Background: Clozapine ameliorates severe schizophrenia but with high risk of toxicity. The relationship between drug dose and blood concentration of clozapine varies appreciably over time within individuals. Pharmacokinetic models have had limited success in countering variability over time. A model that incorporated sex, smoking and fluvoxamine treatment showed clinically useful capacity to predict dosage requirements in the same patients over the ensuing 6 months, but not later than that. Norclozapine is measured as part of clozapine TDM because it shares some activities with clozapine. The ratio of norclozapine to clozapine is therefore an index of metabolic transformation.

Aims: We explored whether incorporating the ratio of norclozapine to clozapine serum concentrations into pharmacokinetic models improved model fitting and thus offered the prospect of a model that better predicted future dose requirements.
Methods
Norclozapine/clozapine ratio was introduced into an existing model that incorporated sex, smoking and fluvoxamine treatment. Improvements in model fitting were assessed by objective function values, goodness of fit plots and visual predictive checks.

Results: Adding norclozapine/clozapine ratio to the existing model significantly improved fitting. Sex, smoking and co-treatment with fluvoxamine remained significantly influential. Models incorporating norclozapine/clozapine ratio were then tested for clinical utility in extending its predictive validity beyond 6 months.

Conclusions: Models incorporating the norclozapine/clozapine ratio model clozapine pharmacokinetics more closely and offer prospects for better prediction of doses needed to meet therapeutic target concentrations later in treatment.

Key words: norclozapine/clozapine ratio, population pharmacokinetics, dosage prediction.

Background
Methotrexate is converted to its active metabolite, methotrexate polyglutamate (MTX-PG1to6) intracellularly, by adding up to 5 glutamate moieties. MTX-PG1to5 can be detected accurately using UPLC-MS/MS, but the pre-analytics is time-intensive. Erythrocyte MTX-PGtotal (the sum of MTX-PG1to6) is useful for therapeutic drug monitoring (TDM) of MTX. We investigated the efficiency of folate deconjugation protocol in converting MTX-PG2to6 to MTX-PG1 quantified on the UPLC-MS/MS.

Methods
Whole blood QCs were prepared by spiking MTX-PG1to6 with a target concentration of 1200 nmol/L MTX-PGtotal, stored at -80ºC. MTX-PG2to6 in the QCs were deconjugated using Folate RBC hemolyzing reagent (Roche), with 90 minute incubation at room temperature. Lysates were further processed per den Boer et al.(1), and individual MTX-PG1to6 were quantified by UPLC-MS/MS. Deconjugation (%) was calculated as the ratio of the measured concentration of MTX-PG1 to the calculated concentration of MTX-PGtotal. Efficiency was high if >70% and moderate between 50–70%

Results
QCs had a mean MTX-PGtotal concentration of 1637 ± 94.7 nmol/L. MTX-PG1 accounted for 70% MTX-PGtotal while MTX-PG2 and MTX-PG3to6 were 20% and 10% respectively.

Conclusion
Folate RBC Hemolyzing reagent deconjugates MTX-PG2to6 to MTX-PG1 with moderate efficiency. A therapeutic window of 60-100 nmol/L MTX-PGtotal in packed erythrocytes is reported for rheumatoid arthritis. Higher MTX-PG1to6 increases MTX-PGtotal and can support both TDM and compliance monitoring. Analysers like the Cobas c-module offer a cost-efficient option for clinical implementation. Optimizing incubation time and temperature may improve deconjugation efficiency.
Keywords: Methotrexate, Methotrexate polyglutamates, therapeutic drug monitoring

Reference
(1)Den Boer E et al., Anal Bioanal Chem. 2013;405(5):1673-81

Background and Purpose: The incidence of breast cancer has surged in the past decade, affecting six out of ten households. Despite advancements, a complete cure remains elusive, necessitating novel therapeutic approaches. This study aims to formulate and evaluate the efficacy of self-dissolving microneedles embedded with capecitabine in treating breast cancer using a DMBA-induced Swiss albino mice model.

Experimental Approach: Breast cancer was induced in Swiss albino mice by administering DMBA orally at a dose of 55 mg/kg for three weeks. Following tumor induction, capecitabine was delivered using a microneedle patch for 14 days. Body weight was measured before and after treatment, accompanied by hematology studies. Tumor size and volume were evaluated at the end of the study. Liver function tests, including SGOT and SGPT, were conducted, and histopathological analysis was performed to assess tissue changes.

Key Results: The developed tumor model was consistent and suitable for evaluating therapeutic efficacy. Mice treated with DMBA showed a significant increase in body weight before capecitabine administration. However, body weight decreased when treated with capecitabine embedded microneedles, indicating effective therapeutic activity. Hematology parameters also increased following DMBA administration but normalized post-treatment. Histopathological analysis confirmed the reduction in tumor volume and improved tissue integrity, supporting the anticancer potential of capecitabine-loaded microneedles.

Conclusion: Capecitabine embedded in self-dissolving microneedles demonstrated effective therapeutic activity against DMBA-induced breast cancer in Swiss albino mice, suggesting its potential as a novel and efficient treatment strategy.
Key words: Breast Cancer, Microneedle, DMBA, Capecitabine

Background: Tamoxifen, a selective estrogen receptor modulator, is widely used in breast cancer therapy. Its effectiveness requires metabolic activation by the enzyme CYP3A4. Calcium channel blockers are often co-administered with tamoxifen in clinical settings, but their potential interactions with tamoxifen metabolism have not been fully investigated.

Aims: To evaluate the effects of the calcium channel blockers nimodipine, nitrendipine, and felodipine on tamoxifen metabolism in vitro and in vivo, particularly their interactions with CYP3A4 and their impact on tamoxifen's pharmacokinetics.

Methods: To evaluate the inhibitory effects of nimodipine, nitrendipine, and felodipine on tamoxifen metabolism, 28 cardiovascular drugs were screened. Kinetic assays identified the inhibition type. Pharmacokinetic studies in rats assessed the impact of pretreatment with these drugs on tamoxifen's AUC, Cmax, and clearance. Additionally, molecular docking studies explored the interactions between the blockers and the active site of CYP3A4.

Results: Kinetic assays revealed competitive inhibition by nimodipine and felodipine in both RLM and HLM, while nitrendipine exhibited non-competitive inhibition in RLM and competitive inhibition in HLM. Pharmacokinetic studies in rats showed that pretreatment with nimodipine and nitrendipine significantly increased tamoxifen's AUC and Cmax, while reducing its clearance. Molecular docking confirmed interactions between these drugs and the active site of CYP3A4.

Conclusions: The study demonstrated that nimodipine, nitrendipine, and felodipine can inhibit tamoxifen metabolism, leading to increased systemic exposure. The observed drug interactions may require careful monitoring and potential dose adjustments of tamoxifen in clinical practice when co-administered with these calcium channel blockers.

Key Words: tamoxifen; calcium channel blockers; pharmacokinetic; molecular docking

Background: Lenvatinib has a high protein binding rate, resulting in high variability of free lenvatinib concentration (FLC) depending on patient conditions and drug interactions.

Aims: In this study, an ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) assay was established to measure FLC in plasma of hepatocellular carcinoma (HCC) patients, and the effect of hepatic dysfunction on FLC was evaluated.

Methods: We studied 31 HCC patients treated orally with lenvatinib. Blood samples were collected at seven time points on the first day of lenvatinib administration to measure total lenvatinib concentrations (TLC) and FLC using UHPLC-MS/MS.

Results: Pharmacokinetic (PK) parameters were calculated, and hepatic function was assessed using Child-Pugh (CP) classification and score. Patients were divided into CP class A (n = 22) and class B (n = 9), and PK parameters were compared between the two groups. The CP class B group had significantly higher trough concentrations (Ctrough) of total and free lenvatinib as well as Ctrough normalized to dose (Ctrough/Dose) compared to the class A group. A significant positive correlation was found between CP score and both Ctrough and Ctrough/Dose of total and free lenvatinib. Interestingly, FLC tended to reflect the effects of hepatic function decline more sensitively than TLC. In addition, plasma albumin concentration correlated significantly with protein binding rate.

Conclusions: FLC may reflect the effects of hepatic dysfunction more sensitively than TLC. Therefore, measuring FLC may be clinically useful in patients with hepatic dysfunction.

Keywords: Free lenvatinib concentration, UHPLC-MS/MS, Hepatic dysfunction

Background: This study aims to assess the accuracy of 2-point and 3-point blood sampling methods for Busulfan.

Methodology: A comprehensive review was conducted using the PubMed database spanning from 1989 to March 2024, with keywords including Busulfan, limited sampling, and therapeutic drug monitoring. Accuracy was evaluated based on the percentage of root mean square error (%RMSE) and the percentage of Mean Prediction Error (%MPE). The analysis included forest plots and a heterogeneity test.

Results: Nine studies comprising 44 results were included in the analysis, revealing no significant heterogeneity. A notable difference in mean accuracy was observed between 2-point and 3-point sampling for both %RMSE (3.177 ± 0.182, 95% CI 2.818 to 3.535, p < 0.001) and %MPE (-52.47 ± 3.916, 95% CI -60.256 to -44.689).

Conclusion: The findings suggest that 3-point sampling offers significantly higher accuracy and is therefore preferable for limited sampling in Busulfan monitoring.

Background: High plasma levels of sunitinib exposure can lead to severe toxicity; therefore, exploring the relationship between  exposure and toxicity  may provide a valuable reference basis for the precise administration of sunitinib.

Aims: This study aims to investigate the relationship between sunitinib exposure and toxicity in a real-world patient cohort.

Methods: We conducted a retrospective observational cohort study involving 39 patients diagnosed with metastatic renal cell carcinoma(mRCC) who were treated with sunitinib. The steady-state trough concentrations of sunitinib and its active metabolite were quantified using HPLC-MS/MS. Sunitinib-induced toxicity was evaluated based on the Common Terminology Criteria for Adverse Events version 4.0.

Results: A total of 33 patients (85%) reported experiencing sunitinib-induced toxicity. The mean exposure in patients with toxicity was significantly higher (87.10 ± 30.85 ng/mL) than in those without toxicity (58.16 ± 22.27 ng/mL) (P = 0.035). However, in individuals experiencing grade 3-4 toxicities, the exposure level (73.78 ± 18.69 ng/mL) was not significantly different from that in the no-toxicity group (55.24 ± 18.72 ng/mL (P = 0.196).

Conclusions: This study confirms a positive association between sunitinib exposure and toxicity in Chinese patients with mRCC in a real-world cohort. The absence of a significant relationship between grade ≥3 toxicity and exposure may be attributed to poor medication adherence, potentially leading to decreased blood concentrations due to self-discontinuation of the drug. In future studies, we will expand the sample size and enhance the management of patient medication adherence.

Key Words: sunitinib; exposure; toxicity; real-world; mRCC

Therapeutic drug monitoring (TDM) is a clinical practice used for optimizing and individualizing drug therapy, and it is essential for ensuring the safety and efficacy of drugs and achieving precision medicine. As a result of the ongoing advancement of TDM, there is an urgent desire for a fast, simple, and easy-to-operate technology to achieve point-of-care testing for TDM. In recent years, aptamers have frequently been employed as biorecognition elements due to their high specificity, good stability, low cost, and ease of modification. Aptamers can be combined with various signal transduction methods (such as optics and electrochemistry) to form aptamer sensors (aptasensors), which are extensively applied in different fields including environmental monitoring, food safety, and clinical diagnosis. Aptamer sensors are increasingly becoming an attractive tool for immediate TDM, yet there is currently no comprehensive review on this topic. Consequently, we intend to provide a thorough overview of the applications of aptamer sensors in point-of-care TDM over the past five years and analyze its development trend, aiming to promote the continuous advancement of point-of-care testing technologies for TDM.

Background: Cholangiocarcinoma (CCA) is a highly aggressive bile duct cancer with limited treatment options and poor prognosis. Current therapies often fail due to late-stage diagnosis and drug resistance, highlighting the need for novel treatments. Natural compounds from Thai traditional plants offer promising anticancer potential.

Aim: This study aims to explore new agents for CCA treatments and evaluating mechanisms of action in CCA cell lines.

Methods: The cytotoxic effects of Thai traditional plants, and chamuangone, a phytochemical from Thai medicinal plants, on CCA using KKU-100 and KKU-452 cell was evaluated through the MTT assay. The effects of chamuangone on cell apoptosis and Ki67 expression were performed using Muse® flow cytometry.

Results: Methanolic extracts of Elaeagnus latifolia barks showed inhibitory cell proliferation of KKU-100 with IC50 values of 134.6 and 219.8 µg/ml for 24 and 48 h. Methanolic extracts of Hylocereus cacti, Averrhoa bilimbi, and Phyllanthus acidus -barks showed inhibitory cell proliferation of KKU-452 with IC50 values of 269.8 and 154.1, 194.5 and 52.27, 169.2 and 157.4 µg/ml for 24 and 48 h. Interesting, chamuangone demonstrated a strong inhibitory effect on CCA cells, with an IC50 value of 1 µg/ml, and effectively induced apoptosis also significantly suppressed the Ki67 expression.

Conclusions: Chamuangone significantly induces cytotoxic effects, highlighting its potential as a more effective component of methanolic extracts from the barks of Thai traditional plants. These findings suggest that chamuangone could serve as a promising therapeutic candidate for cancer drug development.

Keywords: Cholangiocarcinoma, Cell Culture, Apoptosis, Cancer Drug Development, Thai Traditional Plants

Background: Venetoclax (VEN) combined with hypomethylating agents (HMAs) has shown promising activity in patients with myelodysplastic syndromes (MDS).

Aims: The aim of the current study was to evaluate the exposure-efficacy/safety relationships of the VEN + HMAs regimen in Chinese MDS patients.

Methods: This study retrospectively collected the predose (C0) and 6 hours post-oral dosing plasma concentration (C6) of VEN for exposure-response analyses, using receiver operator characteristic (ROC) curves and logistic regression.

Results: The average C0 and C6 of VEN were 1990.60 ± 1591.12ng/mL and 2966.66 ± 1406.96ng/mL, respectively, with large inter-individual variability. The use of azole antifungals was the only significant factor influencing VEN concentration (P < 0.05). Compared to VEN 400mg administered without azole antifungals, concomitant use of azole antifungals with VEN 100mg resulted in a 100.03% and 18.50% increase in VEN C0 and C6, respectively. Based on logistic regression and ROC curve analyses, a plasma threshold of 2858.80ng/mL for C6 was found to be significantly associated with treatment success. Other factors, including C0, demographics, and disease characteristics (e.g., molecular mutations, baseline grade III/IV neutropenia, and prior therapies), were not associated with the probability of marrow remission. In the safety analyses, higher VEN concentrations were not associated with an increased probability of grade ≥ 3 infection or a more serious decrease in platelets and neutrophils.

Conclusions: This study provides insights into the VEN exposure threshold that predicts efficacy but not adverse events, offering valuable guidance for optimizing treatment strategies.

Keywords
venetoclax, hypomethylating agents, myelodysplastic syndromes, exposure-response

Background:

Intravenous busulfan, a key component of pediatric hematopoietic stem cell transplantation (HSCT) conditioning regimens, has a narrow therapeutic range requiring precise dose adjustments. Standard clinical practice involves obtaining five to eight post-infusion plasma concentrations to estimate patient-specific exposure, but frequent sampling increases patient discomfort and venipuncture-related risks.

Aims:

To evaluate the feasibility of limited sampling strategies and assess their predictive accuracy with model-informed precision dosing (MIPD) through a retrospective analysis of pediatric busulfan courses from Children's Healthcare of Atlanta.

Methods:

MIPD of intravenous busulfan was conducted using exemplar one- and two-compartment population pharmacokinetic (PK) models from the literature within the Bayesian Clinical Decision Support System (CDSS) DoseMeRx™. Individualized model performance was assessed by fitting varying subsets of post-peak plasma concentrations from the first dose and comparing their goodness-of-fit and predictive accuracy against models individualized with all first-dose samples for predicting subsequent concentrations after the second dose.

Results:

Most limited sampling strategies demonstrated high predictive accuracy in both one- and two-compartment models, with those incorporating both early and later time points performing best. The optimal strategy included an initial level between three- and four-hours post-infusion, an optional second level between four and six hours, and a final level between five- and nine-hours post-infusion.

Conclusion:

This study demonstrates that a Bayesian CDSS, when combined with a limited sampling strategy, can accurately predict future busulfan plasma concentrations in pediatric HSCT patients, achieving comparable performance to fully-informed PK models currently used in clinical practice.

Keywords: Bayesian, limited sampling, busulfan, pediatric, HSCT