Antibody-drug conjugates (ADCs) are innovative drugs composed of cytotoxic molecules (payload) linked to antibodies, that selectively target and kill cancer cells upon internalization. In vivo, ADCs exist as intact molecules, naked antibodies, or released, unconjugated (linker-)payload. Accurate quantification of these entities is crucial for understanding ADCs pharmacokinetics. Ligand-binding assays are commonly used to measure ADC concentrations and total antibody concentrations, whereas LC-MS/MS is used to analyze the payload. Due to limitations in ligand-binding assays, this review focuses on quantitative LC-MS methods for the different ADC entities. Quantitative LC-MS assays were described for all ADC entities, available from full manuscripts and regulatory reviews of 12 ADCs evaluated by the European Medicine Agency, by January 2025. The review summarized sample pre-treatment, chromatography, mass spectrometry, validation, and stability data for each LC-MS method. Overall, critical details were often missing, particularly concerning sample pre-treatment, validation criteria, and sample stability. In conclusion, LC-MS quantification of ADC entities is feasible but current methods lack sufficient detail. Our review highlights the need for further research to develop reliable LC-MS assays for ADCs. This review may serve as a starting point and outlines key factors to consider in future LC-MS method development.

PURPOSE DPYD -guided fluoropyrimidine dosing effectively limits the risk of severe toxicity while maintaining clinical efficacy. However, recent data suggest that c.1236G>A variant carriers, starting with a 25% reduced dose, have shorter progression-free survival than wild-type patients receiving a full dose. Although overall survival was unaffected, further investigation is warranted. To address this, we retrospectively compared 5-fluorouracil (5-FU) exposure between c.1236G>A variant carriers and DPYD wild-type patients. MATERIALS AND METHODS Pharmacokinetic data from nine clinical trials involving capecitabine-treated patients were pooled. Blood samples were collected before and after administration of capecitabine to assess systemic levels of its metabolites, including 5-FU. Capecitabine dosages were reduced for c.1236G>A variant carriers in accordance with the clinical guidelines at the time of study execution and varied from no reduction (n = 11) to a 25% (n = 16) or 50% (n = 8) reduction. Pharmacokinetic exposure, expressed as area under the plasma concentration-time curve (AUC 0-∞ ), was determined using noncompartmental analysis and dose-normalized to 850 mg/m 2 . RESULTS In total, 35 heterozygous c.1236G>A patients and 66 DPYD wild-type patients were evaluable. Patients carrying c.1236G>A who received a 50% dose reduction had a lower dose-normalized geometric mean 5-FU exposure (234 ng·h/mL coefficient of variation [CV = 43%]) compared with fully dosed c.1236G>A carriers (553 ng·h/mL [CV = 51%]) and fully dosed DPYD wild-type patients (582 ng·h/mL [CV = 48%]; P < .001). All c.1236G>A carriers who received a 50% dose reduction had AUC 0-∞ values below the AUC 0-∞ range observed in the wild-type group. CONCLUSION Our findings indicate that an upfront 25% dose reduction for capecitabine in c.1236G>A carriers is likely more appropriate than the currently recommended 50% dose reduction. We stress the importance of individual dose titration in c.1236G>A carriers to avoid both over- and undertreatment.

Background: Therapeutic Drug Monitoring optimizes oral anticancer drug treatment by measuring plasma levels. Volumetric absorptive microsampling (VAMS) allows home sampling with a minimal blood sample. However, methods for converting whole blood into plasma are required to interpret these results. This study aimed to establish conversion methods for abiraterone, alectinib, cabozantinib, imatinib, olaparib, sunitinib, and their metabolites, while assessing the differences between venous and capillary blood. The feasibility of home sampling was also evaluated. Methods: Plasma and VAMS samples, both from venipuncture-collected whole blood tubes and from a finger prick, were collected from each patient. The VAMS samples were deemed comparable if their concentrations were within ±20% of each other for ≥2/3rd of the patients. The Passing–Bablok regression and conversion factor methods were tested for the plasma and VAMS finger prick samples. The estimated plasma concentrations using both methods were required to be within ±20% of the measured plasma concentrations for ≥2/3rd of the pairs. Results: Overall, 153 patients were enrolled in this study. Conversion methods were applied to the VAMS samples, and the acceptance criteria were met for alectinib-M4, cabozantinib, imatinib, N-desmethyl imatinib, olaparib, sunitinib, and N-desethyl sunitinib but not for abiraterone, D4A, or alectinib. The capillary and venous VAMS concentrations were similar, except for that of D4A. Patients were positive toward home sampling. Conclusions: The established VAMS conversion methods for 7 out of 10 oral targeted anticancer drugs or metabolites met the acceptance criteria. Future studies need to validate the conversion methods with an independent cohort and integrate home sampling via VAMS to provide patients with an alternative to venipuncture at the outpatient clinic.

Understanding the target site pharmacokinetics (PK) of the nitroimidazole analog DNDI-0690, a potential drug for the neglected parasitic disease leishmaniasis, is important due to the diversity of infected tissue sites and potential drug penetration variability. An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for quantifying DNDI-0690 in murine biomatrices (plasma, liver, spleen, skin, and skin microdialysate). The method used three protein precipitation sample preparation procedures, tailored for different biomatrices, utilizing a surrogate biomatrix approach. Murine tissues were enzymatically homogenized with a Collagenase A mixture. Chromatographic detection was performed on a C18 column using gradient elution, coupled to a QTRAP6500 quadrupole MS, operating in positive ionization mode. The method demonstrated accurate and precise quantification of all murine biomatrices on the surrogate biomatrix calibration standards, with a high and reproducible total recovery ranging from 75.9% to 94.2% (CV% ≤ 2.5%). Matrix interferences were mitigated with a deuterated internal standard. Stability experiments demonstrated that DNDI-0690 remained stable in all biomatrices under various conditions. This validated UHPLC-MS/MS method was successfully used to quantify DNDI-0690 in a target site murine infection model, demonstrating its suitability for future target site PK studies involving DNDI-0690.

BACKGROUND:Abiraterone, an active metabolite of abiraterone acetate, is used for the treatment of prostate cancer. Therapeutic drug monitoring (TDM) of abiraterone could improve treatment outcomes. However, its stability in plasma for only 4 hours at room temperature, is making the TDM implementation difficult in clinical practice. Stabilization experiments were performed in our laboratory using esterase inhibitors for the stabilization of abiraterone acetate in preclinical samples. The esterase inhibitor bis(4-nitrophenyl) phosphate (BNPP) stabilizes abiraterone acetate and abiraterone as well. Therefore, we investigated whether the esterase inhibitor BNPP could stabilize abiraterone in fresh human plasma. METHODS:BNPP at 1 and 10 mM were evaluated for its stabilizing effects on abiraterone in fresh human K 2 EDTA plasma. The samples were analyzed using a validated liquid chromatography-mass spectrometry (LC-MS/MS) method. A partial validation assessed BNPP's impact on accuracy, precision, selectivity, and specificity within the fully validated LC-MS/MS method. RESULTS:BNPP at 10 mM, but not 1 mM, effectively prevented abiraterone degradation in fresh human K 2 EDTA plasma, maintaining stability for at least 5 days at room temperature. Partial validation confirmed that all results met the acceptance criteria of the European Medicines Agency guidelines and the US Food and Drug Administration guidance. CONCLUSIONS:We demonstrated that the esterase inhibitor BNPP effectively stabilizes abiraterone in fresh human K 2 EDTA plasma. BNPP had no significant effect on the accuracy, precision, selectivity, or specificity of LC-MS/MS for abiraterone detection. The addition of BNPP to clinical abiraterone samples may be helpful in implementing abiraterone TDM in daily clinical practice.

Leishmaniasis is a neglected parasitic infection that continues to pose a significant global health challenge, with currently limited effective treatment options. DNDI-6174 is a novel orally-active, investigational drug with antileishmanial properties. Herein, a novel ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to quantify DNDI-6174 in relevant murine biomatrices, i.e., K2EDTA plasma and enzymatically-homogenized skin, spleen and liver to support the translational pharmacokinetic-pharmacodynamic model-informed drug development. The chromatographic system consisted of a gradient elution on a standard C18 column connected to a triple quadrupole MS, operating in positive ionization mode. Pre-processing of murine tissues with collagenase A led to a superior homogenization and analyte extraction compared to mechanical disruption. Human K2EDTA plasma served as a surrogate matrix, enabling accurate (bias between -12.0 % and 9.8 %) and precise (relative standard deviation (RSD) ≤ 12.5 %) quantification of DNDI-6174 in the various murine biomatrices. Sample processing with tert-methylbutyl ether resulted in a reproducible recovery between 70.0 % and 93.8 % (RSD ≤ 4.0 %) with an absolute matrix factor between 0.89 and 1.00 for all biomatrices. DNDI-6174 was stable under various conditions, including under tissue homogenization conditions, in all biomatrices investigated. This method was successfully applied in a translational study using a murine cutaneous leishmaniasis skin infection model to assess the target site pharmacokinetics of DNDI-6174, supporting its development as clinical candidate.

BACKGROUND:DPYD-guided dosing enhances safety of fluoropyrimidine-based chemotherapy. However, approximately 23 % of patients still experience severe toxicity unexplained by the four commonly tested DPYD-variant alleles. Elevated pre-treatment uracil levels have been proposed as a surrogate marker for reduced DPD activity and an independent predictor of toxicity. This prospective study evaluated whether uracil-guided dose individualisation can reduce severe fluoropyrimidine-induced toxicity in DPYD wild-type patients. METHODS:Pre-treatment plasma uracil levels were quantified in patients scheduled to receive fluoropyrimidine-based therapy. DPYD wild-type individuals with uracil concentrations > 16 ng/mL (DPYDwt/Uhigh) received a 50 % dose reduction, in accordance with French RNPGx guidelines. The incidence of grade ≥ 3 fluoropyrimidine-related toxicity was compared between dose-reduced DPYDwt/Uhigh patients, DPYDwt patients with uracil ≤ 16 ng/mL (DPYDwt/Unormal), and a historical cohort of DPYDwt/Uhigh patients treated at full dose. Pharmacokinetic data were compared to a second historical cohort. RESULTS:Among 612 evaluable patients, 22 were DPYDwt/Uhigh. The incidence of severe toxicity in the dose-reduced group was significantly lower than in historical full-dose DPYDwt/Uhigh patients (20 % vs 43 %, P = 0.03) and comparable during the first 2 treatment cycles to DPYDwt/Unormal patients (10 % vs 11 %). However, 5-fluorouracil exposure was markedly reduced in nineteen dose-reduced DPYDwt/Uhigh patients (177 vs 381 ng*h/mL), while five subsequently treated fully dosed DPYDwt/Uhigh patients exhibited comparable exposure to historical wild-type controls (456 vs 381 ng*h/mL). No correlation was found between uracil levels and DPD enzyme activity (R=-0.006, P = 0.98). CONCLUSION:Uracil-guided dosing of fluoropyrimidines may reduce toxicity risk but leads to subtherapeutic 5-fluorouracil exposure in DPYD wild-type patients. This indicates that these patients are treated sub-optimally and that uracil is not a reliable predictor of DPD deficiency in DPYD wild-type patients.

Traditional drug‐food interaction studies of oral anticancer agents have a high patient burden. A patient‐friendly alternative approach to studying food effects could be the use of stable isotopically labeled microtracers. A prospective, single‐center, open‐label, crossover, food effect study with the microtracer 2 H 6 ‐alectinib was conducted in patients with ALK‐positive, non‐small cell lung cancer treated with 600 mg alectinib bidaily. On occasion 1 (fed state), patients received 100 μg 2 H 6 ‐alectinib in addition to their usual dose of alectinib and a standardized Dutch breakfast (320–392 kcal and 7.5–7.8 g fat). On occasion 2 (fasted state), patients received 2 H 6 ‐alectinib and alectinib after overnight fasting. Pharmacokinetic (PK) samples were collected up to 8 hours after intake of 2 H 6 ‐alectinib. The effect of food on relative bioavailability (F) and mean transit time of 2 H 6 ‐alectinib was assessed by population PK modeling. Differences in area under the plasma concentration‐time curve (AUC) and maximum concentration ( C max ) between fed and fasted states were estimated by simulations. MTT in the fed state was 3.14 hours (relative standard error (RSE): 16.0%). MTT and F in the fasted state were 28% (RSE: 20.5%) and 35% (RSE: 12.4%) lower, respectively, compared to the fed state. The geometric mean ratio (fed vs. fasted) of AUC and C max was 1.52 (90% confidence interval (CI): 1.25–1.89) and 1.42 (90% CI: 1.16–1.76), respectively. These results showed that the intake of a Dutch breakfast leads to a higher total exposure of alectinib. More importantly, the feasibility of a microtracer food effect study to reduce patient burden was demonstrated.

Home-sampling for therapeutic drug monitoring (TDM) for oral targeted anticancer drugs offers a promising alternative to traditional hospital-based sampling methods, though it presents challenges. This review aims to summarize the state-of-the-art of home-sampling methods for TDM and evaluates the analytical and clinical validation challenges. A comprehensive search was conducted across Embase, Medline, and Scopus. Eligible articles described analytical and/or clinical validation of home-sampling methods for oral targeted anticancer drugs. ASReview was used to process unique references and to identify relevant studies. Of the 39 included articles, 32 detailed on analytical validation experiments, while 27 covered clinical validation experiments. Dried blood spot and volumetric absorptive microsampling were the primary sampling methods. Key challenges were ensuring robust sample collection, sample pretreatment, hematocrit effects, and sample stability, which were generally thoroughly investigated. Clinical validation yielded promising results for most analytes, although external validation remains crucial for confirming reliability. Home-sampling methods for TDM of oral targeted anticancer drugs show promising results for clinical implementation. Methods for well-studied drugs may be clinically implemented immediately, while others require further external validation. Future research should address device-specific challenges and assess patient feasibility to facilitate the routine use of home-sampling in clinical practice.

A combination of targeted anticancer drugs with cytotoxic therapy can potentially overcome multidrug resistance. The multi-target kinase inhibitor sorafenib demonstrates synergistic activity when combined with chemotherapeutics in preclinical models. This phase I trial aimed to assess safety, tolerability, efficacy, and pharmacokinetics of sorafenib with gemcitabine and carboplatin. This single-center, open-label, dose-escalation and dose-expansion study included patients with advanced solid tumors considered for palliative treatment with gemcitabine and carboplatin. The maximum tolerated dose (MTD) was determined using a classic 3 + 3 dose-escalation design. Antitumor activity was evaluated every two treatment cycles. In total, 45 patients received treatment. Of the patients, 49

A reversed-phase liquid chromatography-tandem mass spectrometry method was developed and validated for quantifying nine novel oral targeted anticancer agents mainly indicated for non-small cell lung cancer: adagrasib, capmatinib, ensartinib, entrectinib, larotrectinib, lorlatinib, pralsetinib, selpercatinib and sotorasib in human plasma for therapeutic drug monitoring. Chromatographic separation used an Acquity BEH C18 column with step gradient of 0.1 % formic acid in water and acetonitrile-methanol (50:50, v/v), at a 0.5 mL/min flow rate. Plasma samples were pretreated via precipitation with acetonitrile and diluted in 0.1 % formic acid in water. The reversed-phase chromatography was coupled with tandem mass spectrometry in positive ion mode. The assay was successfully validated over the following ranges: 100 - 10,000 ng/mL for adagrasib, capmatinib, entrectinib, pralsetinib, selpercatinib; 50 - 50,000 ng/mL for ensartinib; 10 - 1000 ng/mL for larotrectinib, lorlatinib; and 10 - 10,000 ng/mL for sotorasib. Accuracy and precision met the predefined criteria. Stability tests confirmed that all analytes were stable in plasma for up to 157 days at -20°C except for entrectinib, which was stable for 35 days at -20°C. At room temperature, the analytes were at least stable in plasma for 7 days, however, for adagrasib, entrectinib and sotorasib, stability for up to 3 days could be demonstrated. We recommend sending these samples on dry ice or refrigerated. After the validation, 74 plasma samples were measured in the application phase and all results but one fell within the validated ranges. This assay allows simultaneous quantification of nine novel targeted therapies and supports therapeutic drug monitoring.

Supplementary Fig. S5. The set of eight genes from the MPAS signature was highly enriched in a GSEA analysis using as input the list of genes sorted on the results from the differential analysis “on treatment” vs. “baseline.”

Supplementary Fig. S2. Vorinostat plasma concentrations over time on days 1 and 14 of the first treatment cycle.

Supplementary Table S3. Results of RNA sequencing.

Supplementary Table S2. Tissue and ctDNA results.

Background: Volumetric Absorptive Microsampling (VAMS) is a useful tool for therapeutic drug monitoring (TDM) of oral targeted anticancer agents. VAMS aims to improve safety and efficacy by enabling at-home blood sample collection by patients. This study aimed to develop and validate an ultra-high performance liquid chromatography–tandem mass spectrometry method for the quantitative determination of abiraterone, alectinib, cabozantinib, imatinib, olaparib, sunitinib, and the metabolites, Δ(4)-abiraterone (D4A), alectinib-M4, imatinib-M1, and N -desethyl sunitinib, in dried whole blood samples using VAMS to support TDM. Methods: After the collection of 10 μL of whole blood sample using the VAMS device, the analytes were extracted from the tip using methanol with shaking, evaporated, and reconstituted in acetonitrile:0.1 mol/L ammonium hydroxide in water (1:1, vol/vol). The extracts were then analyzed using ultra-high performance liquid chromatography–tandem mass spectrometry. Validation experiments based on the ICH M10 guideline were carried out, and stability was evaluated under shipping and storage conditions. VAMS specimens were collected in the outpatient clinic to demonstrate the applicability of the assay. Results: The validated range of the method was considered accurate and precise for all analytes. Accordingly, the validation experiments met the relevant requirements, except for cross-analyte interference. Based on the stability data, shipment can be performed at room temperature within 14 days after sample collection and the VAMS specimen can be stored up to 9 months at −20 and −70°C. Samples from 59 patients were collected at the hospital. Conclusions: The developed method could be used to successfully quantify the concentrations of abiraterone, D4A, alectinib, alectinib-M4, cabozantinib, imatinib, imatinib-M1, olaparib, sunitinib, and N -desethyl sunitinib within the validated range using VAMS. Therefore, the method can be used to estimate the dried whole blood-to-plasma ratios for TDM in the clinic.

Malaria remains a major health concern, aggravated by emerging resistance of the parasite to existing treatments. The World Health Organization recently endorsed the use of artesunate-pyronaridine to treat uncomplicated malaria. However, there is a lack of clinical pharmacokinetic (PK) data of pyronaridine, particularly in special populations such as children and pregnant women. Existing methods for the quantification of pyronaridine in biological matrices to support PK studies exhibit several drawbacks. These include limited sensitivity, a large sample volume required, and extensive analysis time. To overcome these limitations, an ultra-performance reversed-phase liquid chromatography tandem-mass spectrometry method to determine pyronaridine was developed and validated according to international guidelines. The method enabled fast and accurate quantification of pyronaridine in whole blood across a clinically relevant concentration range of 0.500-500 ng/mL (r2 ≥ 0.9963), with a required sample volume of 50 µL. Pyronaridine was extracted from whole blood using liquid-liquid extraction, effectively eliminating the matrix effect and preventing ion enhancement or suppression. The method achieved a satisfactory reproducible sample preparation recovery of 77%, accuracy (as bias) and precision were within ±8.2% and ≤5.3%, respectively. Stability experiments demonstrated that pyronaridine was stable for up to 315 days when stored at -70°C. Adjustments to the chromatographic system substantially reduced carry-over and improved sensitivity compared to prior methods. The method was successfully applied to quantify pyronaridine in whole blood samples from a selection of pregnant malaria patients participating in the PYRAPREG clinical trial (PACTR202011812241529) in the Democratic Republic of the Congo, demonstrating its suitability to support future PK studies. Furthermore, the enhanced sensitivity allows for the determination of pyronaridine up to 42 days post-treatment initiation, enabling assessment of the terminal elimination half-life.

Carboxylesterase 2 (CES2) is expressed mainly in liver and intestine, but most abundantly in intestine. It hydrolyzes carboxylester, thioester, and amide bonds in many exogenous and endogenous compounds, including lipids. CES2 therefore not only plays an important role in the metabolism of many (pro-)drugs, toxins and pesticides, directly influencing pharmacology and toxicology in humans, but it is also involved in energy homeostasis, affecting lipid and glucose metabolism. In this study we investigated the pharmacological and physiological functions of CES2. We constructed Ces2 cluster knockout mice lacking all eight Ces2 genes (Ces2-/- strain) as well as humanized hepatic or intestinal CES2 transgenic strains in this Ces2-/- background. We showed that oral availability and tissue disposition of capecitabine were drastically increased in Ces2-/- mice, and tissue-specifically decreased by intestinal and hepatic human CES2 (hCES2) activity. The metabolism of the chemotherapeutic agent vinorelbine was strongly reduced in Ces2-/- mice, but only marginally rescued by hCES2 expression. On the other hand, Ces2-/- mice exhibited fatty liver, adipositis, hypercholesterolemia and diminished glucose tolerance and insulin sensitivity, but without body mass changes. Paradoxically, hepatic hCES2 expression rescued these metabolic phenotypes but increased liver size, adipose tissue mass and overall body weight, suggesting a "healthy" obesity phenotype. In contrast, intestinal hCES2 expression efficiently rescued all phenotypes, and even improved some parameters, including body weight, relative to the wild-type baseline values. Our results suggest that the induction of intestinal hCES2 may combat most, if not all, of the adverse effects of metabolic syndrome. These CES2 mouse models will provide powerful preclinical tools to enhance drug development, increase physiological insights, and explore potential solutions for metabolic syndrome-associated disorders.

Supplementary Table S1. Representativeness of study participants.