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Suppression of immune functions can be elicited by behavioural conditioning using drugs such as cyclosporin A or rapamycin. Nevertheless, little is known about the underlying mechanisms and generalisability of this phenomenon. Against this background, the present study investigated whether the pharmacological properties of fingolimod (FTY720), an immunosuppressive drug widely applied to treat multiple sclerosis, can be conditioned in rats by means of taste-immune associative learning. For this purpose, a conditioned taste avoidance paradigm was used, pairing the presentation of a novel sweet drinking solution (saccharin or sucrose) as conditioned stimulus (CS) with therapeutically effective doses of FTY720 as unconditioned stimulus (US). Subsequent re-exposure to the CS at a later time point revealed that conditioning with FTY720 induced a mild conditioned taste avoidance only when saccharin was employed as CS. However, on an immunological level, neither re-exposure with saccharin nor sucrose altered blood immune cell subsets or splenic cytokine production. Despite the fact that intraperitonally administered FTY720 could be detected in brain regions known to mediate neuro-immune interactions, the present findings show that the physiological action of FTY720 is not inducible by mere taste-immune associative learning. Whether conditioning generalises across all small-molecule drugs with immunosuppressive properties still needs to be investigated with modified paradigms probably using distinct sensory CS. Moreover, these findings emphasize the need to further investigate the underlying mechanisms of conditioned immunomodulation to assess the generalisability and usability of associative learning protocols as supportive therapies in clinical contexts. [ABSTRACT FROM AUTHOR]

Purpose: Cannabichromene (CBC) is a phytocannabinoid commonly found in cannabis, yet its acute post-dose pharmacokinetics (PK) have not been examined in humans. This is a secondary data analysis from a trial investigating Spectrum Yellow oil, an oral cannabis product used for medical purposes that contained 20 mg cannabidiol (CBD), 0.9 mg Δ9-tetrahydrocannabinol (THC), and 1.1 mg CBC, per 1 mL of oil. Methods: Participants (N = 43) were randomized to one of 5 groups: 120 mg CBD, 5.4 mg THC, and 6.6 mg CBC daily; 240 mg CBD, 10.8 mg THC, and 13.2 mg CBC daily; 360 mg CBD, 16.2 mg THC, and 19.8 mg CBC daily; 480 mg CBD, 21.6 mg THC, and 26.4 mg CBC daily; or placebo. Study medication was administered every 12 h for 7 days. Plasma CBC concentrations were analyzed by a validated two-dimensional high-performance liquid chromatography–tandem mass spectrometry assay. Results: After a single dose and after the final dose, the Cmax of CBC increased by 1.3–1.8-fold for each twofold increase in dose; the tmax range was 1.6–4.3 h. Based on the ratio of administered CBD, THC, and CBC to the plasma concentration, the dose of CBD was 18 times higher than the dose of CBC, yet the AUC0–t of CBD was only 6.6–9.8-fold higher than the AUC0–t of CBC; the dose of THC was similar to the dose of CBC, yet THC was quantifiable in fewer plasma samples than was CBC. Conclusions: CBC may have preferential absorption over CBD and THC when administered together. Trial Registration: Australian New Zealand Clinical Trials Registry #ACTRN12619001450101, registered 18 October 2019. [ABSTRACT FROM AUTHOR]

The endocannabinoid system (ECS) is a complex cell-signaling system. To address the growing need of analytics capturing endocannabinoid levels to investigate the ECS, we developed and validated an assay for the quantitative analysis of 14 endocannabinoids and congeners. A simple extraction using protein precipitation with acetonitrile followed by online-trapping high-performance liquid chromatography–tandem mass spectrometry (LC/LC-MS/MS) was used to monitor the levels of 14 endocannabinoids in plasma. The assay was validated and intra-run and inter-run accuracies and imprecisions as well as matrix effects, recoveries, and sample stabilities were determined. As a proof of concept, a subset of study samples after naturalistic administration of Cannabis flower and concentrate was analyzed. With the exception of N-oleoyl dopamine and oleamide, all endocannabinoids fulfilled the predefined acceptance criteria. Reproducible recoveries and no significant matrix effects were observed. Sample stability was an issue. Analysis of the proof-of-concept study samples revealed a significantly (p = 0.006) higher concentration of docosatetraenoyl ethanolamide in concentrate users (300 ± 13 pg/mL) compared to flower users (252 ± 11 pg/mL). A robust, sensitive high-throughput assay for the quantitation of 14 endocannabinoids and congeners was successfully validated. Our study showed that it is mandatory to (A) appropriately stabilize samples and (B) separate and separately quantify 1-AG and 2-AG; otherwise, study results are unreliable. The analysis of study samples from Cannabis flower users versus Cannabis concentrate users revealed higher levels of docosatetraenoyl ethanolamide and anandamide (n.s.) in high THC concentrate users in accordance with the existing literature, supporting the validity of the assay measurements. [ABSTRACT FROM AUTHOR]

Autosomal dominant polycystic kidney disease (ADPKD) is the most commonly inherited kidney disease. Although children with ADPKD show normal renal function, cyst development is already occurring. In this study, we aimed to identify markers and associated molecular pathways of disease progression in children and young adults with ADPKD. Plasma samples were collected during a 3-year randomized, double-blind, placebo-controlled, phase III clinical trial that was designed to test the efficacy of pravastatin on slowing down ADPKD progression in pediatric patients. Samples from 58 patients were available at baseline and at the 3-year endpoint of the study, respectively. Furthermore, plasma samples from 98 healthy children were used as controls. Metabolomic analysis was performed using liquid chromatography-tandem mass spectrometry and differences in metabolic profiles over time and within study groups were evaluated. While pravastatin therapy led to a decrease in a percent change of total kidney volume (HtTKV) in ADPKD patients, it had minimal effects on metabolite changes. Oxidative stress, endothelial dysfunction, inflammation and immune response were the most affected signaling pathways that distinguished healthy from diseased children. Pathway analysis revealed that metabolites in the arginine metabolism (urea and nitric oxide cycles), asparagine and glutamine metabolism, in the methylation cycle and kynurenine pathway were significantly changed between healthy and children with ADPDK and continued to diverge from the control levels while the disease progressed. Detected metabolite changes were primarily governed by disease progression, and less by pravastatin treatment. Identified metabolic pathways, from arginine and asparagine to kynurenine metabolism could present therapeutic targets and should be further investigated for potential to treat ADPKD progression at an early stage. [ABSTRACT FROM AUTHOR]

Background and Objective: Buprenorphine has been shown to be effective in treating infants with neonatal opioid withdrawal syndrome. However, an evidence-based buprenorphine dosing strategy has not been established in the treatment of neonatal opioid withdrawal syndrome because of a lack of exposure–response data. The aim of this study was to develop an integrated pharmacokinetic and pharmacodynamic model to predict buprenorphine treatment outcomes in newborns with neonatal opioid withdrawal syndrome. Methods: Clinical data were obtained from 19 newborns with a median (range) gestational age of 37 (34–41) weeks enrolled in a pilot pharmacokinetic study of buprenorphine. Sparse blood sampling, comprising three specimens obtained around the second dose of buprenorphine, was performed using heel sticks with dried blood spot technology. Standardized neonatal opioid withdrawal syndrome severity scores (Finnegan scores) were collected every 3–4 h based on symptoms by bedside nursing staff. Mean Finnegan scores were used as a pharmacodynamic marker in the exposure–response modeling. The blood concentration–Finnegan score relationship was described using a physiologic indirect response model with inclusion of natural disease remission. Results: A total of 52 buprenorphine blood concentrations and 780 mean Finnegan scores were available for the pharmacokinetic/pharmacodynamic modeling and exposure–response analysis. A one-compartment model with first-order absorption adequately described the pharmacokinetic data. The buprenorphine blood concentration at 50% of maximum effect for the inhibition of disease progression was 0.77 ng/mL (95% confidence interval 0.32–1.2). The inclusion of natural disease remission described as a function of postnatal age significantly improved the model fit. Conclusions: A buprenorphine pharmacokinetic/pharmacodynamic model was successfully developed. The model could facilitate model-informed optimization of the buprenorphine dosing regimen in the treatment of neonatal opioid withdrawal syndrome. [ABSTRACT FROM AUTHOR]

Background and Objectives: Despite limited evidence, cannabidiol-rich cannabis extracts have been popularly used in pediatrics. With increased use, it is critical to determine basic pharmacokinetic parameters of cannabidiol in these extracts in the pediatric population. The objective of this study was to determine the disposition of oral cannabidiol cannabis extracts and drug interactions in children with pediatric epilepsy. Methods: We conducted a prospective observational study evaluating the disposition of oral cannabidiol in children (< 18 years of age) receiving cannabidiol extracts for epilepsy. Subjects underwent serial blood draws after oral cannabidiol administration. Cannabidiol and metabolites, along with anticonvulsant concentrations were determined. Results: Twenty-nine patients had sufficient pharmacokinetic data and were included in the analysis. Mean age was 9.7 years (standard deviation 4.3) and 17 patients (59%) were male. Median peak plasma cannabidiol concentrations was 13.1 ng/mL (interquartile range 6.8–39.3 ng mL); median time to peak of 2.0 h (interquartile range 2.0–4.0 h). Mean acute elimination half-life of oral cannabidiol was 6.2 h (standard deviation 1.8 h). There was an observed half-life of degradation of 533 days noted for cannabidiol concentrations when stored for 0.6–3.1 years. There was some impact on cannabidiol pharmacokinetic parameters when cannabidiol was co-administered with zonisamide (elimination rate constant and V1) and levetiracetam (elimination rate constant). Conclusions: In pediatric patients using oral cannabidiol-rich cannabis extract for epilepsy, the time to peak concentration of plasma cannabidiol and average acute elimination half-life were shorter than those reported for adults. Co-administration of zonisamide and levetiracetam had some impact on cannabidiol pharmacokinetic parameters. There was an observed degradation of plasma cannabidiol in long-term storage. Clinical registration: ClinicalTrials.gov Identifer no. NCT02447198. [ABSTRACT FROM AUTHOR]

Purpose: Intrauterine growth restriction (IUGR) has been shown to induce the programming of metabolic disturbances and obesity, associated with hypothalamic derangements. The present study aimed at investigating the effects of IUGR on the protein and metabolite profiles of the hypothalamus of adult female rats. Methods: Wistar rats were mated and either had ad libitum access to food (control group) or received only 50% of the control intake (restricted group) during the whole pregnancy. Both groups ate ad libitum throughout lactation. At 4 months of age, the control and restricted female offspring was euthanized for blood and tissues collection. The hypothalami were processed for data independent acquisition mass spectrometry-based proteomics or targeted mass spectrometry-based metabolomics. Results: The adult females submitted to IUGR showed increased glycemia and body adiposity, with normal body weight and food intake. IUGR modulated significantly 28 hypothalamic proteins and 7 hypothalamic metabolites. The effects of IUGR on hypothalamic proteins and metabolites included downregulation of glutamine synthetase, glutamate decarboxylase, glutamate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate, and up-regulation of NADH dehydrogenase and phosphoenolpyruvate. Integrated pathway analysis indicated that IUGR affected GABAergic synapse, glutamate metabolism, and TCA cycle, highly interconnected pathways whose derangement has potentially multiple consequences. Conclusion: The present findings suggested that the effects of IUGR on GABA/glutamate–glutamine cycle may be involved in the programming of obesity and hyperglycemia in female rats. [ABSTRACT FROM AUTHOR]

Drug coated balloons (DCB) are becoming the standard-care treatment for peripheral arterial disease (PAD). DCB use excipients to transfer and retain anti-proliferative drugs, such as paclitaxel. Excipients thus play a vital role in the design and function of DCB, however methods to coat balloons with excipients and anti-proliferative drugs remain unknown. The goal of this study was to thus develop an approach to coat and evaluate DCB for various excipients. An air sprayer method was developed to deposit paclitaxel and various excipients onto non-coated commercially available angioplasty balloons. The coating of the angioplasty balloons was evaluated for drug deposition and coating efficiency using high performance liquid chromatography tandem mass spectrometry. Drug transfer and retention of the coated angioplasty balloons into arterial segments were evaluated ex vivo using harvested pig arteries in a pulsatile flow bioreactor. The air sprayer method successfully delivered varying excipients including bovine serum albumin (BSA), urea and iohexol. The air spray method was configured to coat four angioplasty balloons simultaneously with paclitaxel and iohexol with an average paclitaxel load of 4.0 ± 0.70 µg/mm2. The intra-day (within) and inter-day (between) coating precisions, defined as relative standard deviation (RSD), was 17.2 and 15.5%, respectively. Ex vivo deployment of iohexol-paclitaxel DCB yielded an arterial paclitaxel concentration of 123.4 ± 44.68 ng/mg (n = 3) at 1 h, 126.7 ± 25.27 ng/mg (n = 3) at 1 day, and 12.9 ± 12.88 ng/mg (n = 3) at 7 days. This work provides proof-of-concept of a quick, inexpensive approach to coat commercially available angioplasty balloons with paclitaxel and various excipients. [ABSTRACT FROM AUTHOR]

Introduction: The immunosuppressive therapy with everolimus (ERL) after heart transplantation is characterized by a narrow therapeutic window and a substantial variability in dose requirement. Factors explaining this variability are largely unknown. Objectives: Our aim was to evaluate factors affecting ERL metabolism and to identify novel metabolites associated with the individual ERL dose requirement to elucidate mechanisms underlying ERL dose response variability. Method: We used liquid chromatography coupled with mass spectrometry for quantification of ERL metabolites in 41 heart transplant patients and evaluated the effect of clinical and genetic factors on ERL pharmacokinetics. Non-targeted plasma metabolic profiling by ultra-performance liquid chromatography and high resolution quadrupole-time-of-flight mass spectrometry was used to identify novel metabolites associated with ERL dose requirement. Results: The determination of ERL metabolites revealed differences in metabolite patterns that were independent from clinical or genetic factors. Whereas higher ERL dose requirement was associated with co-administration of sodium-mycophenolic acid and the CYP3A5 expressor genotype, lower dose was required for patients receiving vitamin K antagonists. Global metabolic profiling revealed several novel metabolites associated with ERL dose requirement. One of them was identified as lysophosphatidylcholine (lysoPC) (16:0/0:0). Subsequent targeted analysis revealed that high levels of several lysoPCs were significantly associated with higher ERL dose requirement. Conclusion: For the first time, this study describes distinct ERL metabolite patterns in heart transplant patients and detected potentially new drug-drug interactions. The global metabolic profiling facilitated the discovery of novel metabolites associated with ERL dose requirement that might represent new clinically valuable biomarkers to guide ERL therapy. [ABSTRACT FROM AUTHOR]

Introduction: Although current immunosuppressive protocols have dramatically improved 1-year survival of kidney transplants, there has been less progress in terms of long-term graft survival over the last two decades. The key to avoiding late graft loss is early diagnosis and differentiation between anti-allograft immune processes and immunosuppressant toxicity (IS-Tox). Modern bioanalytical technologies have opened new opportunities for the development of sensitive and specific diagnostic tools. There is an immediate need for biomarkers that are able to differentiate between renal allograft rejection and immunosuppressant toxicity. Objective: To test our hypothesis that changes of metabolite patterns in urine have the potential to serve as a non-invasive combinatorial biomarker that can differentiate between allograft immune reactions and IS-Tox. Methods: We used H-NMR spectroscopy and Luminex multiplexing for metabolic profiling of rat urine and the analysis of protein biomarkers in urine and plasma, respectively, to compare the effects of chronic allograft rejection in a Fisher-to-Lewis rat transplant model with IS-Tox induced by cyclosporine, tacrolimus and/or sirolimus in Lewis rats. Results: Our results showed that, while IS-Tox caused changes in metabolite patterns that are typically associated with proximal tubule damage, rejection caused more profuse changes not specifically focused on a particular kidney region. Moreover, metabolite pattern changes were more sensitive than changes in protein markers that were evident only during the later stages of rejection. Conclusion: The present study provides first proof-of-concept that longitudinal monitoring of urine metabolite markers has the potential to differentiate between early renal allograft rejection and immunosuppressant nephrotoxicity. [ABSTRACT FROM AUTHOR]

Stearoyl-CoA desaturase 1 (SCD1) plays a role in the development of obesity and related conditions, such as insulin resistance, and potentially also in neurological and heart diseases. The activity of SCD1 can be monitored using the desaturation index (DI), the ratio of product (16:1n-7 and 18:1n-9) to precursor (16:0 and 18:0) fatty acids. Here, different analytical strategies were applied to identify the method which best supports SCD1 biology. A novel effective approach was the use of the SCD1-independent fatty acid (16:1n-10) as a negative control. The first approach was based on a simple extraction followed by neutral loss triglyceride fatty acid analysis. The second approach was based on the saponification of triglycerides followed by fatty acid analysis (specific for the position of the double bond within monounsaturated fatty acids (MUFAs)). In addition to the analytical LC-MS assays, different matrices (plasma total triglyceride fraction and the very low-density lipoprotein (VLDL) fraction) were investigated to identify the best for studying changes in SCD1 activity. Samples from volunteers on a high-carbohydrate diet were analyzed. Both ultra HPLC (UHPLC)-MS-based assays showed acceptable accuracies (75-125 % of nominal) and precisions (<20 %) for the analysis of DI-specific fatty acids in VLDL and plasma. The most specific assay for the analysis of the liver SCD activity was then validated for specificity and selectivity, intra- and interday accuracy and precision, matrix effects, dilution effects, and analyte stability. After 3 days of high-carbohydrate diet, only the specific fatty acids in human plasma VLDL showed a significant increase in DI and associated SCD1 activity. [ABSTRACT FROM AUTHOR]

Early detection and prevention is an important goal in acute respiratory distress syndrome research. We determined the concentration of the anti-inflammatory 15-deoxy-Δ-prostaglandin-J2 (15d-PGJ2) and other components of the cyclopentenone prostaglandin cascade in relation to lung inflammation in cytokine (IL-1/LPS)-insufflated rats. We found that 15d-PGJ2 levels increase in the bronchoalveolar lavage (BAL) fluid of rats insufflated with cytokines 2 h before. BAL 15d-PGJ2 increases preceded neutrophil recruitment, lung injury, and oxidative stress in the lungs of cytokine-insufflated rats. 15d-PGJ2 was localized in alveolar macrophages that decreased following cytokine insufflation. 15d-PGJ2 may constitute an early biomarker of lung inflammation and may reflect an endogenous attempt to regulate ongoing inflammation in macrophages and elsewhere after cytokine insufflation. [ABSTRACT FROM AUTHOR]

Background and Objective: Tacrolimus is an immunosuppressive drug used for the prevention of the allograft rejection in kidney transplant recipients. It exhibits a narrow therapeutic index and large pharmacokinetic variability. Tacrolimus is mainly metabolized by cytochrome P450 (CYP) 3A4 and 3A5 and effluxed via ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp), encoded by ABCB1 gene. The influence of CYP3A5*3 on the pharmacokinetics of tacrolimus has been well characterized. On the other hand, the contribution of polymorphisms in other genes is controversial. In addition, the involvement of other efflux transporters than P-gp in tacrolimus disposition is uncertain. The present study was designed to investigate the effects of genetic polymorphisms of CYP3As and efflux transporters on the pharmacokinetics of tacrolimus. Subjects and Methods: A total of 500 blood concentrations of tacrolimus from 102 adult stable kidney transplant recipients were included in the analyses. Genetic polymorphisms in CYP3A4 and CYP3A5 genes were determined. In addition, the genes of efflux transporters including P-gp ( ABCB1), multidrug resistance-associated protein (MRP2/ ABCC2) and breast cancer resistance protein (BCRP/ ABCG2) were genotyped. For ABCC2 gene, haplotypes were determined as follows: H1 (wild type), H2 (1249G>A), H9 (3972C>T) and H12 (−24C>T and 3972C>T). Population pharmacokinetic analysis was performed using nonlinear mixed effects modeling. Results: Analyses revealed that the CYP3A5 expressers ( CYP3A5*1 carriers) and MRP2 high-activity group ( ABCC2 H2/H2 and H1/H2) showed a decreased dose-normalized trough concentration of tacrolimus by 2.3-fold ( p < 0.001) and 1.5-fold ( p = 0.007), respectively. The pharmacokinetics of tacrolimus were best described using a two-compartment model with first order absorption and an absorption lag time. In the population pharmacokinetic analysis, CYP3A5 expressers and MRP2 high-activity groups were identified as the significant covariates for tacrolimus apparent clearance expressed as 20.7 × (age/50) × 2.03 (CYP3A5 expressers) × 1.40 (MRP2 high-activity group). No other CYP3A4, ABCB1 or ABCG2 polymorphisms were associated with the apparent clearance of tacrolimus. Conclusions: This is the first report showing that MRP2/ ABCC2 has a crucial impact on the pharmacokinetics of tacrolimus in a haplotype-specific manner. Determination of the ABCC2 as well as CYP3A5 genotype may be useful for more accurate tacrolimus dosage adjustment. [ABSTRACT FROM AUTHOR]

Background and Objective: Tacrolimus is an immunosuppressive drug used for the prevention of the allograft rejection in kidney transplant recipients. It exhibits a narrow therapeutic index and large pharmacokinetic variability. Tacrolimus is mainly metabolized by cytochrome P450 (CYP) 3A4 and 3A5 and effluxed via ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp), encoded by ABCB1 gene. The influence of CYP3A5*3 on the pharmacokinetics of tacrolimus has been well characterized. On the other hand, the contribution of polymorphisms in other genes is controversial. In addition, the involvement of other efflux transporters than P-gp in tacrolimus disposition is uncertain. The present study was designed to investigate the effects of genetic polymorphisms of CYP3As and efflux transporters on the pharmacokinetics of tacrolimus.Subjects and Methods: A total of 500 blood concentrations of tacrolimus from 102 adult stable kidney transplant recipients were included in the analyses. Genetic polymorphisms in CYP3A4 and CYP3A5 genes were determined. In addition, the genes of efflux transporters including P-gp (ABCB1), multidrug resistance-associated protein (MRP2/ABCC2) and breast cancer resistance protein (BCRP/ABCG2) were genotyped. For ABCC2 gene, haplotypes were determined as follows: H1 (wild type), H2 (1249G>A), H9 (3972C>T) and H12 (-24C>T and 3972C>T). Population pharmacokinetic analysis was performed using nonlinear mixed effects modeling.Results: Analyses revealed that the CYP3A5 expressers (CYP3A5*1 carriers) and MRP2 high-activity group (ABCC2 H2/H2 and H1/H2) showed a decreased dose-normalized trough concentration of tacrolimus by 2.3-fold (p < 0.001) and 1.5-fold (p = 0.007), respectively. The pharmacokinetics of tacrolimus were best described using a two-compartment model with first order absorption and an absorption lag time. In the population pharmacokinetic analysis, CYP3A5 expressers and MRP2 high-activity groups were identified as the significant covariates for tacrolimus apparent clearance expressed as 20.7 × (age/50)(-0.78) × 2.03 (CYP3A5 expressers) × 1.40 (MRP2 high-activity group). No other CYP3A4, ABCB1 or ABCG2 polymorphisms were associated with the apparent clearance of tacrolimus.Conclusions: This is the first report showing that MRP2/ABCC2 has a crucial impact on the pharmacokinetics of tacrolimus in a haplotype-specific manner. Determination of the ABCC2 as well as CYP3A5 genotype may be useful for more accurate tacrolimus dosage adjustment. [ABSTRACT FROM AUTHOR]

Opioids such as morphine are the cornerstone of pain treatment. The challenge of measuring the concentrations of morphine and its active metabolites in order to assess human pharmacokinetics and monitor therapeutic drugs in children requires assays with high sensitivity in small blood volumes. We developed and validated a semi-automated LC-MS/MS assay for the simultaneous quantification of morphine and its active metabolites morphine 3β-glucuronide (M3G) and morphine 6β-glucuronide (M6G) in human plasma and in dried blood spots (DBS). Reconstitution in water (DBS only) and addition of a protein precipitation solution containing the internal standards were the only manual steps. Morphine and its metabolites were separated on a Kinetex 2.6-μm PFP analytical column using an acetonitrile/0.1% formic acid gradient. The analytes were detected in the positive multiple reaction mode. In plasma, the assay had the following performance characteristics: range of reliable response of 0.25-1000 ng/mL ( r > 0.99) for morphine, 1-1,000 ng/mL ( r > 0.99) for M3G, and 2.5-1,000 ng/mL for M6G. In DBS, the assay had a range of reliable response of 1-1,000 ng/mL ( r > 0.99) for morphine and M3G, and of 2.5-1,000 ng/mL for M6G. For inter-day accuracy and precision for morphine, M3G and M6G were within 15% of the nominal values in both plasma and DBS. There was no carryover, ion suppression, or matrix interferences. The assay fulfilled all predefined acceptance criteria, and its sensitivity using DBS samples was adequate for the measurement of pediatric pharmacokinetic samples using a small blood of only 20-50 μL. [ABSTRACT FROM AUTHOR]

Objectives: This prospective, open-label multicenter study was conducted to assess the pharmacokinetics of Biolimus A9 after elution from BioMatrix II coronary stents. Recent clinical trials have demonstrated the efficacy and safety of Biolimus A9 eluted from different stent platforms. To date, the pharmacokinetics of Biolimus A9 in patients following the deployment of BioMatrix II stents has not yet been studied Methods: BioMatrix II stents were implanted into 27 patients with coronary artery disease. The primary endpoints of the study were the systemic concentrations of Biolimus A9 after 28 days and 6 months as measured using a sensitive validated liquid chromatography-tandem mass spectrometry assay. Results: The highest measured blood concentration at any time point was 394 pg/mL. At 28 days and 6 months following stent placement, 51.8 and 100% of patients, respectively, had Biolimus A9 concentrations <10 pg/mL. After 9 months, 100% of the patients were free of major cardiac adverse events (MACE). There was no Biolimus A9 toxicity, no cardiac or non-cardiac deaths, no myocardial infarctions, nor target vessel or target lesion revascularizations during the 9 months of follow-up. No case of acute, subacute, or late stent thrombosis was detected. Conclusions: Compared to other drug-eluting stents, such as Cypher, BioMatrix II results in relatively low systemic exposure, which may be explained by the ablominal coating of the Biomatrix II stent in combination with Biolimus A9's high lipophilicity. [ABSTRACT FROM AUTHOR]

The bacterium Actinoplanes sp. ATCC 53771 is known to perform drug metabolism of several xenobiotics similarly to humans. We identified a cytochrome P450 enzyme from this strain, CYP107E4, and expressed it in Escherichia coli using the pET101 vector. The purified enzyme showed the characteristic reduced-CO difference spectra with a peak at 450 nm, indicating the protein is produced in the active form with proper heme incorporation. The CYP107E4 enzyme was found to bind the drug diclofenac. Using redox enzymes from spinach, the reconstituted system is able to produce hydroxylated metabolites of diclofenac. Production of the human 4′-hydroxydiclofenac metabolite by CYP107E4 was confirmed, and a second hydroxylated metabolite was also produced. [ABSTRACT FROM AUTHOR]

Purpose. Drug delivery to the central nervous system (CNS) is limited by the blood-brain barrier (BBB). Thus, a noninvasive and reversible method to enhance BBB permeation of drugs is highly desirable. In the present work, we studied if ultrasound-induced mild hyperthermia (USHT, 0.4 watts (W)/cm² at 41°C) can enhance drug absorption in BBB endothelial cells, and we elucidated the mechanism of USHT on cellular accumulation. Methods. To accomplish these aims, we studied the effects of hyperthermia (41°C), USHT, P-glycoprotein (P-gp) modulator (PSC 833), and combination of USHT and PSC 833 on accumulation of P-gp substrate (R123) and non-P-gp substrates (sucrose, 2-deoxyglucose, and antipyrine) in monolayers of primary bovine brain microvessel endothelial cells (BBMEC). Results. USHT, through its thermal effect, produces a significant (relative to controls; no USHT) and comparable increase in R123 accumulation with PSC 833. We also demonstrate that USHT increases permeability of hydrophobic (R123 and [[sup 14]C]-antipyrine) and not hydrophilic molecules ([[sup 14]C]-sucrose and 2-[³H]-deoxy-oglucose). The enhanced permeability is reversible and size dependent, as USHT produces a much larger effect on cellular accumulation of [[sup 14]C]-antitpyrine (molecular weight of 188 D) than that of R123 (molecular weight of 380.8 D). Although USHT increases membrane permeability, it did not affect P-gp activity or the activity of glucose transporters. Conclusions. Our results point to the potential use of USHT as a reversible and noninvasive approach to increase BBB permeation of hydrophobic drugs, including P-gp-recognized substrates. [ABSTRACT FROM AUTHOR]

Purpose. Grapefruit juice (GJ) is known to increase the oral bioavailability of many CYP3A-substrates by inhibiting intestinal phase-I metabolism. However, the magnitude of AUC increase is often insignificant and highly variable. Since we earlier suggested that CYP3A and P-glycoprotein (P-gp) form a concerted barrier to drug absorption, we investigated the role of P-gp in GJ-drug interactions. Methods. The transcellular bidirectional flux of drugs that are (i) CYP3A-and/or P-gp substrates (Vinblastine, Cyclosporine, Digoxin, Fexofenadine, Losartan) or that are (ii) primary CYP3A-substrates (Felodipine, Nifedipine) was evaluated across MDCK-MDR1 cell monolayers with or without GJ, verifying monolayer integrity at all times. Results. While both apical-to-basal (A-B) and basal-to-apical (B-A) fluxes of all CYP3A/P-gp substrates tested were increased in the presence of GJ, the resulting net efflux (B-A/A-B) was in all cases significantly greater with GJ than control (Vin, 28.0 vs. 5.1; CsA, 9.9 vs. 2.8; Dig, 22. 9 vs. 14.7, Fex, 22.3 vs. 11.1, Los, 39.6 vs. 26). In contrast, no such GJ flux effect was observed with Pel and Nif, substrates of CYP3A only (2 vs. 1.7 and 1.2 vs. 1.3). Conclusions. GJ significantly activates P-gp-mediated efflux of drugs that are substrates of P-gp, potentially partially counteracting the CYP3A-inhibitory effects of GJ. [ABSTRACT FROM AUTHOR]

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Enteric-coated mycophenolate sodium (EC-MPS) has a mean half-life of 11.7 hours, which encouraged hope of using this drug once daily in a nonadherent adolescent SLE patient. This is a case report on a 17-year-old adolescent with a history of noncompliance who was switched from twice-daily mycophenolate mofetil (MMF) to once-daily EC-MPS. The EC-MPS dose was equimolar to the daily MMF dose (1 g MMF BID and 1.44 g of EC-MPS OD). The active compound of both drugs, mycophenolic acid, was measured using a commercially available EMIT assay. Both drugs were well-tolerated and maintained remission of the SLE. The average of three 12-hour areas under the time–concentration curves (AUC) on 1 g of MMF BID was 59.0 mg×h/L. In contrast, the 24-hour AUC after 1.44 g EC-MPS OD was 283.2 mg×h/L, more than double the expected 118.0 mg×h/L of two MMF dosing intervals. A repeat 24-hour AUC after 1.08 g of EC-MPS was 218.2 mg×h/L. EC-MPS once daily may be a well-tolerated therapeutic option for nonadherent adolescent lupus patients, but may be associated with a significantly higher exposure than the equivalent MMF BID dose. [ABSTRACT FROM AUTHOR]

Cell-based diabetes therapy requires an abundant cell source. Here, we report reversal of diabetes for more than 100 d in cynomolgus macaques after intraportal transplantation of cultured islets from genetically unmodified pigs without Gal-specific antibody manipulation. Immunotherapy with CD25-specific and CD154-specific monoclonal antibodies, FTY720 (or tacrolimus), everolimus and leflunomide suppressed indirect activation of T cells, elicitation of non-Gal pig-specific IgG antibody, intragraft expression of proinflammatory cytokines and invasion of infiltrating mononuclear cells into islets. [ABSTRACT FROM AUTHOR]

The number of cannabis users increased up to 188 million users worldwide in 2017. Smoking and vaping are the most common consumption routes with formation of side-stream smoke/vapor and secondhand exposure to cannabinoids has been described in the literature. External contamination of hair by cannabis smoke has been studied but there are no studies on third-hand cannabis exposure due to deposition of smoke or vapor on surfaces. We tested whether cannabinoids could be detected on surfaces and objects in a room where cannabis is vaporized. Surface samples were collected using isopropanol imbued non-woven wipes from hard surfaces and objects. Each surface was swabbed three times with standardized swabbing protocol including three different patterns. Samples were analyzed using LC-ESI-MS/MS in combination with online extraction. THC was detected on 6 samples out of the 15 collected in the study room at quantifiable levels ranging 348–4882 ng/m2. Negative control samples collected from areas outside the study room were all negative. We demonstrated that surfaces exposed to side-stream cannabis vapor are positive for THC at quantifiable levels. This study represents a first step in understanding how side-stream cannabis vapor deposits in the environment and potentially results in a tertiary exposure for users and non-users. [ABSTRACT FROM AUTHOR]

The study objective was to elucidate the molecular mechanisms underlying the negative effects of mycophenolic acid (MPA) on human intestinal cells. Effects of MPA exposure and guanosine supplementation on nucleotide concentrations in LS180 cells were assessed using liquid chromatography-mass spectrometry. Proteomics analysis was carried out using stable isotope labeling by amino acids in cell culture combined with gel-based liquid chromatography-mass spectrometry and lipidome analysis using 1H nuclear magnetic resonance spectroscopy. Despite supplementation, depletion of guanosine nucleotides (p < 0.001 at 24 and 72 h; 5, 100, and 250 μM MPA) and upregulation of uridine and cytidine nucleotides (p < 0.001 at 24 h; 5 μM MPA) occurred after exposure to MPA. MPA significantly altered 35 proteins mainly related to nucleotide-dependent processes and lipid metabolism. Cross-reference with previous studies of MPA-associated protein changes widely corroborated these results, but showed differences that may be model- and/or method-dependent. MPA exposure increased intracellular concentrations of fatty acids, cholesterol, and phosphatidylcholine (p < 0.01 at 72 h; 100 μM MPA) which corresponded to the changes in lipid-metabolizing proteins. MPA affected intracellular nucleotide levels, nucleotide-dependent processes, expression of structural proteins, fatty acid and lipid metabolism in LS180 cells. These changes may compromise intestinal membrane integrity and contribute to gastrointestinal toxicity. [ABSTRACT FROM AUTHOR]