DMPK perspective on quantitative model analysis for chimeric antigen receptor cell therapy: Advances and challenges.

Chimeric antigen receptor (CAR) cells are genetically engineered immune cells that specifically target tumor-associated antigens and have revolutionized cancer treatment, particularly in hematological malignancies, with ongoing investigations into their potential applications in solid tumors. This review provides a comprehensive overview of the current status and challenges in drug metabolism and pharmacokinetics (DMPK) for CAR cell therapy, specifically emphasizing on quantitative modeling and simulation (M&S). Furthermore, the recent advances in quantitative model analysis have been reviewed, ranging from clinical data characterization to mechanism-based modeling that connects in vitro and in vivo nonclinical and clinical study data. Additionally, the future perspectives and areas for improvement in CAR cell therapy translation have been reviewed. This includes using formulation quality considerations, characterization of appropriate animal models, refinement of in vitro models for bottom-up approaches, and enhancement of quantitative bioanalytical methodology. Addressing these challenges within a DMPK framework is pivotal in facilitating the translation of CAR cell therapy, ultimately enhancing the patients' lives through efficient CAR cell therapies.

Novel Cell Quantification Method Using a Single Surrogate Calibration Curve Across Various Biological Samples.

Quantitative polymerase chain reaction (qPCR) is generally used to quantify transplanted cell therapy products in biological samples. As the matrix effects on PCR amplification and variability in DNA recovery from biological samples are well-known limitations that hinder the assay's performance, a calibration curve is conventionally established for each matrix. Droplet digital PCR (ddPCR) is based on the endpoint assay and advantageous in avoiding matrix effects. Moreover, the use of an external control gene may correct assay fluctuations to minimize the effects caused by inconsistent DNA recovery. In this study, we aimed to establish a novel and robust ddPCR method capable of quantifying human cells across various mouse biological samples using a single surrogate calibration curve in combination with an external control gene and DNA recovery normalization. Acceptable accuracy and precision were observed for quality control samples from different tissues, indicating the excellent quantitative and versatile potential of the developed method. Furthermore, the established method enabled the evaluation of human CD8+ T cell biodistribution in immunodeficient mice. Our findings provide new insights into the use of ddPCR-based quantification methods in biodistribution studies of cell therapy products.

Quantitative Model Analysis and Simulation of Pharmacokinetics and Metastasis-Associated Lung Adenocarcinoma 1 RNA Knockdown Effect After Systemic Administration of Cholesterol-Conjugated DNA/RNA Heteroduplex Oligonucleotide Crossing Blood-Brain Barrier of Mice.

The cholesterol-conjugated heteroduplex oligonucleotide (Chol-HDO) is a double-stranded complex; it comprises an antisense oligonucleotide (ASO) and its complementary strand with a cholesterol ligand. Chol-HDO is a powerful tool for achieving target RNA knockdown in the brains of mice after systemic injection. Here, a quantitative model analysis was conducted to characterize the relationship between the pharmacokinetics (PK) and pharmacodynamics (PD), non-coding RNA metastasis-associated lung adenocarcinoma 1 (Malat1) RNA, of Chol-HDO, in a time-dependent manner. The established PK model could describe regional differences in the observed brain concentration-time profiles. Incorporating the PD model enabled the unique knockdown profiles in the brain to be explained in terms of the time delay after single dosing and enhancement following repeated dosing. Moreover, sensitivity analysis of PK exposure/persistency, target RNA turnover, and knockdown potency identified key factors for the efficient and sustained target RNA knockdown in the brain. The simulation of an adequate dosing regimen quantitatively supported the benefit of Chol-HDO in terms of achieving a suitable dosing interval. This was achieved via sufficient and sustained brain exposure and subsequent strong and sustained target RNA knockdown in the brain, even after systemic injection. The present study provides new insights into drug discoveries and development strategies for HDO in patients with neurogenic disorders. SIGNIFICANCE STATEMENT: The quantitative model analysis presented here characterized the PK/PD relationship of Chol-HDO, enabled its simulation under various conditions or assumptions, and identified key factors for efficient and sustained RNA knockdown, such as PK exposure and persistency. Chol-HDO appears to be an efficient drug delivery system for the systemic administration of desired drugs to brain targets.

Utility of Göttingen minipigs for the prediction of human pharmacokinetic profiles after intravenous drug administration.

Göttingen minipigs are increasingly used to evaluate the pharmacokinetic (PK) profiles of drug candidates. However, their accuracy in predicting human PK parameters is unclear. In this study, we investigated the utility of Göttingen minipigs for predicting human PK profiles. We evaluated the PK parameters of 30 compounds with diverse metabolic pathways after intravenous administration in minipigs. Human total clearance (CLtotal) was corrected using the blood to plasma ratio, and the volume of distribution at steady state (Vd(ss)) was corrected with plasma unbound fraction (fup). CLtotal and Vd(ss) were predicted using single-species allometric scaling using data from minipigs and other reported animal models (monkeys, human liver chimeric mice, and rats). The predicted values were compared with actual values reported in humans. Göttingen minipig were superior to rats because of their better predictability of Vd(ss) and CLtotal, as represented by lower absolute average fold error values. However, their predictability for Vd(ss) was inferior to monkey and human liver chimeric mice. Prediction of CLtotal from blood-based minipig data showed excellent correlation with human data, and comparable predictability with monkey and human liver chimeric mice. Thus, Göttingen minipigs can be used as an optional model for preclinical pharmaceutical research for predicting human CLtotal.

Juice of Citrullus lanatus var. citroides (wild watermelon) inhibits the entry and propagation of influenza viruses in vitro and in vivo.

Vaccines and various anti-influenza drugs are clinically used to prevent and treat influenza infections. However, with the antigenic mismatch of vaccines and the emergence of drug-resistant viral strains, new approaches for treating influenza are warranted. This study focused on natural foods as potential candidates for the development of new treatment options for influenza infections. The screening of plants from the Cucurbitaceae family revealed that the juice of Citrullus lanatus var. citroides (wild watermelon) had the strongest ability to inhibit the replication of influenza virus in Madin-Darby canine kidney cells. The results of a time-of-addition assay indicated that wild watermelon juice (WWMJ) inhibits the adsorption and late stages of viral replication, suggesting that WWMJ contains multiple constituents with effective anti-influenza activity. A viral adsorption analysis showed that WWMJ reduces the amount of viral RNA in the cells at 37°C but not at 4°C, confirming that WWMJ inhibits viral entry into the host cells at 37°C. These results suggest that a mechanism other than the inhibition of viral attachment is involved in the anti-influenza action of WWMJ, which is perhaps responsible for a reduction in internalization of the virus. Administration of WWMJ into the nasal mucosa of BALB/c mice infected with the A/PR/8/34 mouse-adapted influenza virus was seen to significantly improve the survival rate. The findings of this study, therefore, demonstrate the anti-influenza potential of WWMJ in vitro and in vivo, thereby suggesting the candidature of WWMJ as a functional food product that can be used to develop anti-influenza agents and drugs.

Quantitative PCR methodology with a volume-based unit for the sophisticated cellular kinetic evaluation of chimeric antigen receptor T cells.

Although the cellular kinetics of chimeric antigen receptor T (CAR T) cells are expressed in units of copies/µg gDNA, this notation carries the risk of misrepresentation owing to dramatic changes in blood gDNA levels after lymphocyte-depleting chemotherapy and rapid expansion of CAR T cells. Therefore, we aimed to establish a novel qPCR methodology incorporating a spike-in calibration curve that expresses cellular kinetics in units of copies/µL blood, as is the case for conventional pharmacokinetic studies of small molecules and other biologics. Dog gDNA was used as an external control gene. Our methodology enables more accurate evaluation of in vivo CAR T-cell expansion than the conventional approach; the unit "copies/µL blood" is therefore more appropriate for evaluating cellular kinetics than the unit "copies/µg gDNA." The results of the present study provide new insights into the relationship between cellular kinetics and treatment efficacy, thereby greatly benefiting patients undergoing CAR T-cell therapy.