Toxicokinetic Studies Research Articles

A novel, sensitive, and fast ultra-high-performance liquid chromatography tandem mass spectrometry method for TNG908 determination in dog plasma and pharmacokinetic study.

TNG908 is a potent and selective protein arginase methyltransferase 5 (PRMT5) inhibitor that is currently going through phase I/II clinical development for the treatment of non-small cell lung cancer. To facilitate pharmacokinetic and toxicokinetic studies of TNG908, here, we reported an ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the detection of TNG908 in dogs. The dog plasma samples were precipitated by acetonitrile and analyzed using a Waters ACQUITY BEH C18 column combined with a Thermo triple quadrupole mass spectrometer. The mobile phase consisted of 0.1% formic acid solution and acetonitrile, at a flow rate of 0.3mL/min. TNG908 and internal standard were monitored by selective reaction monitoring (SRM) with m/z 410.2 > 150.1 and m/z 394.2 > 278.1, respectively. The method demonstrated excellent linearity over the concentration range of 1-1000 ng/mL, with a correlation coefficient greater than 0.995. Acetonitrile-mediated protein precipitation showed high extraction efficiency and a recovery above 80%. The validated assay was further applied to measure TNG908 in dog plasma after oral and intravenous administration and achieved success. The obtained pharmacokinetic parameters indicated low clearance of TNG908 (3.7 ± 0.8mL/min/kg) and moderate oral bioavailability (>36.4%).

Sample Preparation for Pharmaceuticals using A Bioanalytical Method: A Review

The process of developing a method that will allow a compound of interest to be located and measured in a biological matrix is known as bioanalytical method development. A substance can frequently be measured using a variety of techniques, and selecting an analytical technique requires careful thought. Different extraction techniques, such as liquid-liquid extraction, solid phase extraction (SPE), and protein precipitation, are used to analyse drugs and their metabolites in a biological matrix. Samples from these extraction methods are spiked with calibration (reference) standards and quality control (QC) samples. The process of establishing if a quantitative analytical method is suitable for biomedical applications is known as bioanalytical method. includes all processes that will be presented measuring analytes quantitatively in particular biological samples such blood plasma, serum, or urine. To evaluate a drug's effectiveness and safety, clinical and non-clinical toxicokinetic and pharmacokinetic studies are used. Therefore, in order to produce accurate results, it is crucial that the applied bioanalytical procedures utilised are thoroughly defined, verified, and documented to a suitable quality. An overview of the development and validation of bioanalytical methods is given in this article, along with key considerations for each stage of method validation. The created procedure is then verified. Evaluation and interpretation of bioavailability, bioequivalence, pharmacokinetic, and pharmacodynamic investigations heavily rely on bioanalytical validations. where various parameters are carried out, including accuracy, precision, selectivity, sensitivity, reproducibility, and stability.

Application of newly developed and validated UPLC-MS/MS method for pharmacokinetic study of ROS1/NTRK inhibitor taletrectinib in beagle dog plasma

Taletrectinib is a potent selective ROS and pan-NTRK tyrosine kinase inhibitor (TKI) and has been developed to treat non-small cell lung cancer (NSCLC). To facilitate pharmacokinetic and toxicokinetic studies of taletrectinib, we developed a procedure for ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to detect the plasma level of taletrectinib in dogs. This assay procedure was validated in compliance with FDA guidance. The dog plasma samples were spiked with internal standard (IS), followed by protein precipitation, and analyzed using a Waters ACQUITY BEH C18 column coupled to a Thermo triple quadrupole mass spectrometer. Separation was executed using the acetonitrile-0.1 % formic acid solution with gradient elution, at a flow rate of 0.4 mL/min. Taletrectinib and IS were monitored by multiple reaction monitoring (MRM) with m/z 406.2 > 349.2 and m/z 441.2 > 138.1, respectively. The procedure demonstrated excellent linearity with a correlation coefficient greater than 0.999 within the concentration range of 0.2–200 ng/mL. The inter- and intra-day accuracy ranged from −5.25 % to 5.26 %, and the precision was below 6.39 %. Acetonitrile-mediated protein precipitation showed high extraction efficiency and a recovery above 85 %. The procedure was then applied to quantify taletrectinib in beagle dog plasma after oral and intravenous doses and achieved success. The obtained pharmacokinetic parameters indicated high bioavailability of taletrectinib (>85 %) and extensive tissue distribution (>40 L/kg).

Calcium kinetic rates in Caucasian males and females from birth to adulthood

Calcium plays an important role in bone physiology and its kinetics change over lifetime. The analysis of calcium deposition and release through stable isotope techniques has guided recommendations on nutritional uptake for overall health. In addition, calcium kinetics have great relevance for toxicokinetic studies of bone-seeking elements (e.g, aluminium and lead) since these elements use common uptake and release pathways. While the impact of many factors on calcium kinetics have been investigated individually, a consolidated age- and sex-dependent kinetic description amenable for toxicokinetic modeling, however, is still lacking. Motivated by this need, we systematically reviewed the existing literature on calcium kinetics and assembled a large and consistent dataset. Then, building on the work of O'Flaherty in the 1990s, we formulated age- and sex-dependent functions describing calcium deposition, release, net retention, and mass. This description represents the current knowledge on calcium kinetics in a reference individual of Caucasians as most data was from this population.

Potential of 6′‑hydroxy justicidin B from Justicia procumbens as a therapeutic agent against coronavirus disease 2019

BackgroundSince the onset of the coronavirus disease 2019 (COVID-19) pandemic, remarkable advances have been made in vaccine development to reduce mortality. However, therapeutic interventions for COVID-19 are comparatively limited despite these intensive efforts. Furthermore, the rapid mutation capability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a characteristic of its RNA structure, has led to the emergence of multiple variants, necessitating a shift from a predominantly vaccine-centric approach to one that encompasses therapeutic strategies. 6′-Hydroxy justicidin B (6′-HJB), an arylnaphthalene lignan isolated from Justicia procumbens, a traditional Chinese medicine, is known for its antiviral properties. Hypothesis/PurposeThe aim of the present study was to assess the effectiveness and safety of 6′-HJB against SARS-CoV-2 in order to determine its potential as a therapeutic agent against COVID-19. MethodsThe efficacy of 6′-HJB was evaluated both in vitro using Vero and Calu-3 cell lines and in vivo using ferrets. The safety assessment included toxicokinetics, safety pharmacology, and Good Laboratory Practice (GLP)-compliant toxicity evaluations following single- and repeated-dose toxicity studies in dogs. ResultsThe anti-SARS-CoV-2 efficacy of 6′-HJB was evaluated through dose-response curve (DRC) analysis using immunofluorescence; 6′-HJB demonstrated superior inhibition of SARS-CoV-2 growth and lower cytotoxicity than remdesivir. In SARS-CoV-2-infected ferret, 6′-HJB showed efficacy comparable to that of the positive control, Truvada. Further GLP toxicity studies corroborated the safety profile of 6′-HJB. Single-dose and 4-week repeated oral toxicity studies in Beagle dogs demonstrated minimal harmful effects at the highest dosages. The lethal dose of 6′-HJB exceeded 2,000 mg kg-1 in Beagle dogs. Toxicokinetic and GLP safety pharmacology studies demonstrated no adverse effects of 6′-HJB on metabolic processes, respiratory or central nervous systems, or cardiac functions. ConclusionThis research highlights both the antiviral efficacy and safety profile of 6′-HJB, underscoring its potential as a novel COVID-19 treatment option. The potential of 6′-HJB was demonstrated using modern scientific methodologies and standards.

134. Reduction of mycotoxin exposure with an algae- and clay-based decontaminant in a toxicokinetic study on broiler chickens

134. Reduction of mycotoxin exposure with an algae- and clay-based decontaminant in a toxicokinetic study on broiler chickens

13-Week Repeated Oral Toxicity and Toxicokinetic Studies of Rabeprazole Sodium and Sodium Bicarbonate Combination in Dogs.

The subchronic toxicity and toxicokinetics of a combination of rabeprazole sodium and sodium bicarbonate were investigated in dogs by daily oral administration for 13 consecutive weeks with a 4-week recovery period. The dose groups consisted of control (vehicles), (5 + 200), (10 + 400), and (20 + 800) mg/kg of rabeprazole sodium + sodium bicarbonate, 20mg/kg of rabeprazole sodium only, and 800mg/kg of sodium bicarbonate only. Esophageal ulceration accompanied by inflammation was observed in only one animal in the male (20 + 800) mg/kg rabeprazole sodium + sodium bicarbonate group. However, the severity of the ulceration was moderate, and the site of occurrence was focally extensive; thus, it was assumed to be a treatment-related effect of rabeprazole sodium + sodium bicarbonate. In the toxicokinetics component of this study, systemic exposure to rabeprazole sodium (AUClast and Cmax at Day 91) was greater in males than females, suggesting sex differences. AUClast and Cmax at Day 91 were increased compared to those on Day 1 in a dose-dependent manner. A delayed Tmax and no drug accumulation were observed after repeated dosage. In conclusion, we suggest under the conditions of this study that the no-observed-adverse-effect level (NOAEL) of the combination of rabeprazole sodium + sodium bicarbonate in male and female dogs is (10 + 400) and (20 + 800) mg/kg, respectively.

INTASYL PH-762: PD-1 intratumoral immunotherapy for cutaneous carcinoma.

TPS9620 Background: Immune checkpoint-targeted antibodies directed at PD-1 or PD-L1 block co-inhibitory receptors expressed by anti-tumor T cells, breaking immune tolerance against tumor cells and generating cancer immunity. Intratumoral (IT) immunotherapy aims to use the tumor as a ‘self-vaccine’. Local immune stimulation may induce robust priming of an anti-tumor immune response and generate abscopal tumor responses, mediated by circulating activated anti-tumor immune cells. Local delivery of immunotherapy minimizes systemic exposure and off-target toxicities and may decrease tumor size and improve surgical morbidity. PH-762 is an INTASYL compound designed to precisely silence PD-1 mRNA. INTASYL is a patented, self-delivering RNAi technology platform designed to impart specific properties to small interfering RNAs. PH-762’s unique structural and chemical modifications ensure an optimized cell and tissue uptake profile with IT administration. In vitro investigations have demonstrated efficient uptake of PH-762 by human T cells, silencing of PD-1 mRNA and subsequent protein reduction. Preclinical studies have shown that IT injections of murine-targeted PH-762 (mPH-762) can silence PD-1 mRNA in T cells within the tumor and increase the secretion of IFN-γ. mPH-762 was well tolerated at the maximum administered dose and treatment with mPH-762 provided robust and statistically significant inhibition of tumor growth. Toxicokinetic studies conducted in marmoset monkeys demonstrated that PH-762, when administered intravenously at doses of up to 147 mg/kg, is well-tolerated. No cytokine-release associated cytokines were detected in the plasma of treated monkeys at this dose. Methods: This open-label Phase 1 clinical study (NCT 06014086) is designed to evaluate the safety and tolerability of neoadjuvant use of IT PH-762 in cutaneous squamous cell carcinoma, melanoma, or Merkel cell carcinoma, to determine the pharmacokinetic profile of PH-762 after IT injection, to observe pathologic and immunologic tumor responses, and to determine the recommended dose for development. Escalating dose concentrations of PH-762 (from 1.14 mg/mL through 22.00 mg/mL) are tested serially in cohorts of 3 patients each. Patients receive IT PH-762 once weekly, 4 times over a 3-week period prior to surgical excision, which occurs 5 weeks after the initial injection. Following excision of the tumor, patients are followed for approximately 11 weeks. Tumor changes are evaluated per iRECIST criteria and pathological response. Immunological response in tumor tissue and blood samples are assessed as secondary endpoints. This clinical study, enrolling patients since November 2023, will establish the basis for continued clinical development of PH-762. Clinical trial information: 06014086.

Repeated-dose toxicity and toxicokinetic study of isobutylparaben in rats subcutaneously treated for 13weeks.

Parabens have historically served as antimicrobial preservatives in a range of consumables such as food, beverages, medications, and personal care products due to their broad-spectrum antibacterial and antifungal properties. Traditionally, these compounds were believed to exhibit low toxicity, causing minimal irritation, and possessing limited sensitization potential. However, recent evidence suggests that parabens might function as endocrine-disrupting chemicals (EDCs). Consequently, extensive research is underway to elucidate potential human health implications arising from exposure to these substances. Among these parabens, particular concerns have been raised regarding the potential adverse effects of iso-butylparaben (IBP). Studies have specifically highlighted its potential for inducing hormonal disruption, significant ocular damage, and allergic skin reactions. This study aimed to evaluate the prolonged systemic toxicity, semen quality, and estrus cycle in relation to endocrine disruption endpoints, alongside assessing the toxicokinetic behavior of IBP in Sprague-Dawley rats following a 13-week repeated subcutaneous administration. The rats were administered either the vehicle (4% Tween 80) or IBP at dosage levels of 2, 10, and 50mg/kg/day for 13weeks. Blood collection for toxicokinetic study was conducted on three specified days: day 1 (1st), day 30 (2nd), and day 91 (3rd). Systemic toxicity assessment and potential endocrine effects were based on various parameters including mortality rates, clinical signs, body weights, food and water consumption, ophthalmological findings, urinalysis, hematological and clinical biochemistry tests, organ weights, necropsy and histopathological findings, estrus cycle regularity, semen quality, and toxicokinetic behavior. The findings revealed that IBP induced local irritation at the injection site in males at doses ≥ 10mg/kg/day and in females at 50mg/kg/day; however, systemic toxicity was not observed. Consequently, the no-observed-adverse-effect level (NOAEL) for IBP was determined to be 50mg/kg/day in rats of both sexes, indicating no impact on the endocrine system. The toxicokinetics of IBP exhibited dose-dependent systemic exposure, reaching a maximum dose of 50mg/kg/day, and repeated administration over 13weeks showed no signs of accumulation.

Abstract 1870: LCB36, a bi-specific antibody drug conjugate (BsADC) utilizing ConjuAll conjugation technology and proprietary prodrug payload selectively activated in cancer cells for treating B-cell blood cancers expressing CD20 and/or CD22

Abstract Although frontline chemoimmunotherapy elicits responses in many B-Cell malignancy patients, treatment options remain limited, and clinical outcomes are suboptimal for those with relapsed or refractory diseases. Minimal residual disease (MRD) can lead to disease relapse despite achieving complete remission through targeted therapy, and tumor heterogeneity is responsible for MRD and compromises the efficacy of targeted therapy, especially in B-cell malignancies. Antibody-drug conjugates (ADCs) play a crucial role in oncology indications, and bispecific antibodies (BsAbs) represent one of the fastest-growing classes of next generation antibody therapeutics for cancer therapy. Several advantages of targeting dual tumor-associated antigens (TAAs) with BsAbs and bi-specific antibody drug conjugates (BsADCs) have been proposed, including improved efficacy, heightened tumor cell specificity, and reduced side effects in normal tissues. Notably, the ability of BsADCs to bind to cancer cells expressing even just one antigen has prompted further investigation into their efficacy in heterogeneous cancer models. LCB36 is a BsADC composed of a BsAb targeting both clinically validated CD20 and CD22, which are expressed in B-Cell malignancy, and a proprietary DNA cross-linking PBD prodrug that is site-specifically conjugated to the BsAb using ConjuAll technology. We assessed the expression levels of CD20 and CD22 in the cancer cell lines. While most cells expressed both antigens, some populations showed a low expression level of CD20 and a high expression level of CD22, while others exhibited the opposite pattern, suggesting heterogeneous expression of the two antigens within the same cell line. To confirm the heterogeneity of the two antigens in blood cancer patients, we analyzed single-cell sequencing data [GSE132509]. Each patient had CD20 and CD22 double-positive cells along with CD20 or CD22 single-positive cells, suggesting the possibility of relapse due to MRD following CD20 or CD22 single-targeted therapy. LCB36 demonstrated superior efficacy in CD20 and CD22 double-positive cell lines and cell-line derived xenografts (CDXs) models compared to ADCs targeting either CD20 or CD22, while showing comparable efficacy to single-target ADCs in CD20 or CD22 single-positive cancer cell lines and CDXs. Pharmacokinetic and toxicokinetic studies of LCB36 were analyzed in rats and Cynomolgus monkeys, respectively, using LC-MS/MS bioanalysis. Preliminary toxicity studies in rats and Cynomolgus monkeys demonstrated that LCB36 is well tolerated. In conclusion, LCB36, composed of a BsAb that binds to two clinically validated targets, showed efficacy at well-tolerated doses and has the potential to increase event-free survival periods by reducing MRD. Citation Format: Wihak Kim, Namjeong Choi, Cul-Woong Chung, Hwanhee Oh. LCB36, a bi-specific antibody drug conjugate (BsADC) utilizing ConjuAll conjugation technology and proprietary prodrug payload selectively activated in cancer cells for treating B-cell blood cancers expressing CD20 and/or CD22 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1870.

Toxicokinetic study of scandium oxide in rats

Canada has recently invested in the large-scale exploitation of scandium oxide. However, there are no studies available to date to understand its toxicokinetics in the animal or human body, which is necessary to assess exposure and health risks. The aim of this research was to investigate the toxicokinetics of absorbed scandium oxide (Sc2O3) using the rat as an experimental model. Male Sprague-Dawley rats were injected intravenously with 0.3 or 1 mg Sc2O3/kg body weight (bw). Blood and excreta (urine and feces) were collected sequentially during a 21-day period, and main organs (liver, spleen, lungs, kidneys, brain) were withdrawn at sacrifice on day 21. Inductively coupled plasma-mass spectrometry (ICP-MS) was used for the measurement of Sc element in the different samples. The mean residence time (MRTIV) calculated from the blood profile was 19.7 ± 5.9 h and 43.4 ± 24.6 h at the lower and higher doses, respectively. Highest tissue levels of Sc were found in the lungs and liver; respective lung values of 10.6 ± 6.2% and 3.4 ± 2.3% of the Sc dose were observed at the time of sacrifice while liver levels represented 8.9 ± 6.4% and 4.6 ± 1.1%. Elimination of Sc from the body was not complete after 21 days. Cumulative fecal excretion over the 21-day collection period represented 12.3 ± 1.3% and 5.9 ± 1.0% of the lower and higher Sc doses, respectively, and showed a significant effect of the dose on the excretion; only a small fraction of the Sc dose was recovered in urine (0.025 ± 0.016% and 0.011 ± 0.004% in total, respectively). In addition to an effect of the dose on the toxicokinetics, results highlight the importance of the lung as a site of accumulation and retention of Sc2O3, which raises the question of the risks of effects related to respiratory exposure in workers. The results also question the relevance of urine as a matrix for biological exposure monitoring. A more in-depth inhalation toxicokinetic study would be necessary.

Multigenerational exposure of Ag materials (nano and salt) in soil - environmental hazards in Enchytraeus crypticus (Oligochaeta).

Because of its properties, silver is among the most used metals both as salt and as nanomaterials (NMs), hence reaching the environment. Multigenerational (MG) exposure testing is scarce, and especially so for NMs and soil invertebrates. In this study the MG effects of Ag NMs (Ag NM300K) and Ag salt (AgNO3) were assessed, using Enchytraeus crypticus in LUFA 2.2 soil. Survival, reproduction and internal Ag concentration in the animals were measured throughout 7 generations (5 generations (F0-F4) in spiked soil plus 2 (F5-F6) in clean soil) exposed to sublethal concentrations corresponding to the reproduction EC10 and EC50 obtained in standard toxicity tests (45 and 60 mg Ag per kg soil DW for AgNO3; 20 and 60 mg Ag per kg soil DW for Ag NM300K). MG exposure caused a dose-related decrease in reproduction for both Ag forms. Ag uptake peaked in the F1 (64 days) for AgNO3 and F2 (96 days) for Ag NM300K, after which it decreased. In agreement with toxicokinetic studies, a maximum body Ag concentration was reached (20 mg Ag per kg body DW (AgNO3) and 70 mg Ag per kg body DW (Ag NM300K)) and after which detoxification mechanisms seem to be activated with elimination of Ag accompanied by a decrease in reproduction. Transfer to clean soil allowed Ag to be (fully) eliminated from the animals. This MG study confirmed the effects determined in standard reproduction toxicity tests but further allowed to monitor the dynamics between exposure and effects of the Ag materials, and how the animals seem to cope with Ag for 7 generations by compensating between detoxification and reproductive output.

Improved quantitative determination of (R)- and (S)‐rabeprazole sodium and its metabolites in rat plasma by LC-MS/MS and its application to a toxicokinetic study

There exist two enantiomers: (R)‐ and (S)‐rabeprazole. (R)‐rabeprazole offers specific pharmacokinetic advantages and enhanced therapeutic efficacy, warranting further investigation and development. Here, we developed a simple and rapid chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify rabeprazole enantiomers and their metabolites (rabeprazole sulfoxide and desmethyl rabeprazole enantiomers) and a LC-MS to quantify rabeprazole thioether. As for the chiral LC-MS/MS method, Chiral-AGP column (150 × 4 mm, 5 μm) was used and its mobile phase was acetonitrile (mobile phase A) and 10 mmol/L ammonium acetate (mobile phase B) (linear gradient profile: 0 min, 10 % B; 5 min, 15 % B; 9 min, 15 % B; 9.01 min, 10 % B; 13 min, 10 % B). The multiple reactions monitoring transitions of m/z 360.3 → 242.1, 376.2 → 240.1, 346.2 → 228.2 and 368.2 → 190.2 were opted for quantifying rabeprazole enantiomers, rabeprazole sulfoxide, desmethyl rabeprazole enantiomers and internal standard omeprazole. The analyte samples were prepared by a simple liquid–liquid extraction method. As for the LC-MS method, analytes were separated on a Inertsil® ODS-3 column (4.6 × 150 mm, 5 μm). The mobile phase was acetonitrile-5 mmol/L ammonium acetate water solution (65:35, v/v). ESI+ was used and ion peaks with m/z 344.2 (rabeprazole thioether) and 285.1 (internal standard diazepam) were monitored. Both these 2 methods were validated for specificity, linearity, precision, accuracy, matrix effect and extraction recovery, and, particularly, the stability of analytes under various conditions. We successfully applied these methods to a 13-week toxicokinetic study of rabeprazole in rats after intravenous administration of (R)- (80, 20, 5 mg/kg/d) and racemic (80 mg/kg/d) rabeprazole sodium. The results showed that rabeprazole and its metabolites did not accumulate in rats. However, desmethyl rabeprazole and rabeprazole thioether showed higher exposure and lower clearance rate in the last administration than in the first one. (R)-rabeprazole showed a higher exposure and a slower elimination rate than (S)-rabeprazole in rats. These findings offer experimental evidence and a theoretical foundation for further preclinical investigations and clinical applications of (R)-rabeprazole.

Development and validation of a sensitive UPLC-MS/MS analytical method for GFH009 in rat plasma and its application to toxicokinetics studies.

GFH009 is a potent, highly selective, small molecule that targets and inhibits the activity of the CDK9/cyclin T1 regulatory complex of P-TEFb. This study aimed to develop and validate a highly selective and sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for precise quantification of GFH009 in rat plasma. This method was subsequently employed for conducting toxicokinetic studies of GFH009 in rats. Plasma was prepared using a simple protein precipitation method by acetonitrile. Chromatographic separation of the analytes was achieved on a BEH C18 analytical column with a rapid 3.0 min run time and a flow rate of 0.5ml/min. The calibration curves for plasma samples exhibited excellent linearity over a wide concentration range of 1.0-1,000 ng/ml for GFH009. Intra- and inter-day accuracies were within 92.7-105.7%, and precisions were no more than 6.7%. Furthermore, the analyte demonstrated stability under four different storage conditions, with variations of <15.0%. This study pioneers a methodological innovation by introducing a highly reliable, specific and sensitive analytical method for GFH009 in rat plasma. The successful application of this method in toxicokinetic studies further underscores its significance, offering valuable insights for the methodology of clinical pharmacokinetic research.

Acute and chronic toxicity in Sprague–Dawley rats of orally-administered total lignans from Arctii Fructus, a potential therapeutic drug for diabetes

Arctii Fructus is the dried ripe fruit of Arctium lappa L. (family Asteraceae) and is in the Chinese pharmacopoeia. Previous research showed that the total lignans from Arctii Fructus (TLAF) have pharmacological activities related to diabetes. This study evaluated the acute and chronic (26 weeks) toxicities associated with oral daily administration of TLAF in Sprague–Dawley (SD) rats. An acute-toxicity test showed that TLAF caused 10% mortality at 3,000 mg/kg × 2 (6-h interval), with toxic symptoms, such as dyspnea and tonic convulsions, indicating potential neurotoxicity. A chronic-toxicity study showed no mortality after administration. The no observed adverse-effect level was 1,800 mg/kg (approximately 54 times higher than the human clinical dose) for 26 weeks of TLAF oral administration in SD rats, with toxicity signs of excessive oral and nasal secretions and moist circumferential hair that recovered after TLAF discontinuation. In the toxicokinetic study, the two main components of TLAF, arctigenin plasma level was positively correlated with dose and tended to accumulate after multiple doses. At 1,800 mg/kg, arctiin plasma level increased and tended to accumulate after multiple doses. These results indicated that TLFA has relatively low toxicity and the potential for clinical treatment of diabetes.

Pharmacology of a CpG-Linked Anti-Mir-126 Oligonucleotide Targeting Acute Myeloid Leukemia (AML) in Rats and Non-Human Primates (NHP)

Introduction: Acute myeloid leukemia (AML) is a lethal hematopoietic malignancy, characterized by the accumulation of clonal myeloid progenitor cells arrested in development. AML patients have dismal outcomes with a 5-year overall survival (2011 - 2017) of 29.5% (SEER). The persistence of leukemia stem cells (LSCs), a treatment-resistant primitive cell population able to inititiate and maintain AML, is thought to underlie relapse. Even newly approved drugs do not target LSCs efficiently. MicroRNA (miR)-126 is a non-coding regulatory RNA that contributes to LSC homeostasis in the bone marrow niche. Several lines of evidence validate miR-126 as a target in AML. Thus, we designed and made a novel anti-miR-126 oligonucleotide (oligo) linked to a CpG moiety, termed miRisten. miRisten incorporates modifications such as 2'-fluoro and phosphorothioate substituting phosphodiester in the sugar moiety of the nucleotide to enhance in vivo stability. miRisten eradicates LSCs in mouse models of AML and synergizes with FDA-approved AML drugs (PMID: 29505034, 34372909). Here, the results of IND-enabling toxicokinetic (TK) studies of miRisten in rats and NHP are presented. Objective: To investigate the pharmacological properties of miRisten to establish a safe dosing strategy to achieve prolonged knockdown of miR-126 for a first-in-human trial. Experimental Plan and Results: Good laboratory practice (GLP) TK studies were carried out in Sprague-Dawley rats. miRisten was given IV b.i.d. for 19 days at human equivalent doses (HED) of 1.6 and 4 mg/kg. miRisten was measured in plasma after the first dose. Plasma miRisten C max average was 3622 ± 335 and 6010 ± 175 nM post first doses of 1.6 and 4mg/kg, respectively. Plasma t 1/2 averaged 20.0 ± 3.8 minutes across both doses. The area under the curve (AUC 0 - ¥) were 638.9 ± 32 nMxhr (1.6 mg/kg) and 1313.5 ± 439 (4 mg/kg). A parallel toxicology study was undertaken documenting cage-side observations and body weight; complement activation, blood chemistries, urine and hematology were also evaluated. There were no unscheduled deaths in the toxicology segment. Four single NHP exploratory studies were also carried out (Fig. 1: Treatment schema). In each study, miRisten was given IV as a slow bolus (~5') b.i.d. and PK parameters evaluated in plasma. Study #1 &amp; #2: 1 wk at 4mg/kg; #3: 1 wk at 5 mg/kg; #4: 18 days at 4 mg/kg. In all studies, miRisten dosage was ramped up (1, 2, 4 mg/kg for all and 5 mg/kg at dose 4 for Study #3). Solumedrol was given as premedication for the first 3 doses in all studies to prevent known phosphorothioate-mediated complement activation and cytokine release syndrome. Blood draws for PK were carried out immediately following doses 3 and 12 (Studies 1 - 3) and after the last dose on Day 18 (Study 4). The average area under the curve AUC was 3860 ± 1189 (4 mg/kg) and 3954 ± 1569 nMxhr (5 mg/kg) which was greater than observed in mice given the equivalent HED in bolus doses (~6x, 465.0). The t 1/2 of plasma miRisten was 11.7 min on Day 3 and 12.0 min on Day 7, comparable to mouse (9.6 min) and rat (20 min). Cage-side observations, body weight, complement activation, blood chemistries, urine and hematology were also evaluated. There was no terminal sacrifice of NHP however, all animals tolerated the treatment without observable adverse events. Conclusion: The dose ramp-up in NHP combined with pre-medication with solumedrol were undertaken to prevent possible acute inflammatory reactivity leading potentially to death. There were no unscheduled deaths in any species studied. These data are consistent with the safety of miRisten given IV with a dose ramping regimen combined with solumedrol. The greater AUC in NHP may predict better potency in humans. In mice, miRisten at 1.6 mg/kg HED q.d. effectively eradicated LSCs [PMID 29505034].

Diel Pattern of Microplastic Residues in Zebrafish.

As one of the emerging pollutants, microplastics (MPs) can be taken up by aquatic organisms through ingestion. However, little is known about the uptake pattern in organisms over time and the associated mechanisms of retention patterns. The present study aims to elucidate these patterns in fish, their relationship with light/dark conditions, and examine the uptake kinetic process of small-sized plastic pollutants, especially during the long-neglected dark period. Zebrafish were sampled every 2 h during the light and dark periods after exposure to an environmentally relevant concentration (100 items/L) of MPs. The results demonstrated that MP residues in zebrafish decreased during the dark period rather than increased over time. The MP retention rhythm and the swimming behavior of exposed zebrafish displayed a statistically significant light/dark variation. Moreover, a very strong and statistically significant positive correlation was found between the swimming speed of zebrafish and the number of MP residues in the gastrointestinal tracts of zebrafish. These results clearly demonstrate that fibrous MP residues in the fish have a discernible diel pattern. This work improves the understanding of the dynamic residual process of MPs in organisms and calls for further in-depth circadian toxicokinetic studies to better suit particle pollutants.

Repeat-dose toxicity study of human umbilical cord mesenchymal stem cells in cynomolgus monkeys by intravenous and subcutaneous injection.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are proposed for the treatment of acute lung injury and atopic dermatitis. To advance hUC-MSC entry into clinical trials, the effects of hUC-MSCs on the general toxicity, immune perturbation and toxicokinetic study of hUC-MSCs in cynomolgus monkeys were assessed. hUC-MSCs were administered to cynomolgus monkeys by intravenous infusion of 3.0 × 106 or 3.0 × 107cells/kg or by subcutaneous injection of 3.0 × 107cells/kg twice a week for 3weeks followed by withdrawal and observation for 6weeks. Toxicity was assessed by clinical observation, clinical pathology, ophthalmology, immunotoxicology and histopathology. Moreover, toxicokinetic study was performed using a validated qPCR method after the first and last dose. After 3rd or 4th dosing, one or three the monkeys in the intravenous high-dose group exhibited transient coma, which was eliminated by slow-speed infusion after 5th or 6th dosing. In all dose groups, hUC-MSCs significantly increased NEUT levels and decreased LYMPH and CD3+ levels, which are related to the immunosuppressive effect of hUC-MSCs. Subcutaneous nodules and granulomatous foci were found at the site of administration in all monkeys in the subcutaneous injection group. Other than above abnormalities, no obvious systemic toxicity was observed in any group. The hUC-MSCs was detectable in blood only within 1h after intravenous and subcutaneous administration. The present study declared the preliminary safety of hUC-MSCs, but close monitoring of hUC-MSCs for adverse effects, such as coma induced by intravenous infusion, is warranted in future clinical trials.

Photobac derived from bacteriochlorophyll-a shows potential for treating brain tumor in animal models by photodynamic therapy with desired pharmacokinetics and limited toxicity in rats and dogs

Photobac is a near infrared photosensitizer (PS) derived from naturally occurring bacteriochlorophyll- a, with a potential for treating a variety of cancer types (U87, F98 and C6 tumor cells in vitro). The main objective of the studies presented herein was to evaluate the efficacy, toxicity and pharmacokinetic profile of Photobac in animals (mice, rats and dogs) and submit these results to the United States Food and Drug Administration (US FDA) for its approval to initiate Phase I human clinical trials of glioblastoma, a deadly cancer disease with no long term cure. The photodynamic therapy (PDT) efficacy of Photobac was evaluated in mice subcutaneously implanted with U87 tumors, and in rats bearing C6 tumors implanted in brain. In both tumor types, the Photobac-PDT was quite effective. The long-term cure in rats was monitored by magnetic resonance imaging (MRI) and histopathology analysis. A detailed pharmacology, pharmacokinetics and toxicokinetic study of Photobac was investigated in both non-GLP and GLP facilities at variable doses following the US FDA parameters. Safety Pharmacology studies suggest that there is no phototoxicity, cerebral or retinal toxicity with Photobac. No metabolites of Photobac were observed following incubation in rat, dog, mini-pig and human hepatocytes. Based on current biological data, Photobac-IND received the approval for Phase-I human clinical trials to treat Glioblastoma (brain cancer), which is currently underway at our institute. Photobac has also received an orphan drug status from the US FDA, because of its potential for treating Glioblastoma as no effective treatment is currently available for this deadly disease.

Glycyrrhizin protects against diosbulbin B-induced hepatotoxicity by inhibiting the metabolic activation of diosbulbin B.

Diosbulbin B (DIOB), isolated from herbal medicine Dioscorea bulbifera L. (DB), could induce severe liver injury, and its toxicology was closely associated with CYP3A4-mediated metabolic oxidation of furan moiety to the corresponding cis-enedial reactive metabolite. Glycyrrhizin (GL), the major bioactive ingredient in licorice, can inhibit the activity of CYP3A4. Thus, GL may ameliorate hepatotoxicity of DIOB when GL and DIOB are co-administrated. The study aimed to investigate the protective effect of GL on DIOB-induced hepatotoxicity and the underlying mechanism. Biochemical and histopathological analysis demonstrated that GL alleviated DIOB-induced hepatotoxicity in a dose-dependent manner. In vitro study with mouse liver microsomes (MLMs) demonstrated that GL reduced the formation of metabolic activation-derived pyrrole-glutathione (GSH) conjugates from DIOB. Toxicokinetic studies showed that the pretreatment with GL caused the increase of AUCs and Cmax of DIOB in blood of mice, resulting in accelerating the accumulation of DIOB in the circulation. In addition, the pretreatment with GL alleviated DIOB-induced hepatic GSH depletion. In summary, GL ameliorated DIOB-induced hepatotoxicity, possibly related to the inhibition of the metabolic activation of DIOB. Thus, development of a standardized combination of DIOB with GL may protect patients from DIOB-induced liver injury.