Toxicokinetic Studies Research Articles

The suitability of a polydimethylsiloxane-based (PDMS) microfluidic two compartment system for the toxicokinetic analysis of organophosphorus compounds

Organ-on-a-chip platforms are an emerging technology in experimental and regulatory toxicology (species-specific differences, ethical considerations). They address gaps between in vivo and in vitro models. However, there are still certain limitations considering material, setup and applicability. The current study examined the suitability of a commercially available polydimethylsiloxane-based (PDMS) organ-chip for the toxicokinetic characterization of the highly toxic nerve agent VX and the organophosphate pesticide parathion. The respective concentrations of 1000 µmol/L and 100 µmol/L VX and parathion were chosen deliberately high in order to study concentrations even if high compound absorption by PDMS might occur. Neuronal and liver spheroids, totaling 2 × 106 cells were used to study concentration changes of VX and parathion. In addition, VX enantiomers were quantified. The current study suggests a significant absorption of VX, respectively parathion by PDMS. This might require future investigation of alternative materials or coatings to limit absorption for organophosphorus compounds in toxicokinetic studies.

Использование кроликов в доклинических исследованиях

Rabbits are one of the most commonly used laboratory animal species in biomedical research. A large number of models of various human diseases have been developed on rabbits. Rabbits are widely used to assess the safety of substances and pharmaceuticals. The development of genetically modified rabbits has expanded their use for modeling various pathological conditions. Advantages of the rabbit include its relatively small size and relative ease of housing. Practically any experimental manipulations can be carried out on rabbits (any methods of administering the tested compounds are available, conducting various instrumental studies, multiple collection of blood samples, etc. is possible). In studies of reproductive toxicity, an important advantage of the rabbit relative to larger animals is a short gestation period, good reproductive ability. Rabbits are sensitive to human teratogens and are the preferred non-rodent species for embryofetal development studies. Rabbits are used in immunology for the production of diagnostic antibodies. An important point is that the scientific literature provides a sufficient amount of historical and reference materials on the anatomy, physiology, and pathophysiology of these animals. The disadvantages of rabbits include frequently developed disorders of the gastrointestinal tract. The functional activity of the rabbit’s digestive system is highly dependent on the diet. Changes in the diet should be done gradually. Rabbits need to be able to exercise daily. With the wrong manipulation technique, it is easy to cause injury to the rabbit. Since some diseases may spontaneously develop in laboratory rabbits, this may require either the use of therapy or the exclusion of the animal from the experiment. Therefore, in the case of planning long-term studies on rabbits, it is reasonable to include at least 8 males and 8 females in each group. If recovery groups are needed, the number of animals should be sufficient to evaluate possible long-term effects. When planning and conducting experiments, every effort should be made to implement the principles of 3Rs. Combination designs with pharmacological safety, immunotoxicity, reproductive toxicity, pharmacokinetics, and toxicokinetics studies as part of a toxicity study may help to reduce the number of rabbits in toxicological research. Studies should be designed taking into account the available scientific data on the test compound or on analogue compounds, on the basis of which scientific-based decision can be made to reduce the number of animals in the experiment without compromising the human safety.

ICH M10 guideline - a harmonized global approach to bioanalysis

Bioanalytical methods are used in research on small-molecule and large-molecule drug products to determine analytes and their metabolites in biological matrices such as blood, plasma, serum, urine, feces, saliva, other biological fluids, or tissues. Validation of a bioanalytical method is the essential step before the implementation of the method into routine use in toxicokinetic or pharmacokinetic studies. Harmonization of recommendations for the validation of bioanalytical methods has been advocated for many years. In 2022, The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) finished the work on final version of the ICH M10 guideline, as a combination of four regional guidelines (American, European, Brazilian and Japanise). The document uniforms rules for the performance of the bioanalytical method validation and documentation of sample analysis from clinical and non-clinical studies in most countries around the world, which are submitted to registration authorities.

Toxicokinetics of praseodymium and cerium administered as chloride salts in Sprague-Dawley rats: impacts of the dose and of co-exposure with additional rare earth elements.

We conducted a rat exposure study to assess the impacts of dose and co-exposure with other rare earth elements (REEs) on the toxicokinetics of praseodymium (Pr) and cerium (Ce). We first determined the kinetic profiles of elemental Pr and Ce in blood, urine and feces along with tissue levels at sacrifice on the seventh day following intravenous injection of PrCl3 or CeCl3 at 0.3or1mg/kgbw (of the chloride salts) in adult male Sprague-Dawley rats (n = 5 per group). In blood, Pr and Ce half-lives for the initial phase (t1/2α) increased with increasing doses, while their half-lives for the terminal phase (t1/2β) were similar at both doses. In urine, a minor excretion route, no significant effect of the dose on the cumulative excretion was apparent. In feces, a major excretion route, the fraction of the Pr dose recovered was significantly lower at the 1mg/kg bw dose compared to the 0.3mg/kgbw dose, while no significant dose effect was apparent for Ce. In the liver and spleen, which are the main sites of REEs accumulation, there was a significant effect of the dose only for Ce retention in the spleen (i.e., increased retention of Ce in spleen at higher dose). Results were compared with those of a previous toxicokinetic study with a similar design but an exposure to a quaternary mixture of CeCl3, PrCl3, NdCl3 and YCl3, each administered at 0.3mg/kgbw or 1mg/kgbw. A mixture effect was apparent for the initial elimination phase (t1/2α) of Pr and Ce from blood and forthe fecal excretion of Ce at the 1mg/kg bw. In urine and liver, there was no evident overall mixture effect; in the spleen, there was a higher retention of Pr and Ce in rats exposed to the mixture at the 0.3mg/kgbw, but not at the 1mg/kgbw dose. Overall, this study showed that the dose and mixture exposure are two important factors to consider as determinants of the toxicokinetics of REEs.

Subacute toxicity evaluations of LPM3480392 in rats, a full µ-opioid receptor biased agonist.

Opiates produce analgesia via G-protein signaling, and adverse effects, such as respiratory depression and decreased bowel motility, by β-arrestin pathway. Oliceridine, a G protein-biased MOR agonist, only presents modest safety advantages as compared to other opiates in clinical trials, possibly due to its limited bias. Our previous study shown that LPM3480392, a full MOR biased agonist, is selective for the Gi pathway over the β-arrestin-2. In the present article, we evaluated the subacute toxicity of LPM3480392 in rats. The rats were administered with control article or LPM3480392 0.6, 1.2 or 2.4mg/kg/day for 4 consecutive weeks followed by a 4-week recovery phase. Intravenous infusion was conducted at tail vein at 0.2, 0.4 or 0.8mg/kg/day with a dosing volume of 10mL/kg and 5min/rat/dose, three times a day with an interval of approximately 4h. The concomitant toxicokinetics study was conducted. Two unscheduled rats at 2.4mg/kg/day died with no clear cause. For the scheduled necropsy, the major effects were associated with the MOR agonist-related pharmacodynamic properties of LPM3480392 (e.g., increased activity, increased muscle tone; decreased food consumption and body weight gain; and clinical chemistry changes related with decreased food consumption) in three LPM3480392 groups. In addition, LPM3480392 at 2.4mg/kg/day also induced deep respiration and histopathology changes in testis and epididymis in sporadic individual rats. However, different from other opiates, LPM3480392 presents weak/no immunosuppression and the decreased adrenal gland weight, which may be due to LPM3480392' full MOR bias. At the end of recovery phase, all findings were recovered to some extent or completely. In the toxicokinetics study, the dose-dependent elevation of drug exposure was observed, which partly explained the toxicity of high dose. In summary, LPM3480392 has exhibited good safety characteristics in this subacute toxicity study in rats.

Blood microsampling in cynomolgus monkey and evaluation of plasma PK parameters in comparison to conventional sampling

Microsampling, a reduced volume sampling method, has successfully gained attention at the International Conference on Harmonization (ICH) level and established benefits support its use in Toxicokinetic (TK) studies. These improved sampling techniques are less invasive and in large animal species improve animal welfare (refinement). To evaluate if the plasma concentrations of drugs were influenced by the blood sampling method, the traditional method from femoral vein and microsampling from tail vein in Cynomolgus monkeys were compared. The pharmacokinetic parameters (Cmax, Tmax and AUC) of four drugs (selected based on acid-base and volume of distribution properties) in non-human primate were correlated. The plasma samples were quantified using standard LC-MS/MS methods, qualified to evaluate the precision and accuracy before the analysis of real samples.The results reported in this work demonstrated the suitability of microsampling in supporting PK/TK studies in non-human primates. The data show that the exposure of drugs tested after blood collection using standard procedure from femoral vein and microsampling from tail vein is correlated and is not influenced by acid-base characteristics and volume of distribution.

Simultaneous toxicokinetic studies of aristolochic acid I and II and aristolactam I and II using a newly-developed microdialysis liquid chromatography-tandem mass spectrometry.

Aristolochic acids (AAs) are naturally occurring genotoxic carcinogens linked to Balkan endemic nephropathy and aristolochic acid nephropathy. Aristolochic acid I and II (AA-I and AA-II) are the most abundant AAs, and AA-I has been reported to be more genotoxic and nephrotoxic than AA-II. This study aimed to explore metabolic differences underlying the differential toxicity. We developed a novel microdialysis sampling coupled with solid-phase extraction liquid chromatography-tandem mass spectrometry (MD-SPE-LC-MS/MS) to simultaneously study the toxicokinetics (TK) of AA-I and AA-II and their corresponding aristolactams (AL-I and AL-II) in the blood of Sprague Dawley rats co-treated with AA-1 and AA-II. Near real-time monitoring of these analytes in the blood of treated rats revealed that AA-I was absorbed, distributed, and eliminated more rapidly than AA-II. Moreover, the metabolism efficiency of AA-I to AL-I was higher compared to AA-II to AL-II. Only 0.58% of AA-I and 0.084% of AA-II was reduced to AL-I and AL-II, respectively. The findings are consistent with previous studies and support the contention that differences in the in vivo metabolism of AA-I and AA-II may be critical factors for their differential toxicities.

O31 | An algae‐clay based mycotoxin detoxifier reduces the bioavailability of a mix of mycotoxins in broiler chickens in a toxicokinetic study

Journal of Veterinary Pharmacology and TherapeuticsVolume 46, Issue S1 p. 43-43 ORAL PRESENTATIONS O31 | An algae-clay based mycotoxin detoxifier reduces the bioavailability of a mix of mycotoxins in broiler chickens in a toxicokinetic study First published: 11 June 2023 https://doi.org/10.1111/jvp.13197Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume46, IssueS1Special Issue: 15th International Congress of the European Association for Veterinary Pharmacology and Toxicology held Bruges, Belgium, July 2–5, 2023June 2023Pages 43-43 RelatedInformation

A phase I dose-escalation clinical trial with PLZ4-coated paclitaxel-loaded micelles (PPM) in patients with recurrent or refractory non-myoinvasive bladder cancer.

TPS4615 Background: Bladder carcinoma is one of the top ten cancers in the US with approximately 75% of patients initially presenting with non-myoinvasive bladder cancer (NMIBC). The current standard of care for NMIBC is transurethral resection (TUR) followed by intravesical instillation, usually with Bacille Calmette-Guérin (BCG). Despite combination treatment, 20-80% of patients' disease recurs and 25% progress to invasive stages. The study drug, PLZ4-coated paclitaxel-loaded micelle (PPM), is a solvent-free injectable nanoscale micelle formulation of paclitaxel (PTX). Preclinical data on bladder cancer-specific targeting ligand named PLZ4 has shown potential to specifically deliver chemotherapeutic micelle into bladder cancer cells, both in vitro and in vivo (Zhang, Urol Oncol 2012). In a toxicokinetic study, female Sprague Dawley rats were given intravesicular injections of PPM once weekly for 6 consecutive weeks. No drug related mortality was reported, and no systematic exposure of PPM was observed in toxicokinetic analysis. The purpose of this phase I trial with PPM is to determine the toxicity and recommended Phase II dose. Methods: This is a single arm, open label, Phase I dose-escalation first-in-human trial with an expansion cohort for the treatment of recurrent or refractory NMIBC with PPM. Eligibility includes BCG unresponsive carcinoma in situ or Ta/T1 urothelial carcinoma. Treatment with pembrolizumab is not an inclusion or exclusion criteria as intravesical taxane has comparable efficacy as intravenous pembrolizumab. Up to 29 patients will be recruited at VA Boston Healthcare system. PPM will be given once weekly for six weeks, 50 ml of solution regardless of patient's size or weight will be administered as intravesical instillation. A 3 + 3 design is adopted for this Phase I trial. There are three dose levels at the dose escalation stage: Dose level I: PTX 25 mg or 0.5 mg/ml; Dose Level II: PTX 50 mg or 1.0 mg/ml; Dose Level III: PTX 75 mg or 1.5 mg/ml. At the expansion cohort, up to 12 patients will be treated with PPM at PTX dose of 50 mg or 1.0 mg/ml to determine the efficacy. The primary objective is to evaluate the safety and tolerability of PPM administered through intravesical instillation and determine the recommended Phase II dose of PPM. The secondary objectives are to evaluate the tumor response at 6 weeks after last treatment, determine systemic absorption within 6 hours after intravesical instillation of PPM and assess progression free survival. The efficacy will be determined by urine cytology and cystoscopy after finishing treatment. Molecular correlative studies will be performed. The toxicity will be assessed by Common Terminology Criteria for Adverse Events v5.0. Patients will be followed at 3 month intervals for 2 years or until disease recurrence or progression. This study is currently open for enrollment. NCT05519241. Clinical trial information: NCT05519241 .

Avermectin Trunk Injections: A Promising Approach for Managing the Walnut Husk Fly (Rhagoletis completa)

This study examined the larvicidal effect of trunk-injected abamectin and emamectin benzoate against the walnut husk fly (Rhagoletis completa Cresson, 1929). Walnut trees in two locations in two years were injected with the pesticides at different concentrations. For the toxicokinetic studies, the active ingredient content was measured in the leaves, flowers, husks, and kernels, using a UHPLC-MS/MS analytical method. The walnut husk fly infestation rates were between 3 and 70% and 10 and 34% for abamectin and emamectin benzoate, respectively, and were much lower compared to those measured for the control. The active ingredient content in the walnut husk showed a positive correlation with the larvicidal effect. The injections had a measurable but unsatisfactory insecticidal effect in the second year, when the economic threshold was exceeded. Trace amounts of the active ingredients were detected in the flowers. The residue analysis showed a declining concentration trend in the leaves over time. The largest quantities were detected in the leaves (≤439 ng/g of abamectin; ≤19,079 ng/g of emamectin benzoate), with concentrations in the husks of orders of magnitude lower (≤5.86 ng/g; ≤50.19 ng/g). The measurements showed no active ingredient residue above the MRLs in either fresh or dried kernels. The results indicate that trunk injections of abamectin, as well as trunk injections of emamectin benzoate, have the potential to suppress walnut husk fly populations.

Cytarabine determination from urine for Toxicokinetic and Excretion studies by High- Performance Liquid Chromatography-Tandem Mass Spectrometry

Cytarabine (Cyt) (also known as cytosine arabinoside (ara-C)) used in the treatment of acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL). CYT applied in high doses for treatment can cause renal failure. Monitoring excreted urine drug levels can help kidney failure. For this reason, a method was developed and validated by HPLC-MSMS for urine CYT analysis, which is not included in the literature. In this study, a liquid chromatography (HPLC) with triple quadrupole Mass Spectrometric (MS/MS) method developed for the determination of Cyt from urine for toxicokinetic evaluation. Positive MRM mode selected for the quantification of Cyt. The product and major fragment ion for Cyt 244.0 > 112.0 m/z, for IS 198.0 > 152.0 m/z. The optimal MS parameters for Cyt and IS are as follows Fragmentor 80 V, 70 V, Collision energy, 6, 9 respectively. A novel simple, high-throughput and highly sensitive HPLC-MS/MS method was successfully developed and validated for the determination of Cyt from urine. The developed method has a simple one-step extraction method and a short run time (2.0 minutes) for analysis. The proposed method could be practical and reliable for excretion and toxicokinetic studies and as well as the Therapeutic Drug Monitoring study in humans without an invasive route for Cyt.

Food-grade titanium dioxide translocates across the buccal mucosa in pigs and induces genotoxicity in an in vitro model of human oral epithelium

The whitening and opacifying agent titanium dioxide (TiO2) is used worldwide in various foodstuffs, toothpastes and pharmaceutical tablets. Its use as a food additive (E171 in EU) has raised concerns for human health. Although the buccal mucosa is the first area exposed, oral transmucosal passage of TiO2 particles has not been documented. Here we analyzed E171 particle translocation in vivo through the pig buccal mucosa and in vitro on human buccal TR146 cells, and the effects on proliferating and differentiated TR146 cells. In the buccal floor of pigs, isolated TiO2 particles and small aggregates were observed 30 min after sublingual deposition, and were recovered in the submandibular lymph nodes at 4 h. In TR146 cells, kinetic analyses showed high absorption capacities of TiO2 particles. The cytotoxicity, genotoxicity and oxidative stress were investigated in TR146 cells exposed to E171 in comparison with two TiO2 size standards of 115 and 21 nm in diameter. All TiO2 samples were reported cytotoxic in proliferating cells but not following differentiation. Genotoxicity and slight oxidative stress were reported for the E171 and 115 nm TiO2 particles. These data highlight the buccal mucosa as an absorption route for the systemic passage of food-grade TiO2 particles. The greater toxicity on proliferating cells suggest potential impairement of oral epithelium renewal. In conclusion, this study emphasizes that buccal exposure should be considered during toxicokinetic studies and for risk assessment of TiO2 in human when used as food additive, including in toothpastes and pharmaceutical formulations.

Impact of repeated micro and macro blood sampling on clinical chemistry and haematology in rats for toxicokinetic studies

Non-clinical rodent safety studies are essential in the development of new medicines to assess for potential adverse effects. Typically, toxicokinetic samples are collected from a satellite group. AstraZeneca implemented repeated microsampling of main study animals as standard in the one-month small molecule regulatory toxicology studies. A retrospective analysis of the clinical chemistry and haematology data collected in 52 independent studies from the adult rat controls explored the impact of micro and macro sampling of main study animals. For the majority of variables, the blood sampling technique had no significant impact on the mean or range. For microsampling, a few variables had statistically significant effects on the mean signal but these were considered to have limited biological relevance and would therefore not introduce a meaningful bias to any toxicological evaluation. The macrosampling had the expected effects on the red cell parameters of haemoglobin, haematocrit and red blood count due to the larger blood volume draw. In contrast, microsampling showed no such changes. In conclusion, this large-scale retrospective analysis supports the use of microsampling, for toxicokinetics, of main study animals and enables us to conduct rodent toxicology studies without satellite animals and further reduce the number of animals used in toxicological assessments.

Abstract 3997: Preclinical characterization of ARX517, a next-generation anti-PSMA antibody drug conjugate for the treatment of metastatic castration-resistant prostate cancer

Abstract Prostate cancer is the most common cancer, and the second leading cause of cancer death, among men in the United States. Metastatic castration-resistant prostate cancer (mCRPC) is an advanced stage of disease in which patients ultimately fail androgen-deprivation therapies and exhibit a poor survival rate. Recently, prostate-specific membrane antigen (PSMA) has been validated as a prostate cancer tumor antigen with its over-expression in prostate tumors and low level of expression in select normal tissues. Using an expanded genetic code to create Engineered Precision Biologics (EPBs), Ambrx has developed ARX517, an anti-PSMA targeted next-generation antibody drug conjugate (ADC), for treatment of mCRPC patients. ARX517 is composed of a humanized anti-PSMA antibody site-specifically conjugated to drug linker AS269 (a potent tubulin inhibitor), yielding a drug-to-antibody ratio of 2. After binding to PSMA expressed on the surface of tumor cells, ARX517 is internalized and delivers a cytotoxic payload which inhibits tubulin polymerization and induces cellular apoptosis. In vitro testing of ARX517 in prostate cancer cell lines with variable PSMA expression demonstrated highly specific and potent sub-nanomolar activity in cells with high PSMA expression. To minimize premature payload release, ARX517 employs a non-cleavable PEG linker and stable oxime conjugation chemistry to enhance stability in circulation. ARX517 exhibited a long terminal half-life and high serum exposure in mice. The serum stability of ARX517 should effectively deliver more payload to target tumor cells, and in multiple CDX and PDX prostate cancer models, ARX517 showed dose-dependent anti-tumor activity in both enzalutamide-sensitive and enzalutamide-resistant models. Repeat dose toxicokinetic studies in non-human primates demonstrated ARX517 was tolerated at exposures well above therapeutic exposures in mouse pharmacology studies, indicating a wide therapeutic index. In summary, ARX517 elicited highly specific, potent cell killing in cell lines with high PSMA expression, inhibited tumor growth in enzalutamide-sensitive and enzalutamide-resistant CDX and PDX models, demonstrated a tolerable safety profile in cynomolgus monkeys, and has a clear therapeutic index based on preclinical serum exposure data. The strong preclinical data and recent clinical validation of PSMA as a mCRPC target provide rationale for evaluation of ARX517 as a potential prostate cancer treatment. ARX517 is currently in a Phase 1 dose escalation trial (ARX517-2011 [NCT04662580]) in the United States. Citation Format: Lillian Skidmore, David Mills, Ji Young Kim, Prathap Shastri, Nick A. Knudsen, Jeff Steen, Jay Nelson, Ying Buechler, Feng Tian, Shawn Zhang. Preclinical characterization of ARX517, a next-generation anti-PSMA antibody drug conjugate for the treatment of metastatic castration-resistant prostate cancer. [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 3997.

Abstract 6318: Preclinical characterization of ARX305, a next-generation anti-CD70 antibody drug conjugate for the treatment of CD70-expressing cancers

Abstract CD70 has emerged as an attractive tumor target with its over-expression in multiple solid and hematological cancers, yet tightly controlled and limited expression in normal cells. In the last decade, multiple anti-CD70 antibody drug conjugates (ADCs) have been developed, but most, if not all, of these ADC programs have been discontinued. Using an expanded genetic code to create Engineered Precision Biologics (EPBs), Ambrx has developed ARX305, a CD70-targeted next-generation antibody drug conjugate (ADC) to potentially overcome the challenges associated with earlier ADCs and treat patients with cancers over-expressing CD70. ARX305 is comprised of a proprietary, high-affinity, humanized anti-CD70 antibody stably and site-specifically conjugated to AS269, a potent microtubule inhibitor. After binding to CD70 expressed on target tumor cells, ARX305 internalizes and releases the cytotoxic payload pAF-AS269 which inhibits cellular proliferation. In vitro testing across a panel of solid and liquid tumor cell lines showed ARX305 elicited highly potent and specific cell killing across multiple tumor types with medium to high CD70 expression levels. In multiple in vivo CD70-expressing xenograft or disseminated models, ARX305 induced significant tumor growth inhibition or regression whereas unconjugated antibody exhibited poor activity. ARX305 utilizes stable oxime conjugation chemistry, a non-cleavable PEG linker, and a membrane-impermeable payload to minimize premature payload release in circulation and associated off-target toxicity. These properties of ARX305 result in high serum stability, with rodent pharmacokinetic studies confirming a similar exposure profile of ARX305 compared to unconjugated antibody and a long serum half-life. Repeat dose toxicokinetic studies in cynomolgus monkeys demonstrated ARX305 was tolerated at exposures well above therapeutic exposures in mouse pharmacology studies, indicating a wide therapeutic index. In summary, the highly potent anti-tumor activity in multiple tumor types and wide therapeutic index of ARX305 support clinical evaluation of this next generation anti-CD70 ADC. ARX305 is currently in a Phase 1 dose escalation study in China, and the IND in the United States is open. Citation Format: Lillian Skidmore, Jingjing Zhu, David Mills, Ting Jin, Suqin Li, Keith Tatsukawa, Nick A. Knudsen, Liying Gong, Jay Nelson, Kari Cox, Amha G. Hewet, Jianing Wang, Tim Buss, Feng Tian, Gang Xia, Shawn Zhang. Preclinical characterization of ARX305, a next-generation anti-CD70 antibody drug conjugate for the treatment of CD70-expressing cancers [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 6318.

Implantation of a Vascular Access Button for Chronic Blood Sampling and Drug Administration in the Rabbit

Rabbits are commonly used for pharmacokinetic (PK) and toxicokinetic (TK) studies in the research setting, requiring repetitive venipuncture, which can be challenging in this species. The auricular vessels are commonly used for venipuncture in rabbits. The repetitive access of these delicate vessels can lead to trauma such as hematomas causing venipuncture to become more challenging as the study progresses. In turn, this leads to missed time points or insufficient blood samples. Surgical models for chronic vascular access in rabbits are common throughout the industry. Common models include exteriorized vascular catheters and implanted vascular access ports. However, these implants come with their own complications and restrictions when used in rabbits. Therefore, the authors evaluated the use of a vascular access button (VAB), an implant commonly used in small rodents, as a refinement to the current chronic models in use in the industry. Seventeen rabbits were implanted with either single or dual channel VABs. The catheters were implanted in the femoral artery and/or vein and then tunneled subcutaneously to the button on the dorsal thoracic area. Overall, the results were outstanding, and an established model was created. The average patency rate was 316 days with several implants still patent after 2 years. The authors feel the implantation and use of a vascular access button in rabbits for routine PK studies is an excellent refinement. The rabbits tolerate the buttons extremely well with minimal issues. The patency rate is equal to or better than vascular access ports and when used with the tethering system, provides a hands-off method for blood collection and intravenous administration in rabbits during PK studies.

Determination of protein-bound α-amanitin in mouse plasma: A potential new indicator of poisoning with the mushroom toxin α-amanitin.

Approximately 70%∼90% of mushroom poisoning deaths are caused by the class of mushroom toxins known as amatoxins. However, the rapid elimination of amatoxins from plasma within 48h after mushroom ingestion limits the practical value of plasma amatoxin analysis as a diagnostic indicator of Amanita mushroom poisoning. To increase the positive detection rate and extend the detection window of amatoxin poisoning, we developed a new method to detect protein-bound α-amanitin based on the hypothesis that RNAP II-bound α-amanitin released from the tissue into the plasma could be degraded by trypsin hydrolysis and then detected by conventional liquid chromatography-mass spectrometry (LC‒MS). Toxicokinetic studies on mice intraperitoneally injected with 0.33mg/kg α-amanitin were conducted to obtain and compare the concentration trends, detection rates, and detection windows of both free α-amanitin and protein-bound α-amanitin. By comparing detection results with and without trypsin hydrolysis in the liver and plasma of α-amanitin-poisoned mice, we verified the credibility of this method and the existence of protein-bound α-amanitin in plasma. Under the optimized trypsin hydrolysis conditions, we obtained a time-dependent trend of protein-bound α-amanitin in mouse plasma at 1-12 days postexposure. In contrast to the short detection window (0-4h) of free α-amanitin in mouse plasma, the detection window of protein-bound α-amanitin was extended to 10 days postexposure, with a total detection rate of 53.33%, ranging from the limit of detection to 23.94μg/L. In conclusion, protein-bound α-amanitin had a higher positive detection rate and a longer detection window than free α-amanitin in mice.

Development of an LC-MS/MS Assay and Toxicokinetic Characterization of Hexamethylenetetramine in Rats.

Hexamethylenetetramine, an aldehyde-releasing agent, is used as a preservative in various food, cosmetics, and medical treatments, such as a treatment for urinary tract infections. It has been reported to be allergenic on contact with the skin, with the additional possibility of causing toxicity once absorbed systemically. Despite its potential toxicity, there are no reports on the in vivo bioavailability of hexamethylenetetramine following oral or dermal administration. In this study, we developed a new simple and sensitive LC-MS/MS method for the determination of hexamethylenetetramine in plasma and applied this method to characterize the toxicokinetics. The developed assay had a sufficient specificity and sensitivity for toxicokinetic characterization, and its accuracy and precision were verified. Following iv injection, the plasma concentration of hexamethylenetetramine showed mono exponential decay, with an elimination half-life of about 1.3 h. Following oral administration, the Tmax reached an average of 0.47 h and bioavailability was estimated as 89.93%. After percutaneous administration, it reached Cmax on average at 2.9-3.6 h. Although the absorption rate was relatively slow, its average bioavailability was calculated as 77.19-78.91%. Overall, most of the orally and percutaneously administered hexamethylenetetramine was absorbed into systemic circulation. The derived results in this study are expected to be utilized as the scientific evidence for further toxicokinetic study and risk assessment.

Glycyrrhetinic acid ameliorates diosbulbin B-induced hepatotoxicity in mice by modulating metabolic activation of diosbulbin B.

Exposure to diosbulbin B (DBB), the primary component of the herbal medicine Dioscorea bulbifera L. (DB), can cause liver injury in humans and experimental animals. A previous study found DBB-induced hepatotoxicity was initiated by CYP3A4-mediated metabolic activation and subsequent formation of adducts with cellular proteins. The herbal medicine licorice (Glycyrrhiza glabra L.) is frequently combined with DB used in numerous Chinese medicinal formulas in an effort to protect against DB-elicited hepatotoxicity. Importantly, glycyrrhetinic acid (GA), the major bioactive ingredient in licorice, inhibits CYP3A4 activity. The study aimed to investigate the protection of GA against DBB-induced hepatotoxicity and the underlying mechanism. Biochemical and histopathological analysis showed GA alleviated DBB-induced liver injury in a dose-dependent manner. In vitro metabolism assay with mouse liver microsomes (MLMs) indicated that GA decreased the generation of metabolic activation-derived pyrrole-glutathione (GSH) conjugates from DBB. Toxicokinetic studies demonstrated that GA increased maximal serum concentration (Cmax ) and area under the serum-time curve (AUC) of DBB in mice. In addition, GA attenuated hepatic GSH depletion caused by DBB. Further mechanistic studies showed that GA reduced the production of DBB-derived pyrroline-protein adducts in a dose-dependent manner. In conclusion, our findings demonstrated that GA exerted protective effect against DBB-induced hepatotoxicity, mainly correlated with suppressing the metabolic activation of DBB. Therefore, the development of a standardized combination of DBB with GA may protect patients from DBB-induced hepatotoxicity.

The Non-Human Primate in Safety Assessment of a Bifunctional Long-Acting Insulin Analogue.

Species selection plays a pivotal part during non-clinical safety assessment in drug development. If possible, use of non-human primates (NHPs) should be avoided due to ethical considerations. However, limiting factors as lack of pharmacologic activity in other species could necessitate use of NHPs. LAI-PCSK9i is a bi-functional molecule combining a long-acting insulin analogue with a PCSK9 inhibitor peptide aiming to provide glycaemic control and to reduce plasma LDL concentrations. The NHP was chosen for the safety assessment of LAI-PCSK9i being the most relevant species with basal levels and plasma lipid composition closest to humans, while the dog and initially also the minipig were deemed irrelevant due to lack of pharmacologic activity on LDL-lowering and biological differences in lipid profiles. An in vivo tolerability and toxicokinetic study of LAI-PCSK9i in NHPs showed recurrent and severe hypoglycaemia at very low doses. Therefore, the minipig was re-evaluated and a follow-up study thoroughly assessing blood glucose and cholesterol levels and clinical signs illustrated that minipigs dosed with LAI-PCSK9i, tolerated the compound and LAI-PCSK9i decreased glucose and LDL over time. This work underlines that careful consideration is required when selecting species during safety assessment in drug development. The tolerability issue in NHPs led to the subsequent selection of the minipig for safety evaluation of LAI-PCSK9i although as a suboptimal alternative, which unexpectedly had a measurable pharmacologic response on LDL lowering. In conclusion, the NHPs may be unsuitable as test species for safety assessment of long-acting insulin analogues due to high sensitivity to recurring hypoglycaemic episodes.