Therapeutic Monitoring of Palbociclib, Ribociclib, Abemaciclib, M2, M20, and Letrozole in Human Plasma: A Novel LC-MS/MS Method.

BACKGROUND: Therapeutic drug monitoring (TDM) using cyclin-dependent kinase inhibitors (CDK4/6is) is a novel approach for optimizing treatment outcomes. Currently, palbociclib, ribociclib, and abemaciclib are the available CDK4/6is and are primarily coadministered with letrozole. This study aimed to develop and validate an LC-MS/MS method for the simultaneous analysis of CDK4/6is, 2 active metabolites of abemaciclib (M2 and M20), and letrozole in human plasma for use in TDM studies. METHODS: Sample pretreatment comprised protein precipitation with methanol and dilution of the supernatant with an aqueous mobile phase. Chromatographic separation was achieved using a reversed-phase XBridge BEH C18 column (2.5 µm, 3.0 × 75 mm XP), with methanol serving as the organic mobile phase and pyrrolidine-pyrrolidinium formate (0.005:0.005 mol/L) buffer (pH 11.3) as the aqueous mobile phase. A triple quadrupole mass spectrometer was used for the detection, with the ESI source switched from negative to positive ionization mode and the acquisition performed in multiple reaction monitoring mode. RESULTS: The complete validation procedure was successfully performed in accordance with the latest regulatory guidelines. The following analytical ranges (ng/mL) were established for the tested compounds: 6-300, palbociclib and letrozole; 120-6000, ribociclib; 40-800, abemaciclib; and 20-400, M2 and M20. All results met the acceptance criteria for linearity, accuracy, precision, selectivity, sensitivity, matrix effects, and carryover. A total of 85 patient samples were analyzed, and all measured concentrations were within the validated ranges. The percent difference for the reanalyzed samples ranged from -11.2% to 7.0%. CONCLUSIONS: A simple and robust LC-MS/MS method was successfully validated for the simultaneous quantification of CDK4/6is, M2, M20, and letrozole in human plasma. The assay was found to be suitable for measuring steady-state trough concentrations of the analytes in patient samples.

Therapeutic drug monitoring and pharmacogenetics to tune imatinib exposure in gastrointestinal stromal tumor patients: hurdles and perspectives for clinical implementation.

Tweetable abstract Present evidence supports the use of intensified pharmacologic monitoring of #imatinib including #TherapeuticDrugMonitoring and #PGx to improve outcomes in patients with GI stromal tumor. Future studies need to address emerging questions to facilitate implementation in clinics.

Association of ADME gene polymorphisms on toxicity to CDK4/6 inhibitors in patients with HR+ HER2- metastatic breast cancer.

A wide interindividual variability in therapeutic response to cyclin-dependent kinases 4 and 6 inhibitors (CDKis) palbociclib, ribociclib and abemaciclib, among patients with HR+/HER2- metastatic breast cancer has been reported. This study explored the impact of genetic polymorphisms in ADME genes (responsible for drug absorption, distribution, metabolism, and elimination) on CDKis safety profiles in 230 patients. Selected endpoints include grade 3/4 neutropenia at day 14 of the first treatment cycle, early dose-limiting toxicities (DLTs), and dose reductions within the initial three cycles. Our analysis revealed associations between these endpoints and polymorphisms in CYP3A4, CYP3A5, ABCB1, and ABCG2 genes. Their impact on CDKis plasma concentrations (Ctrough) was also examined. Specifically, ABCB1 c.1236C>T and c.2677C>T polymorphisms correlated significantly with grade 3/4 neutropenia at day 14 (OR 3.94, 95% CI 1.32-11.75; p = 0.014 and OR 3.32, 95% CI 1.12-9.85; p = 0.030). Additionally, ABCB1 c.3435C>T was associated with an elevated risk of early DLTs and dose reductions (OR 3.28, 95% CI 1.22-8.84, p = 0.019; OR 2.60, 95% CI 1.20-5.60, p = 0.015). Carriers of the CYP3A4*22 allele also demonstrated in univariate a higher risk of early DLTs (OR 3.10, 95% CI 1.01-9.56, p = 0.049). Furthermore, individuals with the ABCB1 1236T-3435T-2677T(A) variant haplotype exhibited significant associations with grade 3/4 neutropenia at day 14 (OR 3.36, 95% CI 1.20-9.41; p = 0.021) and early DLTs in univariate (OR 3.08, 95% CI 1.19-7.95; p = 0.020). Homozygous carriers of the ABCB1 T-T-T(A) haplotype tended to have a higher mean ribociclib Ctrough (934.0 ng/mL vs. 752.0 ng/mL and 668.0 ng/mL). Regardless preliminary, these findings offer promising insights into the role of pharmacogenetic markers in CDKis safety profiles, potentially contributing to address the interindividual variability in CDKis responses.

Clinical impact of body mass index on palbociclib treatment outcomes and effect on exposure.

The impact of body mass index (BMI) on treatment outcomes in patients with cancer is gaining increasing attention given the limited data available. The aim of this study was to investigate the contribution of BMI on the safety and efficacy profile of palbociclib in 134 patients with metastatic luminal-like breast cancer treated with palbociclib and endocrine therapy (ET). Normal-weight and underweight patients (BMI<25) were compared with overweight and obese (BMI≥25). Detailed clinical and demographic data were collected. Patients with a BMI<25 had a higher incidence of relevant-hematologic toxicities (p = 0.001), dose reduction events (p = 0.003), and tolerated lower dose intensities (p = 0.023) compared to patients with a BMI≥25. In addition, patients with a BMI<25 had significantly shorter progression-free survival (log-rank p = 0.0332). A significant difference was observed in the subgroup of patients for whom systemic palbociclib concentrations were available: patients with a BMI<25 had a 25% higher median minimum plasma concentrations (Cmin) compared to BMI≥25. This study provides compelling evidence for a clinically relevant contribution of BMI in discriminating a group of patients who experienced multiple toxicities that appeared to affect treatment adherence and lead to poorer survival. BMI could become a valuable tool for personalizing the starting dose of palbociclib to improve its safety and efficacy.

The use of therapeutic drug monitoring to highlight an over-looked drug-drug interaction leading to imatinib treatment failure.

BACKGROUND: Chronic oral anticancer therapies, are increasingly prescribed and present new challenges including the enhanced risk of overlooked drug-drug interactions (DDIs). Lengthy treatments and patients' management by different professionals can lead to serious prescribing errors that therapeutic drug monitoring (TDM) can help identifying thus allowing a more effective and safer treatment of patients with polypharmacy. OBJECTIVES: This report aims to exemplify how an intensified pharmacological approach could help in the clinical monitoring of patients on chronic treatments. METHODS: A patient with gastrointestinal stromal tumor was referred to our clinical pharmacology service due to tumor progression while on imatinib therapy. The investigation was based on TDM, pharmacogenetics, DDI evaluation and Circulating tumor DNA (ctDNA) analysis. The patient underwent repeated blood samplings to measure imatinib and norimatinib plasma concentrations through a validated LC-MS/MS method. Polymorphisms affecting genes involved in imatinib metabolism and transport were investigated using SNPline PCR Genotyping System. Drug-drug interactions were evaluated though Lexicomp. ctDNA analysis was performed on MiSeq platform. RESULTS: TDM analysis revealed that the patient was underexposed to imatinib (Cmin = 406 ng/mL; target Cmin = 1100 ng/mL). Subsequent DDI analysis highlighted a dangerous interaction with carbamazepine, via CYP3A4 and P-gp strong induction, omitted at the time of imatinib treatment start. No relevant pharmacogenetic variants were identified and appropriate compliance to treatment was ascertained. ctDNA monitoring was performed to assess potential tumor-related resistance to imatinib. Carbamazepine was cautiously switched to a non-interacting antiepileptic drug, restoting IMA plasma concentration (i.e. Cmin = 4298 ng/mL). The progression of the disease, which in turn led to the patient's death, was also witnessed by an increasing fraction of ctDNA in plasma. CONCLUSION: The active pharmacological monitoring allowed the identification of a dangerous previously over-looked DDI leading to IMA under-exposure. The switch to a different antiepileptic treatment, reversed the effect of DDI, restoring therapeutic IMA plasmatic concentrations.

LC-MS/MS Method for the Quantification of PARP Inhibitors Olaparib, Rucaparib and Niraparib in Human Plasma and Dried Blood Spot: Development, Validation and Clinical Validation for Therapeutic Drug Monitoring.

Poly (ADP-ribose) polymerase inhibitors (PARPis) are becoming increasingly meaningful in oncology, and their therapeutic drug monitoring (TDM) might be beneficial for patients. Several bioanalytical methods have been reported for PARPis quantification in human plasma, but advantages might be obtained using dried blood spot (DBS) as a sampling technique. Our aim was to develop and validate a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for olaparib, rucaparib, and niraparib quantification in both human plasma and DBS matrices. Additionally, we aimed to assess the correlation between the drug concentrations measured in these two matrices. DBS from patients was obtained using Hemaxis DB10 for volumetric sampling. Analytes were separated on a Cortecs-T3 column and detected with electrospray ionization (ESI)-MS in positive ionization mode. Validation was performed according to the latest regulatory guidelines, in the range (ng/mL) 140-7000 for olaparib, 100-5000 for rucaparib, and 60-3000 for niraparib, within the hematocrit (Hct) range 29-45%. The Passing-Bablok and Bland-Altman statistical analyses revealed a strong correlation between plasma and DBS for olaparib and niraparib. However, due to the limited amount of data, it was challenging to establish a robust regression analysis for rucaparib. To ensure a more reliable assessment, additional samples are required. The DBS-to-plasma ratio was used as a conversion factor (CF) without considering any patient-related hematological parameters. These results provide a solid basis for the feasibility of PARPis TDM using both plasma and DBS matrices.

The Role of Gender Pharmacogenetics in the Personalization of Drug Treatment.

The use of pharmacogenetic guidelines in personalizing treatments has shown the potential to reduce interindividual variability in drug response by enabling genotype-matched dosing and drug selection. However, other important factors, such as patient gender, may interact strongly with pharmacogenetics in determining the individual profile of toxicity and efficacy but are still rarely considered when planning pharmacological treatment. The literature indicates that males and females respond differently to drugs, with women being at higher risk for toxicity and having different plasma exposure to drugs at standard doses. Recent studies have shown that pharmacogenetic variants may have different predictive value in different sexes, as in the case of treatment with opioids, angiotensin-converting enzyme inhibitors, or proton pump inhibitors. Of particular interest is the case of treatment with fluoropyrimidines for cancer. A significant increase in toxicity has been described in female patients, with a more pronounced effect of specific DPYD and TYMS polymorphisms also noted. This manuscript reviews the major findings in the field of sex-specific pharmacogenomics. SIGNIFICANCE STATEMENT: Interindividual variability in drug response is an emerging issue in pharmacology. The genetic profile of patients, as well as their gender, may play a role in the identification of patients more exposed to the risk of adverse drug reactions or poor efficacy. This article reviews the current state of research on the interaction between gender and pharmacogenetics in addressing interindividual variability.

Impact of ABCG2 and ABCB1 Polymorphisms on Imatinib Plasmatic Exposure: An Original Work and Meta-Analysis.

Adequate imatinib plasma levels are necessary to guarantee an efficacious and safe treatment in gastrointestinal stromal tumor (GIST) and chronic myeloid leukemia (CML) patients. Imatinib is a substrate of the drug transporters ATP-binding cassette subfamily B member 1 (ABCB1) and ATP-binding cassette subfamily G member 2 (ABCG2) that can affect its plasma concentration. In the present study, the association between three genetic polymorphisms in ABCB1 (rs1045642, rs2032582, rs1128503) and one in ABCG2 (rs2231142) and the imatinib plasma trough concentration (Ctrough) was investigated in 33 GIST patients enrolled in a prospective clinical trial. The results of the study were meta-analyzed with those of other seven studies (including a total of 649 patients) selected from the literature through a systematic review process. The ABCG2 c.421C>A genotype demonstrated, in our cohort of patients, a borderline association with imatinib plasma trough levels that became significant in the meta-analysis. Specifically, homozygous carriers of the ABCG2 c.421 A allele showed higher imatinib plasma Ctrough with respect to the CC/CA carriers (Ctrough, 1463.2 ng/mL AA, vs. 1196.6 ng/mL CC + AC, p = 0.04) in 293 patients eligible for the evaluation of this polymorphism in the meta-analysis. The results remained significant under the additive model. No significant association could be described between ABCB1 polymorphisms and imatinib Ctrough, neither in our cohort nor in the meta-analysis. In conclusion, our results and the available literature studies sustain an association between ABCG2 c.421C>A and imatinib plasma Ctrough in GIST and CML patients.

Bioanalytical Methods for Poly(ADP-Ribose) Polymerase Inhibitor Quantification: A Review for Therapeutic Drug Monitoring.

BACKGROUND: Therapeutic drug monitoring (TDM) of poly(ADP-ribose) polymerase inhibitors (PARPis) is an exploratory practice aimed at improving the quality of treatment through personalized therapy. Currently, there are 4 European Medicines Agency-approved and US Food and Drug Administration-approved PARPis available clinically whose quantification requires validated analytical methods: olaparib, niraparib, rucaparib, and talazoparib. The purpose of this literature review was to highlight the pharmacological features of PARPis that could support their TDM practice and provide a detailed discussion of the available liquid chromatography coupled with tandem mass spectrometry methods for their quantification. METHODS: Using several Medical Subject Heading terms, the literature was searched using several research engines, including SciFinder, Web of Science, Google Scholar, and PubMed, to find articles published before August 2022. RESULTS: Exposure-efficacy and exposure-safety profiles, drug-drug interactions, and hepatic/renal impairment of PARPis provide the potential rationale to monitor their concentrations through TDM. Several bioanalytical methods for their quantification have been reported and compared, and a great deal of heterogeneity has been found among methods, regarding both their analytical and regulatory aspects. CONCLUSIONS: In addition to reducing toxicity and increasing the efficacy of PARPis therapy, TDM could be beneficial to thoroughly investigate the exposure-response relationships of PARPis and to establish pharmacokinetic thresholds for clinical decisions. Based on the comparison of published bioanalytical methods, their transferability and validation both play a key role in method selection. For future use in clinical TDM, we anticipate that bioanalytical methods should address every analytical need more thoroughly and should be validated with standardized guidelines.

A fast and validated LC-MS/MS method to quantify lenvatinib in dried blood spot.

A new LC-MS/MS method for the quantification of lenvatinib (LENVA) in venous Dried Blood Spot (DBS) samples has been presented. This method is characterized by a short run time (4 min), requires a volumetric sampling of 10 µL and extraction of the entire spot to avoid hematocrit (Hct) and spot volume effects. The quantification method was successfully validated in the range of 5.00-2000 ng/mL on two different DBS filter papers (Whatman 31 ET CHR and Whatman 903) according to European Medicines Agency (EMA) and Food and Drug Administration (FDA) guidelines, European Bioanalysis Forum (EBF), and International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT) recommendations. During the validation process, the following parameters were evaluated: recovery (≥ 77% for both filter papers), absence of matrix effect, process efficiency (close to 72% for Whatman 31 ET CHR and close to 77% for Whatman 903), Hct effect (CV ≤ 6.3% and accuracy within 96-112%), linearity (r ≥ 0.998 for Whatman 31 ET CHR and r ≥ 0.999 for Whatman 903), intra- and inter-day precision (CV ≤ 8.8%) and accuracy (92.8-108%), selectivity and sensitivity, reproducibility with incurred samples reanalysis (ISR), and stability. This method was applied to quantify venous DBS samples from patients with hepatocellular carcinoma treated with LENVA enrolled in a cross-validation study (CRO-2018-83). A good correlation between LENVA plasma concentration determined by standard procedure and the new developed DBS LENVA method (R2 ≥ 0.996) has been observed.

CYP2D6 and CYP2C8 pharmacogenetics and pharmacological interactions to predict imatinib plasmatic exposure in GIST patients.

AIMS: Patients on treatment with oral fixed dose imatinib are frequently under- or overexposed to the drug. We investigated the association between the gene activity score (GAS) of imatinib-metabolizing cytochromes (CYP3A4, CYP3A5, CYP2D6, CYP2C9, CYP2C19, CYP2C8) and imatinib and nor-imatinib exposure. We also investigated the impact of concurrent drug-drug-interactions (DDIs) on the association between GAS and imatinib exposure. METHODS: Serial plasma samples were collected from 33 GIST patients treated with imatinib 400 mg daily within a prospective clinical trial. Imatinib and nor-imatinib Ctrough were quantified by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Genetic polymorphisms with a functional impact on imatinib-metabolizing cytochromes were identified and a GAS was calculated for each gene. A DDI-adjusted GAS was also generated. RESULTS: Imatinib and nor-imatinib Ctrough were measured in 161 plasma samples. CYP2D6 GAS and metabolizer status based on genotype were associated with imatinib and (imatinib + nor-imatinib) Ctrough . CYP2D6 poor and intermediate metabolizers were predicted to have a lower nor-imatinib/imatinib metabolic ratio than normal metabolizers (0.197 and 0.193 vs. 0.247, P = .0205), whereas CYP2C8*3 carriers had a higher ratio than CYP2C8*1/*1 patients (0.263 vs. 0.201, P = .0220). CYP2C9 metabolizer status was inversely related to the metabolic ratio with an effect probably driven by the linkage disequilibrium between CYP2C9*2 and CYP2C8*3. The CYP2D6 DDI-adjusted GAS was still predictive of imatinib exposure. CONCLUSIONS: These findings highlight that CYP2D6 plays a major role in imatinib pharmacokinetics, but other players (i.e., CYP2C8) may influence imatinib exposure. These findings could drive the selection of patients more susceptible to imatinib under- or overexposure who could be candidates for personalized treatment and intensified monitoring strategies.

An Integrated Pharmacological Counselling Approach to Guide Decision-Making in the Treatment with CDK4/6 Inhibitors for Metastatic Breast Cancer.

A wide inter-individual variability in the therapeutic response to cyclin-dependent kinases 4 and 6 inhibitors (CDKis) has been reported. We herein present a case series of five patients treated with either palbociclib or ribociclib referred to our clinical pharmacological counselling, including therapeutic drug monitoring (TDM), pharmacogenetics, and drug-drug interaction analysis to support clinicians in the management of CDKis treatment for metastatic breast cancer. Patients' plasma samples for TDM analysis were collected at steady state and analyzed by an LC-MS/MS method for minimum plasma concentration (Cmin) evaluation. Under and overexposure to the drug were defined based on the mean Cmin values observed in population pharmacokinetic studies. Polymorphisms in selected genes encoding for proteins involved in drug absorption, distribution, metabolism, and elimination were analyzed (CYP3A4, CYP3A5, ABCB1, SLCO1B1, and ABCG2). Three of the five reported cases presented a CDKi plasma level above the population mean value and were referred for toxicity. One of them presented a low function ABCB1 haplotype (ABCB1-rs1128503, rs1045642, and rs2032582), possibly causative of both increased drug oral absorption and plasmatic concentration. Two patients showed underexposure to CDKis, and one of them was referred for early progression. In one patient, a CYP3A5*1/*3 genotype was found to be potentially responsible for more efficient drug metabolism and lower drug plasma concentration. This intensified pharmacological approach in clinical practice has been shown to be potentially effective in supporting prescribing oncologists with dose and drug selection and could be ultimately useful for increasing both the safety and efficacy profiles of CDKi treatment.

"A new LC-MS/MS method for the simultaneous quantification of abemaciclib, its main active metabolites M2 and M20, and letrozole for therapeutic drug monitoring".

Abemaciclib (ABEMA) is the last CDKi approved for the treatment of breast cancer. Adverse reactions to this drug are not experienced in the same manner by the entire patient population but in case of severe toxicity dose reductions and therapy discontinuation are required, suggesting that a TDM-guided treatment could be beneficial for these patients. ABEMA is extensively metabolized by the liver. The most abundant active metabolites are M2 and M20. This CDKi is administered together with anti-estrogen drugs, such as letrozole (LETRO). The aim of this work was to develop and validate a LC-MS/MS method for the simultaneous quantification of ABEMA, M2, M20, and LETRO. The chromatographic separation of the analytes was obtained using a SIL-20AC XR auto-sampler coupled to LC-20AD UFLC Prominence XR pumps (Shimadzu, Tokyo, Japan). The chromatographic column employed was an XTerra MS C18, (3,5 µm, 125 Å, 50x2.1 mm) coupled with a Security Guard Cartridge (MS C18, 125 Å, 3.9x5 mm) provided by Waters. Detection was performed by an API 4000 QTrap (SCIEX) mass spectrometer. The presented analytical method was fully validated according to EMA and FDA guidelines on bioanalytical method validation. Linearity was confirmed on 10 independent tests (R2 within 0.997-1.000) over the concentration ranges of 40-800 ng/mL for ABEMA, 10-200 ng/mL for M2 and M20, 20-400 ng/mL for LETRO. The method was applied to analyze plasma samples from patients enrolled in a clinical trial, collected at Cmin. Incurred sample reanalysis was performed on a set of 30 samples, confirming the reproducibility of the analytical method.

Population pharmacokinetic modelling of imatinib in healthy subjects receiving a single dose of 400 mg.

PURPOSE: Imatinib is indicated for treatment of CML, GIST, etc. The population pharmacokinetics (popPK) of imatinib in patients under long-term treatment are reported in literature. Data obtained from bioequivalence trials for healthy subjects were used to evaluate the influence of demographic and pharmacogenetic factors on imatinib pharmacokinetics (PK) in a collective without concurrent drugs, organ dysfunction, inflammation etc. In addition, the differences in PK between the healthy subjects and a patient cohort was examined to identify possible disease effects. METHODS: 26 volunteers were administered orally with single dose of 400 mg imatinib. 16-19 plasma samples per volunteer were collected from 0.5 up to 72 h post-dose. The popPK was built and post hoc estimates were compared with previously published PK parameters evaluated by non-compartmental analysis in the same cohort. The predictivity of the model for data collected from 40 patients with gastrointestinal stromal tumors at steady state was evaluated. RESULTS: The popPK was best described by a two-compartment transit model with first-order elimination. No significant covariates were identified, probably due to the small cohort and the narrow range of demographic covariates; CYP3A5 phenotypes appeared to have some influence on the clearance of imatinib. Good agreement between non-compartment and popPK analyses was observed with the differences of the geometric means/ median of PK estimates below 10%. The model indicated lower clearance for patients compared to healthy volunteers (p value < 0.01). CONCLUSION: The two-compartment transit model adequately describes the absorption and distribution of imatinib in healthy volunteers. For patients, a lower clearance of imatinib compared to healthy volunteer was estimated by the model. The model can be applied for dose individualization based on trough concentrations assuming no significant differences in absorption between patients and healthy volunteers.

A rapid, simple and sensitive LC-MS/MS method for lenvatinib quantification in human plasma for therapeutic drug monitoring.

Lenvatinib (LENVA) is an oral antineoplastic drug used for the treatment of hepatocellular carcinoma and thyroid carcinoma. LENVA therapeutic drug monitoring (TDM) should be mandatory for a precision medicine to optimize the drug dosage. To this end, the development of a sensitive and robust quantification method to be applied in the clinical setting is essential. The aim of this work was to develop and validate a sensitive, rapid, and cost-effective LC-MS/MS method for the quantification of LENVA in human plasma. On this premise, sample preparation was based on a protein precipitation and the chromatographic separation was achieved on a Synergi Fusion RP C18 column in 4 min. The method was completely and successfully validated according to European Medicines Agency (EMA) and Food and Drug Administration (FDA) guidelines, with good linearity in the range of 0.50-2000 ng/mL (R≥0.9968). Coefficient of variation (CV) for intra- and inter-day precision was ≤11.3% and accuracy ranged from 96.3 to 109.0%, internal standard normalized matrix effect CV% was ≤2.8% and recovery was ≥95.6%. Successful results were obtained for sensitivity (signal to noise (S/N) ratio >21) and selectivity, dilution integrity (CV% ≤ 4.0% and accuracy 99.9-102%), and analyte stability under various handling and storage conditions both in matrix and solvents. This method was applied to quantify LENVA in patient's plasma samples and covered the concentration range achievable in patients. In conclusion, a sensitive and robust quantification method was developed and validated to be applied in the clinical setting.

A new dried blood spot LC-MS/MS method for therapeutic drug monitoring of palbociclib, ribociclib, and letrozole in patients with cancer.

Therapeutic drug monitoring (TDM) is strongly suggested to define the proper drug dosage to overcome inter- and intra-patient variability in drug exposure, which is typically observed with oral anticancer agents, such as palbociclib (PALBO), ribociclib (RIBO) and letrozole (LETRO), all approved for the treatment of HR+, HER2- locally advanced or metastatic breast cancer (BC). Optimal TDM implementation requires a blood sampling organization that can be hampered by limited availability of health and laboratory personnel. Dried Blood Spot (DBS) sampling is proposed to overcome such limitations. The aim of this work was the development of a new LC-MS/MS method to analyze DBS samples containing PALBO, RIBO, and LETRO. Analytes extraction from DBS was performed by adding a methanolic solution containing the corresponding internal standards. LC-MS/MS analysis was performed using a LC Nexera (Shimadzu) system coupled with an API 4000 QTrap (SCIEX) mass spectrometer. The chromatographic separation was performed on a Luna Omega Polar C18 column (Phenomenex). The method was applied to 38 clinical samples collected by finger prick. The influence of hematocrit and spot size, sample homogeneity, stability, and correlation between finger prick and venous DBS measurement were assessed. The analytical validation was performed according to EMA and FDA guidelines. The analytical range of the method was 1 to 250 ng/mL for PALBO, 40 to 10000 ng/mL for RIBO, and 2 to 500 ng/mL for LETRO, where linearity was assessed, obtaining mean coefficients of determination (R2) of 0.9979 for PALBO, 0.9980 for RIBO, and 0.9987 for LETRO). The LC-MS/MS method runtime was 6.6 min. Incurred sample reanalysis demonstrated reproducibility, as the percentage difference between the two quantifications was lower than 20% for 100% of PALBO, 81.8% of RIBO, and 90.9% of LETRO paired samples. Intra- and inter-day precision (CV (%)) was lower than 11.4% and intra- and inter-day accuracy was between 90.0 and 106.5%. DBS sample stability at room temperature was confirmed for 2.5 months. A positive correlation was observed between DBS and plasma concentrations for the 3 drugs, Lin's concordance correlation coefficients obtained by DBS normalization applying a selected strategy were 0.958 for PALBO, 0.957 for RIBO, and 0.963 for LETRO. In conclusion, a fast, easy, and reproducible DBS LC-MS/MS method for the simultaneous quantification of palbociclib; ribociclib and letrozole was developed to be used in clinical practice.

Dried Blood Spot Technique Applied in Therapeutic Drug Monitoring of Anticancer Drugs: a Review on Conversion Methods to Correlate Plasma and Dried Blood Spot Concentrations.

BACKGROUND: Anticancer drugs are notoriously characterized by a low therapeutic index, the introduction of therapeutic drug monitoring (TDM) in oncologic clinical practice could therefore be fundamental to improve treatment efficacy. In this context, an attractive technique to overcome the conventional venous sampling limits and simplify TDM application is represented by dried blood spot (DBS). Despite the significant progress made in bioanalysis exploiting DBS, there is still the need to tackle some challenges that limit the application of this technology: one of the main issues is the comparison of drug concentrations obtained from DBS with those obtained from reference matrix (e.g., plasma). In fact, the use of DBS assays to estimate plasma concentrations is highly dependent on the chemical-physical characteristics of the measured analyte, in particular on how these properties determine the drug partition in whole blood. METHODS: In the present review, we introduce a critical investigation of the DBS-to-plasma concentration conversion methods proposed in the last ten years and applied to quantitative bioanalysis of anticancer drugs in DBS matrix. To prove the concordance between DBS and plasma concentration, the results of statistical tests applied and the presence or absence of trends or biases were also considered.

An SPR investigation into the therapeutic drug monitoring of the anticancer drug imatinib with selective aptamers operating in human plasma.

The anticancer drug imatinib is often involved in therapeutic drug monitoring (TDM) studies aimed at improving the treatment of several forms of leukemia and gastrointestinal stromal tumors (GIST). To further implement the TDM of imatinib in clinical practice, we developed a detection assay by using an ssDNA aptamer, which demonstrated excellent selectivity and was not affected by interference from the components of human plasma samples. The efficient binding of imatinib to the aptamer was demonstrated by means of surface plasmon resonance (SPR) analysis, which allowed the development of a quantitative assay in the concentration range between 400 and 6000 ng mL-1 (0.7-10 µM), where a lower limit of quantification (LLOQ) of 400 ng mL-1 was achieved. The precision of the assay was found to be within 12.0%, whereas the accuracy was in a range between 97.1 and 101.5%. The sample preparation procedure displayed a recovery in the range of 48.8-52.8%. Solid validation data were collected according to the regulatory guidelines and the method was compared with standard analytical techniques, leading to the development of a feasible aptasensor for the TDM of patients administered with imatinib.

Development and validation of a selective SPR aptasensor for the detection of anticancer drug irinotecan in human plasma samples.

In this work, a surface plasmon resonance (SPR)-based assay for the quantification of antineoplastic drug irinotecan in human plasma samples has been developed for the first time. The selective binding of irinotecan with an aptamer receptor, operating in human plasma, allowed to set-up a novel analytical methodology to detect the drug in the analytical range of interest by using SPR as detection technique. After hybridizing the aptamer to the sensing platform and optimizing the sample preparation procedure, a quantitative assay was validated according to FDA regulatory guidelines. The analytical working range was found between 100 and 7500 ng mL-1 with negligible interferences from plasma components and co-medication associated with the administration of irinotecan. The utility of the new SPR assay was confirmed by analyzing plasma samples in parallel with LC-MS as reference technique, providing a new analytical tool for the therapeutic drug monitoring of irinotecan in patients under chemotherapy regimens.

A LC-MS/MS method for therapeutic drug monitoring of sorafenib, regorafenib and their active metabolites in patients with hepatocellular carcinoma.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of sorafenib (SORA), its N-oxide active metabolite and of regorafenib (REGO) and its two active metabolites regorafenib N-oxide and N-desmethyl regorafenib N-oxide in hepatocellular carcinoma patients' plasma. A proper analytes' separation was obtained with Synergi Fusion RP column (4 µm, 80 Å, 50 × 2.0 mm) using a gradient elution of 10 mM ammonium acetate with 0.1% formic acid (mobile phase A) and methanol:isopropanol (90:10, v/v, mobile phase B) containing 0.1% formic acid. The analysis was then performed by electrospray ionization in negative mode coupled with a triple quadrupole mass spectrometry, API 4000QT, monitoring two transitions for each analyte, one for the quantification and the other for confirmation. The method could be easily applied to the clinical practice thanks to the short run (7 min), the low amount of patient plasma necessary for the analysis (5 µL) and the fast sample processing based on protein precipitation. The method was therefore fully validated according to FDA and EMA guidelines. The linearity was assessed (R2≥0.998) over the concentration ranges of 50-8000 ng/mL for SORA and REGO, and 30-4000 ng/mL for their metabolites, that appropriately cover the therapeutic plasma concentrations. The presented method also showed adequate results in terms of intra- and inter-day accuracy and precision (CV ≤ 7.2% and accuracy between 89.4% and 108.8%), recovery (≥85.5%), sensitivity, analytes stability under various conditions and the absence of the matrix effect. Once the validation was successfully completed, the method was applied to perform the Cmin quantification of SORA, REGO and their metabolites in 54 plasma samples collected from patients enrolled in a clinical study ongoing at the National Cancer Institute of Aviano.