Monitoring Tacrolimus Concentrations in Whole Blood and Peripheral Blood Mononuclear Cells: Inter- and Intra-Patient Variability in a Cohort of Pediatric Patients

Tacrolimus (TAC) is a first-choice immunosuppressant for solid organ transplantation, characterized by high potential for drug-drug interactions, significant inter- and intra-patient variability, and narrow therapeutic index. Therapeutic drug monitoring (TDM) of TAC concentrations in whole blood (WB) is capable of reducing the incidence of adverse events. Since TAC acts within lymphocytes, its monitoring in peripheral blood mononuclear cells (PBMC) may represent a valid future alternative for TDM. Nevertheless, TAC intracellular concentrations and their variability are poorly described, particularly in the pediatric context. Therefore, our aim was describing TAC concentrations in WB and PBMC and their variability in a cohort of pediatric patients undergoing constant immunosuppressive maintenance therapy, after liver transplantation. TAC intra-PBMCs quantification was performed through a validated UHPLC–MS/MS assay over a period of 2–3 months. There were 27 patients included in this study. No significant TAC changes in intracellular concentrations were observed (p = 0.710), with a median percent change of −0.1% (IQR −22.4%–+46.9%) between timings: this intra-individual variability was similar to the one in WB, −2.9% (IQR −29.4–+42.1; p = 0.902). Among different patients, TAC weight-adjusted dose and age appeared to be significant predictors of TAC concentrations in WB and PBMC. Intra-individual seasonal variation of TAC concentrations in WB, but not in PBMC, have been observed. These data show that the intra-individual variability in TAC intracellular exposure is comparable to the one observed in WB. This opens the way for further studies aiming at the identification of therapeutic ranges for TAC intra-PBMC concentrations.

The Rate of Cellular Energy Production of Muscle Cells Is Attenuated By Carnitine Intracellular Deficiency Caused By Imatinib Treatment

Introduction: Previous works identified that imatinib intake through the carnitine-specific OCTN2 (SLC22A5) transporter resulted in a significant decrease of carnitine intracellular concentrations in chronic myeloid leukemia (CML) and muscle cell lines. On contrary, even high doses of carnitine in preincubation did not influence imatinib cell intake capacity. Specifically performed inhibition of OCTN2 activity by vinorelbine resulted in block of carnitine cell intake, while imatinib intake was only slightly reduced (13-30%). This observation is in line with the knowledge that imatinib is transported also through other known SLC transporters. OCTN2 transporter is the major transporter for carnitine, an essential compound in cell energy metabolism. Presented work follows a hypothesis that non-equal competition between imatinib and carnitine intake through OCTN2 can lead to the carnitine intracellular deficiency, which can be in CML patients manifested by a disruption of skeletal muscle mitochondrial density and cause side effects like fatigue, muscle pain and cramps reported up to 80% of patients treated with imatinib (Kekale et al., 2015). Methods: Muscle cell HTB-153 (human rhabdomyosarcoma, ATCC HS 729), CML cell line KCL-22 (DSMZ ACC 519) were used for in vitro experiments. Intracellular concentration of imatinib, carnitine and metabolites were measured by chromatographic separation using XBridge Amide column (50x2.1mm, 3.5µm; Waters, Milford (MA), USA) and ZIC-pHILIC column (50x2.1mm, 5 µm; Merck, Darmstadt, Germany) coupled to tandem mass spectrometer (QTRAP 4000; Sciex, USA). Results: Carnitine, resp. L-carnitine transports long-chain fatty acids to mitochondria and its high rate is required especially in energetically demanding tissues such as skeletal and cardiac muscles. The concentrations of citric acid cycle (CAC) metabolites (citrate, malate, alpha-ketoglutarate, succinate, fumarate, 2-hydroxyglutarate, cis-aconitate), glycolysis (phosphoenolpyruvate, 3- phosphoglycerate, lactate), production of ATP, ADP and AMP were measured in HTB-153 cells 3 and 24 hours after imatinib treatment in vitro. The significant decrease of malate (CAC), lactate (glycolysis) and ATP levels were found at both time points after imatinib treatment compared to baseline. The same observations were found in KCL-22, which was used for comparison as BCR-ABL1 positive cell line. Additionally, significant decrease of succinate and 2-hydroxyglutarate (CAC) was detected in KCL-22 after imatinib treatment. Next, HTB-153 was incubated with imatinib (1-8 µM) for 24 hours and carnitine (8 µM) was supplied for last 3 hours of incubation, i.e., after 21 hours of imatinib treatment start. No significant changes were found in any metabolites of CAC and glycolysis. Production of ATP, ADP and AMP was not changed as well. Conclusions: Imatinib treatment of muscle (rhabdomyosarcoma) and CML cell lines caused a significant decrease of intracellular concentrations of carnitine. Significant decrease of ATP levels and of certain metabolites of CAC and glycolysis outlined that cells struggle from attenuated mitochondria energy production after imatinib treatment. This has not happened, if carnitine was supplied to the culture for final 3 hours of 24 hours incubation with imatinib. Observed data strongly support the hypothesis that decreased carnitine intake to the muscle cells due to competition with imatinib transport through OCTN2 attenuated mitochondria energy production. Interestingly, the clinical trial NCT03426722 (Chae H et al. 2019) showed that L-carnitine could effectively relieve imatinib-related muscle cramps and significantly increase QoL in patients with advanced gastrointestinal stromal tumor. Supported by GACR18-18407S, MZCR00023736 Disclosures No relevant conflicts of interest to declare.

Relationship between plasma and intracellular concentrations of bedaquiline and its M2 metabolite in South African patients with rifampin-resistant TB

Bedaquiline is recommended for the treatment of all patients with rifampin-resistant tuberculosis (RR-TB). Bedaquiline accumulates within cells, but its intracellular pharmacokinetics have not been characterized, which may have implications for dose optimization. We developed a novel assay using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure the intracellular concentrations of bedaquiline and its primary metabolite M2 in patients with RR-TB in South Africa. Twenty-one participants were enrolled and underwent sparse sampling of plasma and peripheral blood mononuclear cells (PBMCs) at months 1, 2 and 6 of treatment and at 3 and 6 months after bedaquiline treatment completion. Intensive sampling was performed at month 2. We used non-compartmental analysis to describe plasma and intracellular exposures and a population pharmacokinetic model to explore the relationship between plasma and intracellular pharmacokinetics and the effects of key covariates. Bedaquiline concentrations from month 1 to month 6 of treatment ranged from 94.7 – 2540 ng/mL in plasma and 16.2 – 5478 ng/mL in PBMCs and concentrations of M2 over the six-month treatment period ranged from 34.3 – 496 ng/mL in plasma and 109.2 – 16764 ng/mL in PBMCs. Plasma concentrations of bedaquiline were higher than M2, but intracellular concentrations of M2 were considerably higher than bedaquiline. In the pharmacokinetic modeling, we estimated a linear increase in the intracellular-plasma accumulation ratio for bedaquiline and M2, reaching maximum effect after 2 months of treatment. The typical intracellular-plasma ratio 1 and 2 months after start of treatment was 0.61 (95%CI: 0.42-0.92) and 1.10 (95%CI: 0.74-1.63) for bedaquiline and 12.4 (95%CI: 8.8-17.8) and 22.2 (95%CI: 15.6-32.3) for M2. The intracellular-plasma ratio for both bedaquiline and M2 was decreased by 54% (95%CI: 24-72%) in HIV-positive patients compared to HIV-negative patients. Bedaquiline and M2 were detectable in PBMCs 6 months after treatment discontinuation. M2 accumulated at higher concentrations intracellularly than bedaquiline, supporting in vitro evidence that M2 is the main inducer of phospholipidosis.

Synchrotron-based X-ray fluorescence microscopy reveals accumulation of polymyxins in single human alveolar epithelial cells

Intravenous administration of the last-line polymyxins results in poor drug exposure in the lungs and potential nephrotoxicity; while inhalation therapy offers better pharmacokinetics/pharmacodynamics for pulmonary infections by delivering the antibiotic to the infection site directly. However, polymyxin inhalation therapy has not been optimized and adverse effects can occur. This study aimed to quantitatively determine the intracellular accumulation and distribution of polymyxins in single human alveolar epithelial A549 cells. Cells were treated with an iodine-labeled polymyxin probe FADDI-096 (5.0 and 10.0 μM) for 1, 4, and 24 h. Concentrations of FADDI-096 in single A549 cells were determined by synchrotron-based X-ray fluorescence microscopy. Concentration- and time-dependent accumulation of FADDI-096 within A549 cells was observed. The intracellular concentrations (mean ± SEM, n ≥ 189) of FADDI-096 were 1.58 ± 0.11, 2.25 ± 0.10, and 2.46 ± 0.07 mM following 1, 4 and 24 h of treatment at 10 μM, respectively. The corresponding intracellular concentrations following the treatment at 5 μM were 0.05 ± 0.01, 0.24 ± 0.04, and 0.25 ± 0.02 mM (n ≥ 189). FADDI-096 was mainly localized throughout the cytoplasm and nuclear region over 24 h. The intracellular zinc concentration increased in a concentration- and time-dependent manner. This is the first study to quantitatively map the accumulation of polymyxins in human alveolar epithelial cells and provides crucial insights for deciphering the mechanisms of their pulmonary toxicity. Importantly, our results may shed light on the optimization of inhaled polymyxins in patients and the development of new-generation safer polymyxins.

Using a Riboswitch Sensor to Detect Co2+/Ni2+ Transport in E. coli

Intracellular concentrations of essential mental ions must be tightly maintained to avoid metal deprivation and toxicity. However, their levels in cells are still difficult to monitor. In this report, the combination of a Co2+Ni2+-specific riboswitch and an engineered downstream mCherry fluorescent protein allowed a highly sensitive and selective whole-cell Co2+/Ni2+ detection process. The sensors were applied to examine the resistance system of Co2+/Ni2+in E. coli, and the sensors were able to monitor the effects of genetic deletions. These results indicate that riboswitch-based sensors can be employed in the study of related cellular processes.

Azithromycin Concentrations During Long-Term Regimen in Patients With MALT Lymphoma

IntroductionIn view of the antineoplastic effects of the macrolide clarithromycin in mucosa associated lymphatic tissue (MALT)-lymphoma, we performed a pilot study assessing levels of azithromycin in plasma, peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes (PMN) of MALT-lymphoma patients to determine the pharmacokinetics and potential influences of respective concentrations on the therapeutic outcome.Material and methodsIn total 16 patients with MALT-lymphoma received 1.5g of oral azithromycin once-weekly over 6 months. Blood was sampled directly prior to the following dose every 4 weeks during treatment. Drug levels were analysed by high performance liquid chromatography in plasma and intracellularly in PBMC and PMN. They were correlated with patients’ age, weight and body-mass-index and compared between patients responsive or unresponsive to treatment.ResultsMean azithromycin plasma levels of all patients were 58.97±30.48 ng/ml, remaining stable throughout the treatment period. Correlation analysis of plasma azithromycin showed no significance. Intracellular PBMC concentrations were 6648±8479 ng/ml, without any significant difference between responders and non-responders. Mean PMN levels were 39274±25659 ng/ml and significantly higher in patients unresponsive to treatment (t=2.858, p=0.017).ConclusionOur drug regime led to continuously high plasma and exceedingly high intracellular concentrations of azithromycin in PBMC and PMN. Age, weight or body-mass-index had no significant influence on plasma levels and thence should not be considered in dosage finding. High AZM levels in PBMC did not lead to a better treatment response, whereas enrichment in PMN suggested a poorer outcome. The threshold for immunomodulatory effects on lymphoma cells might not have been reached. Additionally, the finding of stable plasma and intracellular concentrations over months with high-dose azithromycin administered in intervals might also be important for the further design of azithromycin-based trials against MALT-lymphoma.

Intracellular homocysteine metabolites in SLE: plasma S-adenosylhomocysteine correlates with coronary plaque burden

Background and aimsWe hypothesised that intracellular homocysteine and homocysteine metabolite levels in patients with SLE are disproportionately elevated compared with the levels seen in healthy subjects and that they are independently associated with coronary plaque in SLE.MethodsA liquid chromatography–tandem mass spectrometry absolute quantification assay was used for the determination of six analytes in both plasma and peripheral blood mononuclear cells (PBMCs): homocysteine (Hcy), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), cystathionine (Cysta) and 5-methyltetrahydrofolate (5m-THF). We then compared intracellular (PBMC) and extracellular (plasma) Hcy and Hcy metabolite (SAM, SAH, Met, Cysta and 5m-THF) concentrations in 10 patients with SLE and in 10 age, sex and ethnicity matched controls. Subjects with a history of diabetes mellitus, cardiovascular disease, hypertension, alcohol consumption in excess of 3 units per day, anaemia, renal insufficiency (serum creatinine >1.5 mg/dL) and pregnancy were excluded. All patients with SLE had two coronary CT angiography studies as screening for occult coronary atherosclerotic disease.ResultsPlasma from patients with SLE had higher levels of Hcy (p<0.0001), SAH (p<0.05), SAM (p<0.001) and lower levels of Met (p<0.05) and Cysta (p<0.001) compared with controls. PBMC intracellular concentrations from patients with SLE had higher levels of Cysta (p<0.05), SAH (p<0.05), SAM (p<0.001) and lower levels of 5m-THF (p<0.001). Plasma SAH showed a positive correlation with total coronary plaque, calcified plaque and non-calcified plaque (p<0.05).ConclusionIntracellular concentrations of Hcy metabolites were significantly different between patients with SLE and controls, despite similar intracellular Hcy levels. Plasma SAH was positively correlated with total coronary plaque, calcified plaque and non-calcified plaque.