The novel ghrelin receptor inverse agonist PF-5190457 administered with alcohol: preclinical safety experiments and a phase 1b human laboratory study.

Rodent studies indicate that ghrelin receptor blockade reduces alcohol consumption. However, no ghrelin receptor blockers have been administered to heavy alcohol drinking individuals. Therefore, we evaluated the safety, tolerability, pharmacokinetic (PK), pharmacodynamic (PD) and behavioral effects of a novel ghrelin receptor inverse agonist, PF-5190457, when co-administered with alcohol. We tested the effects of PF-5190457 combined with alcohol on locomotor activity, loss-of-righting reflex (a measure of alcohol sedative actions), and on blood PF-5190457 concentrations in rats. Then, we performed a single-blind, placebo-controlled, within-subject human study with PF-5190457 (placebo/0 mg b.i.d., 50 mg b.i.d., 100 mg b.i.d.). Twelve heavy drinkers during three identical visits completed an alcohol administration session, subjective assessments, and an alcohol cue-reactivity procedure, and gave blood samples for PK/PD testing. In rats, PF-5190457 did not interact with the effects of alcohol on locomotor activity or loss-of-righting reflex. Alcohol did not affect blood PF-5190457 concentrations. In humans, all adverse events were mild or moderate and did not require discontinuation or dose reductions. Drug dose did not alter alcohol concentration or elimination, alcohol-induced stimulation or sedation, or mood during alcohol administration. Potential PD markers of PF-5190457 were acyl-to-total ghrelin ratio and insulin-like growth factor-1. PF-5190457 (100 mg b.i.d.) reduced alcohol craving during the cue-reactivity procedure. This study provides the first translational evidence of safety and tolerability of the ghrelin receptor inverse agonist PF-5190457 when co-administered with alcohol. PK/PD/behavioral findings support continued research of PF-5190457 as a potential pharmacological agent to treat alcohol use disorder.

Tacrolimus Concentration in Saliva of Kidney Transplant Recipients: Factors Influencing the Relationship with Whole Blood Concentrations.

OBJECTIVE: The objective of this study was to examine the association between tacrolimus concentration in oral fluids and in whole blood and to investigate the various factors that influence this relationship. PATIENTS AND METHODS: Forty-six adult kidney transplant recipients were included in the study. Study A (ten patients) included the collection of several paired oral fluid samples by passive drool over a 12-h post-dose period. Study B (36 patients) included the collection of oral fluids pre-dose and at 2 h after the tacrolimus dose under three conditions: un-stimulated, after stimulation with a tart candy, and after mouth rinsing. The tacrolimus concentration in oral fluids was measured by a specially developed sensitive and specific liquid chromatography mass spectrometry method. A salivary transferrin concentration of >1 mg/dL was used as a cut-off value for oral fluid blood contamination. RESULTS: Rinsing the oral cavity before sampling proved to provide the most suitable sampling strategy giving a correlation coefficient value of 0.71 (p = 0.001) between the tacrolimus concentration in oral fluids and the tacrolimus concentration in whole blood at trough. Mean and 95% confidence interval of tacrolimus concentration in oral fluids at the pre-dose concentration for samples collected after mouth rinsing was 584 (436, 782) pg/mL. The ratio of the tacrolimus concentration in oral fluids to the tacrolimus concentration in whole blood (*100) was 11% (95% confidence interval 9-13) for all sampling times. Oral fluid pH or weight of a saliva sample did not influence the tacrolimus concentration in oral fluids. Tacrolimus distribution into oral fluids exhibited a delay with a pronounced counter-clockwise hysteresis with respect to the time after dose. A multivariate analysis of variance revealed that the tacrolimus concentration in oral fluids is related to the tacrolimus concentration in whole blood and tacrolimus plasma-binding proteins including albumin and cholesterol. CONCLUSION: An optimal sampling strategy for the determination of the tacrolimus concentration in oral fluids was established. Measuring the tacrolimus concentration in oral fluids appears to be a feasible and non-invasive method for predicting the concentration of tacrolimus in whole blood.

Dysregulation of Δ4-3-oxosteroid 5ß-reductase in diabetic patients: Implications and mechanisms.

Aldo-keto reductase family 1 member D1 (AKR1D1) is a Δ4-3-oxosteroid 5ß-reductase required for bile acid synthesis and steroid hormone metabolism. Both bile acids and steroid hormones, especially glucocorticoids, play important roles in regulating body metabolism and energy expenditure. Currently, our understanding on AKR1D1 regulation and its roles in metabolic diseases is limited. We found that AKR1D1 expression was markedly repressed in diabetic patients. Consistent with repressed AKR1D1 expression, hepatic bile acids were significantly reduced in diabetic patients. Mechanistic studies showed that activation of peroxisome proliferator-activated receptor-α (PPARα) transcriptionally down-regulated AKR1D1 expression in vitro in HepG2 cells and in vivo in mice. Consistently, PPARα signaling was enhanced in diabetic patients. In summary, dysregulation of AKR1D1 disrupted bile acid and steroid hormone homeostasis, which may contribute to the pathogenesis of diabetes. Restoring bile acid and steroid hormone homeostasis by modulating AKR1D1 expression may represent a new approach to develop therapies for diabetes.

Development and validation of a sensitive and selective LC-MS/MS method for determination of tacrolimus in oral fluids.

Tacrolimus is a commonly used immunosuppressive agent in organ transplant recipients. Therapeutic drug monitoring (TDM) of tacrolimus is essential to adjust the dose and achieve optimal immunosuppression level. Routine TDM is practiced using whole blood samples obtained through venipuncture. However, tacrolimus concentration that is present in oral fluid (OF) can theoretically represent the free or pharmacologically active form of tacrolimus. In this study, we report the development and validation of a rapid, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification of tacrolimus in OF. Chromatographic separation were achieved on an Acquity UPLC BEH C18 column by a gradient elution using 2mM ammonium acetate/0.1% (v/v) formic acid in water (mobile phase A) and in methanol (mobile phase B) with a 2.2min chromatographic run time. Tacrolimus was extracted from OF with acetonitrile as the precipitating solvent. Both extraction and chromatography was optimized to provide optimal sample cleanness, negligible matrix effect, and optimal specificity. The lower limit of quantification (LLOQ) for the assay was set at 10pg/mL using a 50µL aliquot of OF obtained by passive drool. The method demonstrated adequate accuracy and precision with accuracy between 94.5-103.6%, and coefficient of variation ranging from 4 to 9.8%. Tacrolimus was stable in OF for up to one month at -80°C and the extracted matrix was stable up to 48h in auto-sampler at 20°C. The method showed high reproducibility as confirmed by incurred sample reanalysis test. This assay was employed in several clinical pharmacokinetic studies and could successfully measure the concentration of tacrolimus in OF.

Alternative matrices for therapeutic drug monitoring of immunosuppressive agents using LC-MS/MS.

Immunosuppressive drugs used in solid organ transplants typically have narrow therapeutic windows and high intra- and intersubject variability. To ensure satisfactory exposure, therapeutic drug monitoring (TDM) plays a pivotal role in any successful posttransplant maintenance therapy. Currently, recommendations for optimum immunosuppressant concentrations are based on blood/plasma measurements. However, they introduce many disadvantages, including poor prediction of allograft survival and toxicity, a weak correlation with drug concentrations at the site of action and the invasive nature of the sample collection. Thus, alternative matrices have been investigated. This paper reviews tandem-mass spectrometry (LC-MS/MS) methods used for the quantification of immunosuppressant drugs utilizing nonconventional matrices, namely oral fluids, fingerprick blood and intracellular and intratissue sampling. The advantages, disadvantages and clinical application of such alternative mediums are discussed. Additionally, sample extraction techniques and basic chromatography information regarding these methods are presented in tabulated form.

Development and validation of an UPLC-MS/MS assay for quantitative analysis of the ghrelin receptor inverse agonist PF-5190457 in human or rat plasma and rat brain.

PF-5190457 is a ghrelin receptor inverse agonist that is currently undergoing clinical development for the treatment of alcoholism. Our aim was to develop and validate a simple and sensitive assay for quantitative analysis of PF-5190457 in human or rat plasma and rat brain using liquid chromatography-tandem mass spectrometry. The analyte and stable isotope internal standard were extracted from 50 µL plasma or rat brain homogenate by protein precipitation using 0.1% formic acid in acetonitrile. Chromatography was carried out on an Acquity UPLC BEH C18 (2.1 mm × 50 mm) column with 1.7 µm particle size and 130 Å pore size. The flow rate was 0.5 mL/min and total chromatographic run time was 2.2 min. The mobile phase consisted of a gradient mixture of water: acetonitrile 95:5% (v/v) containing 0.1% formic acid (solvent A) and 100% acetonitrile containing 0.1% formic acid (solvent B). Multiple reaction monitoring was carried out in positive electro-spray ionization mode using m/z 513.35 → 209.30 for PF-5190457 and m/z 518.47 → 214.43 for the internal standard. The recovery ranged from 102 to 118% with coefficient of variation (CV) less than 6% for all matrices. The calibration curves for all matrices were linear over the studied concentration range (R(2) ≥ 0.998, n = 3). The lower limit of quantification was 1 ng/mL in rat or human plasma and 0.75 ng/g in rat brain. Intra- and inter-run mean percent accuracies were between 85 and 115% and percent imprecision was ≤15%. The assays were successfully utilized to measure the concentration of PF-5190457 in pre-clinical and clinical pharmacology studies of the compound.

More potent lipid-lowering effect by rosuvastatin compared with fluvastatin in everolimus-treated renal transplant recipients.

BACKGROUND: Dyslipidemia is a risk factor for premature cardiovascular morbidity and mortality in renal transplant recipients (RTRs). Pharmacotherapy with mTOR inhibitors aggravates dyslipidemia, thus necessitating lipid-lowering therapy with fluvastatin, pravastatin, or atorvastatin. These agents may not sufficiently lower lipid levels, and therefore, a more potent agent like rosuvastatin maybe needed. METHODS: We have aimed to assess the lipid-lowering effect of rosuvastatin as compared with fluvastatin in RTR receiving everolimus. Safety was assessed as the pharmacokinetic (PK) interaction potential of a rosuvastatin/everolimus combination in RTR. A 12-hour everolimus PK investigation was performed in 12 stable RTR receiving everolimus and fluvastatin (80 mg/d). Patients were then switched to rosuvastatin (20 mg/d), and a follow-up 12/24-hour PK investigation of everolimus/rosuvastatin was performed after 1 month. All other drugs were kept unchanged. RESULTS: In RTR already receiving fluvastatin, switching to rosuvastatin further decreased LDL cholesterol and total cholesterol by 30.2±12.2% (P<0.01) and 18.2±9.6% (P<0.01), respectively. Everolimus AUC0-12 was not affected by concomitant rosuvastatin treatment, 80.3±21.3 µg*h/L before and 78.5±21.9 µg*h/L after, respectively (P=0.61). Mean rosuvastatin AUC0-24 was 157±61.7 ng*h/mL, approximately threefold higher than reported in the literature for nontransplants. There were no adverse events, and none of the patients had or developed proteinuria. CONCLUSION: Rosuvastatin showed a superior lipid-lowering effect compared to fluvastatin in stable RTR receiving everolimus. The combination of everolimus/rosuvastatin seems to be as safe as the everolimus/fluvastatin combination.