BackgroundTacrolimus is a core immunosuppressant in organ transplantation, but its narrow therapeutic window and significant pharmacokinetic variability hinder precision dosing. Although CYP3A5-guided strategies have established clinical relevance for tacrolimus initial dose adjustment, they do not fully account for the marked interindividual variability in tacrolimus exposure, highlighting the need for complementary models to decode more complex genetic regulation. This study aimed to identify candidate genetic modulators of tacrolimus metabolism and develop an integrated predictive framework for individualized therapy.MethodsUsing 46 BXD recombinant inbred mouse strains, we characterized transcriptomics and machine learning, and validated key genes. We then constructed a clinical model using data from 168 renal transplant recipients.ResultsWe identified 19 genomic loci associated with tacrolimus pharmacokinetic traits and supported DBP/CYP2A6 as candidate modulators associated with tacrolimus disposition. The clinical prediction model, incorporating these genes and clinical variables, achieved robust AUROC.ConclusionsThese findings support a polygenic contribution to tacrolimus metabolism and provide an experimental and computational framework for identifying candidate modulators relevant to individualized dosing. The BXD mouse platform offers a systems-genetics approach for mechanistic discovery that may inform future translational studies on tacrolimus precision dosing.

Background The clinical utility of polymyxin B (PB), a last-resort antibiotic for multidrug-resistant Gram-negative infections, is severely compromised by acute kidney injury (AKI). However, both early diagnostic approaches and the mechanistic understanding of PB-induced AKI remain limited. To address these gaps, we conducted a large-scale study in a clinical cohort and a rat model using an integrated analytical approach. Methods We conducted the first integrated mass spectrometry analysis of 83 patients receiving PB-based regimens (60 non-AKI cases and 23 AKI cases), with longitudinal plasma samples collected (n = 207). Key findings were validated across species using male Sprague-Dawley rats. Targeted quantification of plasma polymyxin B1 (PB1), PB2, PB3, isoleucine-polymyxin B1 (ILE-PB1), and PB6 was performed using ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS), alongside spatial mapping of their renal distribution using airflow-assisted desorption electrospray ionization-mass spectrometry imaging (AFADESI-MSI); and metabolite alterations were profiled via untargeted metabolomics. Multivariable logistic regression models were constructed, and the area under the receiver operating characteristic curve (AUC) was used to evaluate their diagnostic performance. Results A therapeutic drug monitoring panel of PB1, PB2, PB3, and ILE-PB1 improved risk stratification over the conventional index (AUC 0.703 vs. 0.561). Additionally, a plasma metabolite panel comprising creatine, PC(16:1/22:5), and LysoPC(18:0) discriminated PB-induced AKI from non-AKI with an AUC of 0.815, outperforming creatinine alone (AUC 0.740), the conventional kidney function marker. Notably, creatine alterations preceded serum creatinine elevations. Importantly, we further demonstrated that PB induces nephrotoxicity through a dual mechanism involving upregulation of renal fatty acid (FA) uptake and synthesis pathways, concomitant with profound suppression of tricarboxylic acid (TCA) cycle activity. Conclusions Both panels enable more accurate prediction of PB-induced AKI and outperform traditional markers, thereby facilitating clinical risk stratification. Mechanistically, PB nephrotoxicity is driven by dual metabolic perturbations—enhanced renal fatty acid metabolism and impaired tricarboxylic acid cycle activity—providing potential therapeutic targets.

IntroductionSuraxavir Marboxil (GP681) is a prodrug metabolized to GP1707D07, which inhibits influenza viral replication by targeting cap-dependent endonuclease through a single oral dose. This study assesses the in vivo drug-drug interaction (DDI) potential between GP681 (including its major metabolite GP1707D07, a substrate of CYP3A4) and itraconazole in healthy Chinese subjects, along with the safety profiles during co-administration. Additionally, it evaluates the impact of CYP1A2, CYP2C19, and CYP3A4 gene polymorphisms on GP1707D07 metabolism.MethodsThe study enrolled twelve healthy adult subjects to receive the treatments consisting of GP681 monotherapy and GP681-itraconazole co-administration in a fixed-sequence. Single nucleotide polymorphisms (SNPs) in CYP gene loci were also analyzed.ResultsCo-administration of itraconazole increased the GP1707D07 AUC0-∞ by about 2.5 folds and Cmax by about 1.4 folds compared with GP681 administered alone. Differences in system exposure were more pronounced during the terminal elimination phase than the early stage of GP1707D07 metabolism. No significant increase in adverse events was observed during co-administration. Using random forest algorithm, we estimated effects of cytochrome P450 enzymes followed the order of CYP 3A4 > CYP 1A2 > CYP 2C19. We also hypothesized CYP 3A4 rs4646437 A>G, CYP 3A4 rs2246709 G>A, and CYP 2C19 rs12768009 A>G to be mutations that enhanced enzyme activity, while CYP1A2 rs762551 C>A weakened it.DiscussionThe pharmacokinetic changes of GP1707D07 during itraconazole co-administration are insufficient to warrant clinical action. Random forest algorithm enhances the understanding of pharmacogenetic variants involved in GP1707D07 metabolism and may serve as a potent tool for assessing gene polymorphism data in small clinical samples.Clinical Trial Registrationclinicaltrials.gov, identifier NCT05789342.

(3-Lactam/(3-lactamase inhibitors (BL/BLIs) are widely used in critically ill patients. Recent research has shown the importance of therapeutic drug monitoring (TDM) of BLs, but few studies have highlighted the importance of detecting BLIs in critically ill patients. In our laboratory, we have developed and validated a simple and robust method for the determination of ceftazidime, cefoperazone, piperacillin, avibactam, sulbactam and tazobactam in human plasma by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Sample preparation was by protein precipitation of 100 mu L of sample, followed by chromatographic separation on an ACQUITY UPLC (R) BEH C18 column (2.1 x 50 mm, 1.7 mu m) and mass spectrometric detection using a SHIMADZU 8050CL in multiple reaction monitoring (MRM) mode. The method was fully validated for selectivity, carry-over, linearity, lower limit of quantification, matrix effect, extraction recovery, stability and dilution integrity. The results of the TDM could provide feedback to clinicians and allow timely adjustment of dosing regimens in critically ill patients. The method is suitable for routine TDM and has been successfully applied to the clinical determination of 81 plasma concentrations in 44 patients.

Multiple myeloma (MM) is the most aggressive and prevalent primary malignant tumor within the blood system, and can be classified into grades RISS-I, II, and III. High-grade tumors are associated with decreased survival rates and increased recurrence rates. To better understand metabolic disorders and expand the potential targets for MM, we conducted large-scale untargeted metabolomics on plasma samples from MM patients and healthy controls (HC). Our study included 33 HC, 38 newly diagnosed MM patients (NDMM) categorized into three RISS grades (grade I: n = 5; grade II: n = 19; grade III: n = 8), and 92 MM patients post-targeted therapy with bortezomib-based regimens. Simultaneously, MM cell lines were employed for validation studies. Metabolites were analyzed and identified using ultra high liquid chromatography coupled with Q Orbitrap mass spectrometry (UPLC-HRMS), followed by verification through a self-built database. Compared with HC participants, a total of 70 metabolites were identified as undergoing significant changes in NDMM. These metabolites were significantly enriched in citrate cycle, choline metabolism, glycerophospholipid metabolism, and sphingolipid metabolism, etc. Notably, a panel of circulating plasma metabolite biomarkers, including lactic acid and leucine, has emerged not only as diagnostic indicators but also as valuable tools for tumor surveillance, aiding in the assessment of disease stage and prognostic evaluation. Moreover, 14 differential metabolites were identified in both MM cell lines and MM patients. Among these, intracellular levels of lactate and leucine significantly decreased in vitro, aligning with the plasma results. Our findings on key metabolites and metabolic pathways provide novel insights into the exploration of diagnostic and therapeutic targets for MM. A prospective study is essential to validate these discoveries for future MM patient care.

Given the increasing use of bevacizumab in combinatorial drug therapy for a multitude of different cancer types, there is a need for therapeutic drug monitoring to analyze the possible correlation between drug trough concentration, and therapeutic effect and adverse reactions. An ultra-performance liquid chromatography tandem-mass spectrometry method was then developed and validated to determine bevacizumab levels in human plasma samples. Chromatographic separation was achieved on a Shimadzu InertSustainBio C18 HP column, whereas subsequent mass spectrometric analysis was performed using a Shimadzu 8050CL triple quadrupole mass spectrometer equipped with an electro-spray ionization source in the positive ion mode. In total, three multiple reaction monitoring transitions of each of the surrogate peptides were chosen with 'FTFSLDTSK' applied as the quantification peptide whereas 'VLIYFTSSLHSGVPSR' and 'STAYLQMNSLR' were designated as the verification peptides using the Skyline software. This analytical method was then fully validated, with specificity, linearity, lower limit of quantitation, accuracy, precision, stability, matrix effect and recovery calculated. The linearity of this method was developed to be within the concentration range 5-400 mu g/ml for bevacizumab in human plasma. Subsequently, eight patients with non-small cell lung cancer (NSCLC) were recruited and injected with bevacizumab over three periods of treatment to analyze their steady-state trough concentration and differences. To conclude, the results of the present study suggest that bevacizumab can be monitored in a therapeutic setting in patients with NSCLC.

The association between polymorphisms of the human ATP binding cassette subfamily B member 1 (ABCB1) gene and opioid response has attracted intense attention recently. As the ABCB1 gene encodes for the transporter P-glycoprotein in the brain and intestine involved in the pharmacokinetics of opioids, we investigated the effects of ABCB1 genetic polymorphisms on doses of opioids for pain relief and determined which pharmacokinetic process was affected in cancer pain patients. Sixty-eight cancer pain patients admitted for intrathecal therapy (ITT) were included. The association between ABCB1 genetic polymorphisms (C3435T, C1236T, G2677T/A and A61G) and systemic doses of opioids before ITT were investigated. Concentrations of oxycodone in plasma and cerebrospinal fluid (CSF) were determined by HPLC-MS/MS in 17 patients treated with oral oxycodone before ITT, and the influences of ABCB1 genetic polymorphisms on plasma-concentration to oral-dose ratios and CSF-concentration to plasma-concentration ratios of oral oxycodone were further analyzed. ABCB1 C3435T and G2677T/A polymorphisms were significantly associated with systemic doses of opioids before ITT, which coincided with the influences of ABCB1 C3435T and G2677T/A polymorphisms on the ratios of plasma-concentration to oral-dose. However, no significant difference was found in ratios of CSF-concentration to plasma-concentration among ABCB1 SNP genotypes. The present study provided the first evidence that ABCB1 C3435T and G2677T/A polymorphisms affect opioid requirement in cancer pain patients via altering transportation function of P-glycoprotein in the intestine, which will further expand our knowledge about pharmacokinetics of opioids and could contribute to the individualization of opioids use.

Amino acid metabolic remodeling is a hallmark of cancer, driving an increased nutritional demand for amino acids. Amino acids are pivotal for energetic regulation, biosynthetic support, and homeostatic maintenance to stimulate cancer progression. However, the role of phenylalanine in multiple myeloma (MM) remains unknown. Here, we demonstrate that phenylalanine levels in MM patients are decreased in plasma but elevated in bone marrow (BM) cells. After the treatment, phenylalanine levels increase in plasma and decrease in BM. This suggests that changes in phenylalanine have diagnostic value and that phenylalanine in the BM microenvironment is an essential source of nutrients for MM progression. The requirement for phenylalanine by MM cells exhibits a similar pattern. Inhibiting phenylalanine utilization suppresses MM cell growth and provides a synergistic effect with Bortezomib (BTZ) treatment in vitro and murine models. Mechanistically, phenylalanine deprivation induces excessive endoplasmic reticulum stress and leads to MM cell apoptosis through the ATF3–CHOP–DR5 pathway. Interference with ATF3 significantly affects phenylalanine deprivation therapy. In conclusion, we have identified phenylalanine metabolism as a characteristic feature of MM metabolic remodeling. Phenylalanine is necessary for MM proliferation, and its aberrant demand highlights the importance of low-phenylalanine diets as an adjuvant treatment for MM.

Background Multiple myeloma (MM) is the most aggressive and prevalent primary malignant tumor within the blood system, and can be classified into grades RISS-I, II, and III. High-grade tumors are associated with decreased survival rates and increased recurrence rates. To better understand metabolic disorders and expand the potential targets for MM, we conducted large-scale untargeted metabolomics on plasma samples from MM patients and healthy controls (HC). Methods Our study included thirty-three HC, thirty-eight newly diagnosed MM patients (NDMM) categorized into three RISS grades (grade I: n = 5; grade II: n = 19; grade III: n = 8), and ninety-two MM patients post-targeted therapy with bortezomib-based regimens. Metabolites were analyzed and identified using ultra high liquid chromatography coupled with Q Orbitrap mass spectrometry (UPLC-HRMS), followed by verification through a self-built database. Results Compared with HC participants, seventy metabolites, primarily associated with the citrate cycle, amino acids and glycerophospholipid/sphingolipid metabolism, and nine metabolic pathways (citrate cycle, choline metabolism, glyceropholipid metabolism, sphingolipid metabolism, valine, leucine and isoleucine biosynthesis, etc.) exhibited significant changes in NDMM. Notably, lactic acid and leucine have emerged not only as diagnostic biomarkers but also as markers for tumor monitoring in staging and prognosis, respectively. Conclusion Our findings on key metabolites and metabolic pathways provide novel insights into the exploration of diagnostic and therapeutic targets for MM. A prospective study is essential to validate these discoveries for future MM patient care.

[This corrects the article DOI: 10.1016/j.apsb.2024.04.021.].

BACKGROUND AND AIMS:A few researches have reported the exposure-efficacy/toxicity relationships of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). On account of the large interpatient pharmacokinetic variability, therapeutic drug monitoring (TDM) seems promising for optimizing dosage regimen and improving treatment efficacy and safety. Therefore, a rapid and convenient ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of icotinib, osimertinib, gefitinib and O-demesthyl gefitinib in human plasma for TDM. MATERIALS AND METHODS:Icotinib-D4 and osimertinib-13CD3 were used as the internal standards (ISs). The samples were prepared by protein precipitation using acetonitrile. Chromatographic separation was achieved on a 40 ℃ Shimadzu Shim-pack Scepter C18-120 column (2.1 ×50 mm, 3.0 µm, Japan) by a Shimadzu 30 A solvent management system. Detection was carried out using a Shimadzu LC-MS 8050CL triple quadrupole mass spectrometer coupled with an electrospray ionization source in positive mode. RESULTS:This analytical method was fully validated with selectivity, carry-over, linearity, lower limit of quantification, accuracy (from 92.68% to 106.62%) and precision (intra- and inter-day coefficients of variation ranged from 0.92% to 9.85%), matrix effect, extraction recovery, stability and dilution integrity. The calibration curves were developed to be within the concentration ranges of 200-4000 ng/mL for icotinib, 50-1000 ng/mL for osimertinib, gefitinib and O-desmethyl gefitinib in human plasma which meet the needs of routine TDM. CONCLUSIONS:The proposed method was used in 100 patients with non-small cell lung cancer for monitoring plasma concentration of the mentioned EGFR-TKIs. The trough concentrations of ICO were distributed between 226.42 ng/mL and 3853.36 ng/mL, peak concentrations were between 609.20 ng/mL and 2191.54 ng/mL. The trough concentrations of OSI were distributed between 110.48 ng/mL and 1183.13 ng/mL. The trough concentrations of GEF were distributed between 117.71 ng/mL and 582.74 ng/mL, while DeGEF was distributed from 76.21 ng/mL to 1939.83 ng/mL with two less than 20 ng/mL. The results of therapeutic drug monitoring aimed to investigate exposure-efficacy/toxicity relationship and improve the efficacy and safety of targeted therapies.

The determination of intracellular tacrolimus concentration in peripheral blood mononuclear cells (PBMCs) is crucial for assessing the effect-site concentration of tacrolimus. Analytical methods previously reported required a minimum of 3 mL of whole blood sample for measuring the tacrolimus concentration. In this study, we developed a highly sensitive method using EASY nLC 1200 combined with Q Exactive orbitrap mass spectrometer for detecting tacrolimus in PBMCs, requiring only 0.5-2 mL of sample. Furthermore, we compared two primary normalization methods for PBMCs tacrolimus concentration using Passing-Bablok regression, Bland-Altman analysis, Spearman's rank correlation, and Mountain plot. The newly established method was employed to compare tacrolimus concentrations in whole blood and PBMCs among 194 lung transplant recipients. The developed method exhibited high sensitivity with a lower limit of quantitation at 5 pg/mL, and excellent intra- and inter-days accuracy and precision. The comparison between different normalization methods for PBMCs tacrolimus concentration revealed a strong correlation between PBMCs count and intracellular protein amount within these cells. This finding suggests that both PBMCs count and intracellular protein amount can be used for normalizing intracellular tacrolimus levels and can be mutually converted. However, a weaker correlation was observed between PBMCs and whole-blood tacrolimus concentrations in lung transplant recipients, warranting further investigation. The method reported herein enables the quantification of PBMCs tacrolimus concentration using smaller volumes of whole blood samples, which has significant implications for both patients and laboratory personnel.

Tacrolimus and voriconazole are usually used simultaneously in lung transplantations. Voriconazole can increase tacrolimus concentrations by inhibiting the cytochrome P450 (CYP) enzyme, which poses a great challenge for dose adjustment. The aim of this study is to clarify the correlation between voriconazole exposure and tacrolimus trough concentrations (C-0), and to establish a population pharmacokinetic model including voriconazole trough concentrations (VOZ) as a covariate for dose optimization. All data were retrospectively collected from lung transplantation patients who were subjected to therapeutic drug monitoring of tacrolimus and voriconazole. The correlation between C-0 and VOZ or voriconazole daily doses was analyzed by Spearman's correlation. A total of 52 patients accounting for 351 pairs of tacrolimus and voriconazole trough concentrations were included. C-0 and C-0/daily dose of tacrolimus (DD) had a significant correlation with VOZ (P < .01) rather than voriconazole daily doses. A linear one-compartment model with first-order absorption and elimination was used as the basic model for population pharmacokinetic analysis. Body weight (WT), DD, VOZ, and hematocrit (HCT) were included as covariates in the final model. With the increase in voriconazole concentrations, the apparent total clearance (clearance/bioavailability, CL/F) of tacrolimus decreased significantly. The simulation results showed that the highest proportion of C-0 within the target range can only reach <50% when the optimal initial drug regimen was given. Therefore, both tacrolimus and voriconazole concentrations need to be continuously monitored during treatments in lung transplantation patients, and the tacrolimus dose can be optimized according to VOZ based on the established pharmacokinetic model.

Therapeutic drug monitoring is an essential tool when managing the therapeutic use of immunosuppressant cyclosporine A (CsA) in cases with solid organ transplantation. In China, the concentration of CsA is primarily measured using immunoassays. However, existing literature recommends mass spectrometry as the current gold standard for the quantitation of CsA. In the present study, it was attempted to develop a novel application to determine CsA concentrations by using ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS). This technique was then compared with a commercially available chemiluminescent microparticle immunoassay (CMIA) and it was investigated how clinical factors may contribute to quantitation differences between the two methods. An UPLC-Orbitrap-MS method was developed to determine CsA concentrations and this method was validated using guidelines put forward by the Food and Drug Administration from the US. In total, 127 blood samples were acquired from patients undergoing kidney transplantation and analyzed by UPLC-HRMS and CMIA assays. The novel method provided sensitive, accurate and precise results. The mean CsA concentration measured by CMIA was significantly higher than that measured by UPLC-HRMS (85.70±48.99 vs. 67.06±34.56 ng/ml, P<0.0001). Passing Bablok analysis yielded a slope of 1.34 (95% CI: 1.22-1.47) and an intercept of -2.54 (95% CI: -10.29-5.52). A group of samples with a higher metabolic ratio (hydroxylated CsA/CsA>1) exhibited larger discrepancies, while a group of samples taken from patients with a longer post-transplantation time (>10 years) featured narrow 95% CIs from -15.32 to 65.69%, as determined by Bland-Altman analysis. In summary, a reliable, accurate and rapid UPLC-HRMS method for CsA analysis was successfully developed. The measurement of CsA by the CMIA assay in renal transplant patients should be further evaluated with a specific focus on positive bias.

目的:利用超高效液相色谱-串联质谱法,比较不同品牌(赛默飞和岛津)的蛋白沉淀板测定人血浆霉酚酸的效果.方法:含霉酚酸的血浆样本经分别2种蛋白沉淀板进行前处理,以霉酚酸-d3为内标,采用Acquity UPLC? BEH-C18柱(2.1 mm×50 mm,1.7 μm)分离,流动相包括水(含0.1％甲酸,2 mmol·L-1醋酸铵,V/V)和乙腈(含0.1％甲酸,V/V),梯度洗脱,流速0.2 mL·min-1.正离子检测模式(+ESI)下,采用多离子反应监测(MRM)模式进行分析,霉酚酸的离子对为m/z 321.2-207.1,内标的离子对为324.0-210.0.比较各蛋白沉淀板的准确度、精密度、基质效应、提取回收率等指标.结果:霉酚酸的线性范围为0.312 5～20μg·mL-1,定量下限为0.312 5 μg·mL-1.各品牌的蛋白沉淀板所获得的准确度、精密度、基质效应和提取回收率均符合要求.结论:这2种蛋白沉淀板操作简便、快速、准确、且精密度良好,方法学考察结果没有显著性差异,均适用于人血浆中霉酚酸的分析,可用于临床治疗药物监测.

目的 结合治疗药物监测及GPⅡb/Ⅲa基因多态性检测,分析利奈唑胺谷浓度影响因素及其致血小板减少影响因素,为利奈唑胺使用患者个体化治疗提供理论支持.方法 选取我院30例利奈唑胺使用患者,超高效液相串联质谱法(ultra performance liquid chromatography tandem mass spectrometry,UPLC-MS/MS)检测利奈唑胺谷浓度,测序检测GPⅡb/Ⅲa基因多态性.t检验、卡方检验、线性回归等分析利奈唑胺谷浓度、血小板减少影响因素,多重线性回归分析血小板变化率影响因素.结果 影响利奈唑胺谷浓度的主要因素为用药剂量和估算肾小球滤过率(estimated glomerular filtration rate,eGFR),建议肾功能不全患者加强对利奈唑胺谷浓度的监测.利奈唑胺致血小板减少与患者性别、eGFR、用药剂量及利奈唑胺谷浓度显著相关,以性别、eGFR、用药时间、GPⅡb/Ⅲa基因型CC为自变量建立的多重线性回归方程具有统计学意义,可以解释32.5％的血小板减少原因.结论 性别、eGFR、利奈唑胺谷浓度、GPⅡb/Ⅲa基因多态性、用药时间和用药剂量与利奈唑胺致血小板减少存在相关性,可以通过多重线性回归方程预测血小板减少的发生,指导利奈唑胺个体化治疗.

目的:考察美罗培南骨水泥药柱的体外缓释规律,为临床使用提供参考.方法:用模具将美罗培南加入骨水泥中制成含美罗培南10％(W/W)的骨水泥植入剂,浸泡于37℃的改良模拟体液中,采用等距浸出法设定取样时间点,应用超高效液相色谱-串联质谱法对浸出液浓度进行检测,观察美罗培南骨水泥植入剂的释放规律.结果:美罗培南骨水泥药柱释药峰值出现在2 h,之后释药速率逐渐降低,19 h累计释放量达总体释放量的92.37％,在24 d内累积释放率达6.00％,体外释放曲线符合Weibull方程,r=0.9151.结论:美罗培南骨水泥具有一定的缓释特性,释放主要集中在前72 h内,后期释放少.本研究结果可以为临床使用美罗培南治疗骨感染提供参考.