to directly predict AP's therapeutic targets and pathways; main pharmacological effects of AP were speculated based on IPA results and confirmed by pharmacodynamics experiments. Rodent toxicity studies were then performed through administration of a single dose of 10g/kg or daily doses of 2, 1, or 0.5g/kg for 8weeks to evaluate the safety of APE, and a similar repeated-dose study was performed using dogs with doses equal to half of the above-mentioned doses. Thus, repeated-dose toxicity studies were performed with both rodents and nonrodents. The IPA analysis and confirmatory pharmacodynamics experiments revealed that the main pharmacological effect of APE was anti-inflammation, which might be achieved by influencing various targets (e.g., AR, AKT, and BAX) and pathways (IL-8). In the single-dose toxicity test, no death or abnormal consequences were observed, and maximum tolerated dose of APE was 10g/kg. Results from the repeated-dose toxicity tests did not reveal any obvious toxic effects from the repeated daily intragastric administration of APE at 1g/kg for 8weeks. In conclusion, APE at a dose of 1g/kg did not exert any adverse effects, and administration of APE could be beneficial for the inflammatory diseases' treatment. PRACTICAL APPLICATION: Andrographis paniculata (Burm.f.) Nees is a plant that exerts clearing and detoxification effects and is widely used around the world, but a comprehensive analysis of its efficacy and safety is needed.

Acute and 28-Day Repeated Dose Subacute Toxicological Evaluation of Coroprotect Dry Syrup in Rodents

Introduction: Ayurvedic medications are extensively used across the world for illness prevention and treatment. The active ingredients of Coroprotect dry syrup are Jethimadh ext, Ajma ext., Tulsi ext., Pipper ext., Sunth ext., Kalamari ext., Vasa ext., Taj ext., Lavang ext., Nagarvel ext., Haridra ext., Kakamachi ext., Pushkarmool ext., and Phudina ful, etc. Because there is no scientific proof that this formulation is safe, a comprehensive toxicity study in wistar rats was conducted. Objective: In Rodents, acute and subacute toxicity of coroprotect dry syrup was studied after a single and repeated 28-day oral dosage Administration. Method: Six Wistar female rats were given a single dose of coroprotect dry syrup at 2000 mg/kg by oral gavage, while doses of 100, 200 and 500 mg/kg/day were given over the course of 28 days in a repeated-dose subacute toxicity study. Results: In an acute toxicity investigation involving coroprotect dry syrup administration, no therapies fatalities or toxic symptoms were found. In the repeated dosage study, there were no substantial differences in body weight fluctuations, food/water ingestion, hematology, or clinical biochemistry content among the control as well as coroprotect dry syrup groups. There were no gross pathological abnormalities or variations in relative organ weights between the control and coroprotect dry syrup groups. Histopathological investigation revealed no abnormalities after therapy with coroprotect dry syrup. Conclusion: In a repeated dosage toxicity study in rodents, the coroprotect dry syrup was determined to be safe at all dose levels.

Early detection of urinary bladder carcinogens in rats by immunohistochemistry for γ-H2AX: a review from analyses of 100 chemicals.

In safety evaluations of chemicals, there is an urgent need to develop short-term methods to replace long-term carcinogenicity tests. We have reported that immunohistochemistry for γ-H2AX, a well-established biomarker of DNA damage, can detect bladder carcinogens at an early stage using histopathological specimens from 28-day repeated-dose oral toxicity studies in rats. Given the markedly low level of γ-H2AX formation in the bladder urothelium of untreated rats, an increase in γ-H2AX-positive cells following chemical exposure can be relatively easy to identify. Among the 100 compounds examined to date, bladder carcinogens can be detected with high sensitivity (33/39; 84.6%) and specificity (58/61; 95.1%). As expected, γ-H2AX formation levels tended to be high following exposure to genotoxic bladder carcinogens, whereas nongenotoxic bladder carcinogens also increased the number of γ-H2AX-positive cells, probably through secondary DNA damage associated with sustained proliferative stimulation. γ-H2AX formation in the bladder urothelium reflects species differences in susceptibility to bladder carcinogenesis between rats and mice and shows a clear dose-dependency associated with the intensity of tumor development as well as high reproducibility. Some of the bladder carcinogens that showed false-negative results in the evaluation of γ-H2AX alone could be detected by combined evaluation with immunostaining for bladder stem cell markers, including aldehyde dehydrogenase 1A1. This method may be useful for the early detection of bladder carcinogens, as it can be performed by simple addition of conventional immunostaining using formalin-fixed paraffin-embedded tissues from 28-day repeated-dose toxicity studies in rodents, which are commonly used in safety evaluations of chemical substances.

Acute and 28-Day Repeated Dose Subacute Toxicological Evaluation of Coroprotect Tablet in Rodents

Introduction: Ayurvedic medications are extensively used across the world for illness prevention and treatment. The components found in the Coroprotect tablet are Ashwagandha ext., Suddhashilajit ext., Giloyext., Dadim ext., Haridra ext., Saragavo ext., Brahmi ext., Papaya ext., Methiext., Punarnava ext., Kalmegh ext.etc. all are found to be safe and used in Ayurvedic system since many years. Objective: In Rodents, acute and subacute toxicity of coroprotect tablet was studied after a single and repeated 28-day oral dosage administration. Method: In acute toxicity testing, 2000 mg/kg single dose of coroprotect tablet have been employed, whereas doses of 100, 200, 400mg/kg have been utilized in subacute toxicity testing over a 28-day period in rodents. Result: With coroprotect tablet administration, no treatment-related fatalities or toxic signshave been identified in an acute toxicity investigation. There have been no significant variations in body weight changes, water/food intake, haematology, or clinical biochemistry content among the control and coroprotect tablet groups in the repeated dosage research. Between the control and coroprotect pill groups, no gross pathological abnormalities or differences in relative organ weights were found. With coroprotect tablet therapy, histopathological investigation indicated no abnormalities. Conclusion: In a repeated dosage toxicity study in rodents, the coroprotect tablet was determined to be safe at all dose levels.

Evaluation of the Antidote Activity of Nimbu swarasa (Citrus limon Linn.) on Jayapala Seed (Croton tiglium Linn.) Induced Toxicity in Wistar Albino Rats

Abstract: Background: Seeds of Jayapala (Croton tiglium Linn.) is a toxic compound which is commonly used in several Ayurvedic formulations following proper detoxification methods as mentioned in the Ayurvedic classics. Adarsha nigantu, one of the treatises of Ayurveda explains Nimbu swarasa (Lemon juice) as an antidote for Jayapala. The present study was aimed to determine the antidote activity of Nimbu swarasa (Citrus limon linn.) on Jayapala (Croton tiglium Linn.) seed induced toxicity on Wistar albino rats. Materials and Methods: A sub acute toxicity study was conducted on Wistar rats following the repeated dose 28 day oral toxicity study in rodents, 407 OECD guidelines which included administration of Jayapala seeds alone in one group and Jayapal seeds along with Nimbu swarasa in different doses in the other groups. Different haematological and biochemical parameters along with histopathology of important organs were carried out to access the antidote effect of Nimbu swarasa. Observation and Results: The repeated administration of Jayapala seed powder caused significant elevation in the serum liver enzymes like SGOT and SGPT (*P<0.5) and marked rise in the serum creatinine level. The co-administration of antidote Nimbu swarasa significantly attenuated Jayapala seed induced increase in the liver enzymes and reduced serum creatinine level. Histopathological examination of colon and jejunum revealed moderate epithelial cell erosions in Jayapala seeds administered group, whereas Nimbu swarasa caused mild to moderate protection in a dose dependant manner. Sections of spleen showed remarkable increase in the proportion of white pulp in toxicant group, whereas the Nimbu swarasa at higher dose level showed marked decrease in the white pulp proportion. The hepatic sections of toxicant group displayed mild cell infiltration and fatty changes in the hepatocyte with mild diffused necrosis whereas the sections of co-administration of antidotes showed normal cytoarchitecture. Conclusion: All the above findings are suggestive of Nimbu swarasa being an effective antidote against Jayapala induced toxicity. Key words: Antidote, Jayapala seeds, Nimbu swarasa, Haematological parameters, Histopathological examination, Toxicity.

Neuropathology Evaluation in Juvenile Toxicity Studies in Rodents: Comparison of Developmental Neurotoxicity Studies for Chemicals With Juvenile Animal Studies for Pediatric Pharmaceuticals.

The developmental neuropathology examination in juvenile toxicity studies depends on the nature of the product candidate, its intended use, and the exposure scenario (eg, dose, duration, and route). Expectations for sampling, processing, and evaluating neural tissues differ for developmental neurotoxicity studies (DNTS) for chemicals and juvenile animal studies (JAS) for pediatric pharmaceuticals. Juvenile toxicity studies typically include macroscopic observations, brain weights, and light microscopic evaluation of routine hematoxylin and eosin (H&E)-stained sections from major neural tissues (brain, spinal cord, and sciatic nerve) as neuropathology endpoints. The DNTS is a focused evaluation of the nervous system, so the study design incorporates perfusion fixation, plastic embedding of at least one nerve, quantitative analysis of selected brain regions, and sometimes special neurohistological stains. In contrast, the JAS examines multiple systems, so neural tissues undergo conventional tissue processing (eg, immersion fixation, paraffin embedding, H&E staining only). An "expanded neurohistopathology" (or "expanded neuropathology") approach may be performed for JAS if warranted, typically by light microscopic evaluation of more neural tissues (usually additional sections of brain, ganglia, and/or more nerves) or/and special neurohistological stains, to investigate specific questions (eg, a more detailed exploration of a potential neuroactive effect) or to fulfill regulatory requests.

POS-442 DISCOVERY OF CHK-336: A FIRST-IN-CLASS, LIVER-TARGETED, SMALL MOLECULE INHIBITOR OF LACTATE DEHYDROGENASE FOR THE TREATMENT OF PRIMARY HYPEROXALURIA

Primary hyperoxalurias (PH) 1-3 are a group of autosomal recessive disorders involving excess hepatic oxalate production and result in frequent kidney stone formation, progressive CKD and in its most severe form, PH1 can lead to ESRD at a young age. Few therapeutic options currently exist for these patients. Lactate dehydrogenase (LDH) catalyzes the final and only committed step in hepatic oxalate synthesis and therefore represents a potential therapeutic target for all forms of PH. Herein we describe the profile of a potent, highly selective small molecule LDH inhibitor which effectively lowers urinary oxalate in a mouse PH1 model and demonstrates an excellent preclinical pharmacokinetic and safety profile. CHK-336 was evaluated in biochemical and cellular LDH activity assays across species and in vivo models of hyperoxaluria, including a novel PH1 mouse generated by Agxt deletion using CRISPR/Cas9. Additional characterization of drug properties, including off-target activities, ADME, safety pharmacology and toxicity in rodents was also performed. CHK-336 demonstrates potent and selective inhibition of LDH in human enzyme assays (IC50 = 0.4 nM) and in cryopreserved hepatocyte assays across species (IC50 = 80 – 131 nM). To minimize the potential for extra-hepatic LDH inhibition, a liver-targeted tissue distribution profile was engineered into the molecule. CHK-336 demonstrates exceptional liver-targeting across species at therapeutic doses (liver/plasma ratio, ~150 (rat, mouse) to >750 (cyno)) and active uptake into rat, cyno and human hepatocytes. Liver targeting is mediated by OATP-uptake into hepatocytes and tight binding to LDH results in a long liver half-life via target-mediated drug disposition that supports once-daily oral dosing. Furthermore, CHK-336 demonstrates a favourable off-target safety and ADME profile with minimal risk of important drug/drug interactions. In the PH1 mouse model, CHK-336 produced significant and dose-dependent reductions in urinary oxalate at low, once-daily oral doses to levels observed in wild-type mice. Wide safety margins were established in rodent toxicity studies to support continued development of CHK-336. By potently blocking LDH, the terminal step in hepatic oxalate synthesis, along with the engineering of a liver-targeted tissue distribution profile, CHK-336 is a promising oral small molecule development candidate with the potential to treat patients with hyperoxaluria.

Toxicological evaluation of banana and milk combination as incompatible diet - An experimental exploration of Samyoga viruddha concept

BackgroundFood is the basic requirement and an essential part of all living beings which sustains life. A lot of emphasize has been given to food in the classics of Ayurveda and a detailed description of food which are both wholesome and unwholesome have been described. Viruddhahara (incompatible food) is a unique concept explained in Ayurveda which, in long run, may be harmful to the body. Objective(s)Intake of Kadaliphala (Musa paradisiaca. Linn, a variety of banana) and cow milk is an example of Samyoga Viruddha (incompatibility with reference to combination of things). This combination is routinely consumed by people. The present study was planned to evaluate the toxicological implication of their combination on wistar rats. Materials and methodsA subacute toxicity study was conducted on Wistar rats following the repeated dose 28-day oral toxicity study in rodents, 407 - OECD guidelines. Different haematological and biochemical parameters along with histopathology of important organs were carried out to assess the toxicological implication of the combination. ResultsRepeated administration of the combination of Kadaliphala (Banana) and cow milk showed statistically significant increase in SGOT & urea and statistically significant decrease in creatinine. Significant decrease was observed in the food intake, faecal wet weight and faecal water in the 7th day of study, food conversion ratio in the 14th day of study, in the food intake, faecal wet weight and faecal water in the 21st day of study and significant decrease in the food conversion ratio in the 28th day of study. In histopathological examination, the test drug administered group showed mild to moderate myocarditis in the sections from heart. In sections from liver of two rats of test group, diffused micro fatty changes were observed. In sections from spleen; mild to moderate increase in the white pulp portion was observed. ConclusionMarked variation in SGOT, urea and creatinine levels and alteration in sections of heart, liver and spleen are indicative of mild toxicological implications of the combination of banana and milk. Continuous intake of this incompatible combination may hence prove harmful to the body.