Investigating the Relations Between Caffeine-Derived Metabolites and Plasma Lipids in 2 Population-Based Studies.

OBJECTIVE: To investigate the relations between caffeine-derived metabolites (methylxanthines) and plasma lipids by use of population-based data from 2 European countries. METHODS: Families were randomly selected from the general population of northern Belgium (FLEMENGHO), from August 12, 1985, until November 22, 1990, and 3 Swiss cities (SKIPOGH), from November 25, 2009, through April 4, 2013. We measured plasma concentrations (FLEMENGHO, SKIPOGH) and 24-hour urinary excretions (SKIPOGH) of 4 methylxanthines-caffeine, paraxanthine, theobromine, and theophylline-using ultra-high-performance liquid chromatography-tandem mass spectrometry. We used enzymatic methods to estimate total cholesterol, high-density lipoprotein cholesterol, and triglyceride levels and the Friedewald equation for low-density lipoprotein cholesterol levels in plasma. We applied sex-specific mixed models to investigate associations between methylxanthines and plasma lipids, adjusting for major confounders. RESULTS: In both FLEMENGHO (N=1987; 1055 [53%] female participants) and SKIPOGH (N=990; 523 [53%] female participants), total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels increased across quartiles of plasma caffeine, paraxanthine, and theophylline (total cholesterol levels by caffeine quartiles in FLEMENGHO, male participants: 5.01±0.06 mmol/L, 5.05±0.06 mmol/L, 5.27±0.06 mmol/L, 5.62±0.06 mmol/L; female participants: 5.24±0.06 mmol/L, 5.15±0.05 mmol/L, 5.25±0.05 mmol/L, 5.42±0.05 mmol/L). Similar results were observed using urinary methylxanthines in SKIPOGH (total cholesterol levels by caffeine quartiles, male participants: 4.54±0.08 mmol/L, 4.94±0.08 mmol/L, 4.87±0.08 mmol/L, 5.27±0.09 mmol/L; female participants: 5.12±0.07 mmol/L, 5.21±0.07 mmol/L, 5.28±0.05 mmol/L, 5.28±0.07 mmol/L). Furthermore, urinary caffeine and theophylline were positively associated with high-density lipoprotein cholesterol in SKIPOGH male participants. CONCLUSION: Plasma and urinary caffeine, paraxanthine, and theophylline were positively associated with plasma lipids, whereas the associations involving theobromine were less clear. We postulate that the positive association between caffeine intake and plasma lipids may be related to the sympathomimetic function of methylxanthines, mitigating the overall health-beneficial effect of caffeine intake.

Validation of a high-throughput method for the quantification of flavanol and procyanidin biomarkers and methylxanthines in plasma by UPLC-MS.

Nutritional biomarkers are critical tools to objectively assess intake of nutrients and other compounds from the diet. In this context, it is essential that suitable analytical methods are available for the accurate quantification of biomarkers in large scale studies. Recently, structurally-related (-)-epicatechin metabolites (SREMs) and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone metabolites (gVLMs) were identified as biomarkers of intake of flavanols and procyanidins, a group of polyphenol bioactives. This study aimed at validating a high throughput method for the quantification of SREMs and gVLMs in plasma along with methylxanthines (MXs), dietary compounds known to interact with flavanol and procyanidin effects. To accomplish this, a full set of authentic analytical standards were used to optimize a micro solid phase extraction method for sample preparation coupled to HPLC-MS detection. Isotopically-labelled standards for all analytes were included to correct potential matrix effects on quantification. Average accuracies of 101%, 93% and 103% were obtained, respectively, for SREMs, gVLMs and MXs. Intra- and inter-day repeatability values were <15%. The method showed linear responses for all analytes (>0.993). Most SREMs and gVLMs had limits of quantifications <5 nM while limits of quantification of MXs were 0.2 µM. All analytes were stable under different tested processing conditions. Finally, the method proved to be suitable to assess SREMs, gVLMs and MXs in plasma collected after single acute and daily intake of cocoa-derived test materials. Overall, this method proved to be a valid analytical tool for high throughput quantification of flavanol and procyanidin biomarkers and methylxanthines in plasma.

Association of caffeine and related analytes with resistance to Parkinson disease among LRRK2 mutation carriers: A metabolomic study.

OBJECTIVE: To identify markers of resistance to developing Parkinson disease (PD) among LRRK2 mutation carriers (LRRK2+), we carried out metabolomic profiling in individuals with PD and unaffected controls (UC), with and without the LRRK2 mutation. METHODS: Plasma from 368 patients with PD and UC in the LRRK2 Cohort Consortium (LCC), comprising 118 LRRK2+/PD+, 115 LRRK2+/UC, 70 LRRK2-/PD+, and 65 LRRK2-/UC, and CSF available from 68 of them, were analyzed by liquid chromatography with mass spectrometry. For 282 analytes quantified in plasma and CSF, we assessed differences among the 4 groups and interactions between LRRK2 and PD status, using analysis of covariance models adjusted by age, study site cohort, and sex, with p value corrections for multiple comparisons. RESULTS: Plasma caffeine concentration was lower in patients with PD vs UC (p < 0.001), more so among LRRK2+ carriers (by 76%) than among LRRK2- participants (by 31%), with significant interaction between LRRK2 and PD status (p = 0.005). Similar results were found for caffeine metabolites (paraxanthine, theophylline, 1-methylxanthine) and a nonxanthine marker of coffee consumption (trigonelline) in plasma, and in the subset of corresponding CSF samples. Dietary caffeine was also lower in LRRK2+/PD+ compared to LRRK2+/UC with significant interaction effect with the LRRK2+ mutation (p < 0.001). CONCLUSIONS: Metabolomic analyses of the LCC samples identified caffeine, its demethylation metabolites, and trigonelline as prominent markers of resistance to PD linked to pathogenic LRRK2 mutations, more so than to idiopathic PD. Because these analytes are known both as correlates of coffee consumption and as neuroprotectants in animal PD models, the findings may reflect their avoidance by those predisposed to develop PD or their protective effects among LRRK2 mutation carriers.

Detection of caffeine and its main metabolites for early diagnosis of Parkinson's disease using micellar electrokinetic capillary chromatography.

Caffeine (CA) is a common xanthine alkaloid found in tea leaves, coffee beans, and other natural plants, and is the most widely used psychotropic substance in the world. Accumulating evidence suggests that low plasma levels of CA and its metabolites may serve as reliable diagnostic markers for early Parkinson's disease (PD) patients. In this study, we demonstrated a new MEKC method for determining CA and its three main downstream metabolites, paraxanthine (PX), theobromine (TB), and theophylline (TP), in human plasma. Plasma samples were collected, and analyzed using MEKC, after SPE. The running buffer was composed of 35 mM phosphate, pH of 10.5, and 25 mM SDS. The separation voltage was 15 kV and the detection wavelength was at 210 nm. Under the optimum conditions, four distinct analytes were completely separated and detected in less than 12 min. Method limits of detection were as low as 7.5 ng/mL for CA, 5.0 ng/mL for TB, and 4.0 ng/mL for both PX and TP. The recoveries were between 88.0% and 105.9%. This method was successfully applied to 27 human plasma samples. The results indicate that the plasma concentrations of the four analytes are significantly lower in patients with early PD than in control subjects (p < 0.05). The area under curve was improved to 0.839 when CA and its three main metabolites were included, suggesting that MEKC testing of CA, TP, TB, and PX may serve as a potential method for early diagnosis of PD.

Alterations of Proximal Tubular Secretion in Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND AND OBJECTIVES: In autosomal dominant polycystic kidney disease (ADPKD), the GFR often remains normal despite significant nephron loss. Proximal tubular secretory clearance may be reduced in ADPKD before detectable changes in GFR. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We used targeted mass spectrometry to quantify secretory solutes from blood and urine samples from 31 patients with ADPKD and preserved GFR (mean eGFR =111±11 ml/min per 1.73 m2) and 25 healthy control individuals as well as from 95 patients with ADPKD and reduced GFR (mean eGFR =53±21 ml/min per 1.73 m2) and 92 individuals with non-ADPKD CKD. We used linear regression to compare the fractional excretion of each solute between ADPKD and control groups. Among 112 patients with ADPKD, we used linear regression to determine associations of solute fractional excretion with height-adjusted total kidney volume. RESULTS: After adjusting for demographics, clinical characteristics, and kidney function measures, the fractional excretions of three secretory solutes were lower in patients with ADPKD and preserved GFR compared with healthy individuals: 52% lower cinnamoylglycine excretion (95% confidence interval, 24% to 70%), 53% lower tiglylglycine excretion (95% confidence interval, 23% to 71%), and 91% lower xanthosine excretion (95% confidence interval, 83% to 95%). In addition to lower excretions of tiglylglycine and xanthosine, patients with ADPKD and reduced GFR also demonstrated 37% lower dimethyluric acid excretion (95% confidence interval, 21% to 50%), 58% lower hippurate excretion (95% confidence interval, 48% to 66%), 48% lower isovalerylglycine excretion (95% confidence interval, 37% to 56%), and 31% lower pyridoxic acid excretion (95% confidence interval, 16% to 42%) compared with patients with non-ADPKD CKD and comparable eGFR. Among patients with ADPKD, solute fractional excretions were not associated with differences in kidney volume. CONCLUSIONS: Patients with ADPKD and preserved and reduced GFR demonstrate lower tubular secretory solute excretion compared with healthy controls and patients with non-ADPKD CKD. Our results suggest that tubular secretion is impaired in ADPKD independent of GFR.

Caffeine protects against experimental acute pancreatitis by inhibition of inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release.

OBJECTIVE: Caffeine reduces toxic Ca2+ signals in pancreatic acinar cells via inhibition of inositol 1,4,5-trisphosphate receptor (IP3R)-mediated signalling, but effects of other xanthines have not been evaluated, nor effects of xanthines on experimental acute pancreatitis (AP). We have determined effects of caffeine and its xanthine metabolites on pancreatic acinar IP3R-mediated Ca2+ signalling and experimental AP. DESIGN: Isolated pancreatic acinar cells were exposed to secretagogues, uncaged IP3 or toxins that induce AP and effects of xanthines, non-xanthine phosphodiesterase (PDE) inhibitors and cyclic adenosine monophosphate and cyclic guanosine monophosphate (cAMP/cGMP) determined. The intracellular cytosolic calcium concentration ([Ca2+]C), mitochondrial depolarisation and necrosis were assessed by confocal microscopy. Effects of xanthines were evaluated in caerulein-induced AP (CER-AP), taurolithocholic acid 3-sulfate-induced AP (TLCS-AP) or palmitoleic acid plus ethanol-induced AP (fatty acid ethyl ester AP (FAEE-AP)). Serum xanthines were measured by liquid chromatography-mass spectrometry. RESULTS: Caffeine, dimethylxanthines and non-xanthine PDE inhibitors blocked IP3-mediated Ca2+ oscillations, while monomethylxanthines had little effect. Caffeine and dimethylxanthines inhibited uncaged IP3-induced Ca2+ rises, toxin-induced Ca2+ release, mitochondrial depolarisation and necrotic cell death pathway activation; cAMP/cGMP did not inhibit toxin-induced Ca2+ rises. Caffeine significantly ameliorated CER-AP with most effect at 25 mg/kg (seven injections hourly); paraxanthine or theophylline did not. Caffeine at 25 mg/kg significantly ameliorated TLCS-AP and FAEE-AP. Mean total serum levels of dimethylxanthines and trimethylxanthines peaked at >2 mM with 25 mg/kg caffeine but at <100 µM with 25 mg/kg paraxanthine or theophylline. CONCLUSIONS: Caffeine and its dimethylxanthine metabolites reduced pathological IP3R-mediated pancreatic acinar Ca2+ signals but only caffeine ameliorated experimental AP. Caffeine is a suitable starting point for medicinal chemistry.

Safety and efficacy of cocoa flavanol intake in healthy adults: a randomized, controlled, double-masked trial.

BACKGROUND: Evidence from dietary intervention studies shows that the intake of flavanols and procyanidins can be beneficial for cardiovascular health. Nevertheless, there is a clear need for advancing our understanding with regard to safe amounts of intake for these bioactives. OBJECTIVE: The aim was to investigate in healthy adults the effects of cocoa flavanol (CF) intake amount and intake duration on blood pressure, platelet function, metabolic variables, and potential adverse events (AEs). DESIGN: This investigation consisted of 2 parts. Part 1 was an open-label, intake-amount escalation study, in which 34 healthy adults (aged 35-55 y) consumed escalating amounts of CFs, ranging from 1000 to 2000 mg/d over 6 wk. Primary outcomes were blood pressure and platelet function, select metabolic variables, and the occurrence and severity of AEs. Secondary outcomes included plasma concentrations of CF-derived metabolites and methylxanthines. On the basis of the outcomes of study part 1, and assessing the same outcome measures, part 2 of this investigation was a controlled, randomized, double-masked, 2-parallel-arm dietary intervention study in which healthy participants (aged 35-55 y) were asked to consume for 12 consecutive weeks up to 2000 mg CFs/d (n = 46) or a CF-free control (n = 28). RESULTS: Daily intake of up to 2000 mg CFs/d for 12 wk was not associated with significant changes in blood pressure or platelet function compared with CF-free controls in normotensive, healthy individuals who exhibited a very low risk of cardiovascular disease. There were no clinically relevant changes in the metabolic variables assessed in either of the groups. AEs reported were classified as mild in severity and did not significantly differ between study arms. CONCLUSION: The consumption of CFs in amounts up to 2000 mg/d for 12 wk was well tolerated in healthy men and women. This trial was registered at clinicaltrials.gov as NCT02447770 (part 1) and NCT02447783 (part 2).

Automated reference region extraction and population-based input function for brain [(11)C]TMSX PET image analyses.

[(11)C]TMSX ([7-N-methyl-(11)C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthine) is a selective adenosine A2A receptor (A2AR) radioligand. In the central nervous system (CNS), A2AR are linked to dopamine D2 receptor function in striatum, but they are also important modulators of inflammation. The golden standard for kinetic modeling of brain [(11)C]TMSX positron emission tomography (PET) is to obtain arterial input function via arterial blood sampling. However, this method is laborious, prone to errors and unpleasant for study subjects. The aim of this work was to evaluate alternative input function acquisition methods for brain [(11)C]TMSX PET imaging. First, a noninvasive, automated method for the extraction of gray matter reference region using supervised clustering (SCgm) was developed. Second, a method for obtaining a population-based arterial input function (PBIF) was implemented. These methods were created using data from 28 study subjects (7 healthy controls, 12 multiple sclerosis patients, and 9 patients with Parkinson's disease). The results with PBIF correlated well with original plasma input, and the SCgm yielded similar results compared with cerebellum as a reference region. The clustering method for extracting reference region and the population-based approach for acquiring input for dynamic [(11)C]TMSX brain PET image analyses appear to be feasible and robust methods, that can be applied in patients with CNS pathology.

Endothelial nitric oxide synthase-enhancing G-protein coupled receptor antagonist inhibits pulmonary artery hypertension by endothelin-1-dependent and endothelin-1-independent pathways in a monocrotaline model.

This study investigates whether endothelin-1 (ET-1) mediates monocrotaline (MCT)-induced pulmonary artery hypertension (PAH) and right ventricular hypertrophy (RVH), and if so, whether the G-protein coupled receptor antagonist KMUP-1 (7-{2-[4-(2-chlorobenzene)piperazinyl]ethyl}-1,3-dimethylxanthine) inhibits ET-1-mediated PA constriction and the aforementioned pathological changes. In a chronic rat model, intraperitoneal MCT (60 mg/kg) induced PAH and increased PA medial wall thickening and RV/left ventricle + septum weight ratio on Day 21 after MCT injection. Treatment with sublingual KMUP-1 (2.5 mg/kg/day) for 21 days prevented these changes and restored vascular endothelial nitric oxide synthase (eNOS) immunohistochemical staining of lung tissues. Western blotting analysis demonstrated that KMUP-1 enhanced eNOS, soluble guanylate cyclase, and protein kinase G levels, and reduced ET-1 expression and inactivated Rho kinase II (ROCKII) in MCT-treated lung tissue over long-term administration. In MCT-treated rats, KMUP-1 decreased plasma ET-1 on Day 21. KMUP-1 (3.6 mg/kg) maximally appeared at 0.25 hours in the plasma and declined to basal levels within 24 hours after sublingual administration. In isolated PA of MCT-treated rats, compared with control and pretreatment with l-NG-nitroarginine methyl ester (100 µM), KMUP-1 (0.1-100 µM) inhibited ET-1 (0.01 µM)-induced vasoconstriction. Endothelium-denuded PA sustained higher contractility in the presence of KMUP-1. In a 24-hour culture of smooth muscle cells (i.e., PA smooth muscle cells or PASMCs), KMUP-1 (0.1-10 µM) inhibited RhoA- and ET-1-induced RhoA activation. KMUP-1 prevented MCT-induced PAH, PA wall thickening, and RVH by enhancing eNOS and suppressing ET-1/ROCKII expression. In vitro, KMUP-1 inhibited ET-1-induced PA constriction and ET-1-dependent/independent RhoA activation of PASMCs. In summary, KMUP-1 attenuates ET-1-induced/ET-1-mediated PA constriction, and could thus aid in the treatment of PAH caused by MCT.

Xanthine derivatives quantification in serum by capillary zone electrophoresis.

A capillary electrophoresis method was developed to quantify caffeine and theophylline, xanthine derivatives with bronchodilator activity. Buffer concentration, pH and applied voltage were optimized using a central composite design-face centred. Separation conditions were: silica capillary tube, 75 µm (i.d.) and 61 cm (total length); absorbance detection, 280 nm; borate buffer, 20 mM, pH 9.0; applied voltage, 25 kV and 1 psi injection/8 s. Validation was performed in blank serum following the International Conference Harmonization guidelines: resolution (peaks without overlapping), linear range (0.125-50 µg/mL; r(2) > 0.9999), limits of detection and quantification (10; 20 and 33; 66 ppb for caffeine and theophylline, respectively), intra- and inter-day precision (Relative standard deviation lower than 1.9%) and accuracy (98-101%). Migration times were <8 min. This method is simple, specific and suitable and reaches high label claims (98.7-100.4%) in pharmaceutical formulations analysis. Moreover, the method was applied to the monitoring of the analytes in serum of patients.

Bioappearance and pharmacokinetics of bioactives upon coffee consumption.

Habitual consumption of medium amounts of coffee over the whole life-span is hypothesized to reduce the risk to develop diabetes type 2 (DM2) and Alzheimer's disease (AD). To identify putative bioactive coffee-derived metabolites, first, pooled urine from coffee drinkers and non-coffee drinkers were screened by UPLC-HDMS. After statistical data analysis, trigonelline, dimethylxanthines and monomethylxanthines, and ferulic acid conjugates were identified as the major metabolites found after coffee consumption. For quantitative analysis of these markers in body fluids, targeted methods based on stable-isotope dilution and UPLC-MS/MS were developed and applied to plasma samples from a coffee intervention study (n = 13 volunteers) who consumed a single cup of caffeinated coffee brew after a 10-day washout period. Chlorogenic acid-derived metabolites were found to be separated into two groups showing different pharmacokinetic properties. The first group comprised, e.g., ferulic acid and feruloyl sulfate and showed early appearance in the plasma (~1 h). The second group contained particularly chlorogenic acid metabolites formed by the intestinal microflora, appearing late and persisting in the plasma (>6 h). Trigonelline appeared early but persisted with calculated half-life times ~5 h. The plasma levels of caffeine metabolites significantly and progressively increased 2-4 h after coffee consumption and did not reach c max within the time frame of the study. The pharmacokinetic profiles suggest that particularly trigonelline, caffeine, its metabolites, as well as late appearing dihydroferulic acid, feruloylglycine and dihydroferulic acid sulfate formed from chlorogenic acid by the intestinal microflora accumulate in the plasma due to their long half-life times during habitual consumption of several cups of coffee distributed over the day. Since some of these metabolites have been reported to show antioxidant effects in vivo, antioxidant-response-element activating potential, and neuroprotective properties, respectively, some of these key metabolites might account for the inflammation- and DM2/AD risk reducing effects reported for habitual life time consumption of coffee.

Mechanism of allopurinol induced TPMT inhibition.

Up to 1/5 of patients with wildtype thiopurine-S-methyltransferase (TPMT) activity prescribed azathioprine (AZA) or mercaptopurine (MP) demonstrate a skewed drug metabolism in which MP is preferentially methylated to yield methylmercaptopurine (MeMP). This is known as thiopurine hypermethylation and is associated with drug toxicity and treatment non-response. Co-prescription of allopurinol with low dose AZA/MP (25-33%) circumvents this phenotype and leads to a dramatic reduction in methylated metabolites; however, the biochemical mechanism remains unclear. Using intact and lysate red cell models we propose a novel pathway of allopurinol mediated TPMT inhibition, through the production of thioxanthine (TX, 2-hydroxymercaptopurine). In red blood cells pre-incubated with 250 µM MP for 2h prior to the addition of 250 µM TX or an equivalent volume of Earle's balanced salt solution, there was a significant reduction in the concentration of MeMP detected at 4h and 6h in cells exposed to TX (4 h, 1.68, p=0.0005, t-test). TX acts as a direct TPMT inhibitor with an apparent Ki of 0.329 mM. In addition we have confirmed that the mechanism is relevant to in vivo metabolism by demonstrating raised urinary TX levels in patients receiving combination therapy. We conclude that the formation of TX in patients receiving combination therapy with AZA/MP and allopurinol, likely explains the significant reduction of methylated metabolites due to direct TPMT inhibition.

Simultaneous pharmacokinetic model for rolofylline and both M1-trans and M1-cis metabolites.

Rolofylline is a potent, selective adenosine A1 receptor antagonist that was under development for the treatment of patients with acute congestive heart failure and renal impairment. Rolofylline is metabolized primarily to the pharmacologically active M1-trans and M1-cis metabolites (metabolites) by cytochrome P450 (CYP) 3A4. The aim of this investigation was to provide a pharmacokinetic (PK) model for rolofylline and metabolites following intravenous administration to healthy volunteers. Data included for this investigation came from a randomized, double-blind, dose-escalation trial in four groups of healthy volunteers (N=36) where single doses of rolofylline, spanning 1 to 60 mg ,were infused over 1-2 h. The rolofylline and metabolite data were analyzed simultaneously using NONMEM. The simultaneous PK model comprised, in part, a two-compartment linear PK model for rolofylline, with estimates of clearance and volume of distribution at steady-state of 24.4 L/h and 239 L, respectively. In addition, the final PK model contained provisions for both conversion of rolofylline to metabolites and stereochemical conversion of M1-trans to M1-cis. Accordingly, the final model captured known aspects of rolofylline metabolism and was capable of simultaneously describing the PK of rolofylline and metabolites in healthy volunteers.

Identification and dynamic analysis of the purine alkaloids in rat plasma after oral administration of green tea by liquid chromatography hybrid ion trap time-of-flight mass spectrometry.

A liquid chromatography hybrid ion trap time-of-flight mass spectrometric (LC-IT-TOF-MS) method was developed and validated for identification and simultaneous determination of the potential bioactive components from green tea in rat plasma. The plasma samples were extracted by liquid-liquid extraction with ethyl acetate and separated on Shim-pack XR-ODS II column by a gradient elution within a runtime of 8.0 min. The mobile phase consisted of A (0.1% formic acid in acetonitrile) and B (0.1% formic acid in water) at a flow rate of 0.4 ml/min. Two prototype components and one metabolite were successfully identified as caffeine, theobromine and theophylline according to their retention times, accurate molecule weight, and major fragment ions. Then they were determined with the addition of two internal standards, hypoxanthine and paracetamol. The linear range was 10-10,000 ng/ml for caffeine, 2.0-2000 ng/ml for theobromine and 1.0-1000 ng/ml for theophylline, respectively. Intra-day and inter-day precision were within 6.0% and 10.9%, and accuracy was less than 4.8% and 6.5%, respectively. The validated method was successfully applied to investigate the dynamic change rules of caffeine, theobromine and theophylline in rat plasma after oral administration of caffeine, theobromine and green tea extract. The comparative analysis of the pharmacokinetic parameters indicated that there were obvious differences between green tea extract administration and single substances administration.

Development and validation of a sensitive LC-MS/MS method for the simultaneous quantitation of theophylline and its metabolites in rat plasma.

A rapid, specific, and reliable LC-MS/MS-based bioanalytical method was developed and validated in rat plasma for the simultaneous quantitation of theophylline and its four metabolites: 1,3-dimethyluric acid (1,3-DMU), 3-methylxanthine (3-MX), 1-methylxanthine (1-MX), and 1-methyluric acid (1-MU). Chromatographic separation of these analytes was achieved on a Gemini C18 column (50 mm × 4.60 mm, 5 µm) using reversed phase chromatography. The analytes were monitored by electrospray ionization in negative ion multiple reaction monitoring mode. Modification of collision energies was performed in parallel with chromatographic separation to further eliminate interference peaks. The method was validated from 0.05 to 30 µg/mL for 1-MX, 1,3-DMU, 1-MU, and theophylline and from 0.1 to 30 µg/mL for 3-MX using 0.2 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD) of less than 13% and with relative error (RE) values of -8.8% to 9.7%. The method was successfully applied for the quantitation of theophylline and its metabolite in rat plasma samples.

Cardioprotective effects of saponins from Panax japonicus on acute myocardial ischemia against oxidative stress-triggered damage and cardiac cell death in rats.

AIM: To study the cardioprotective effects of saponins from Panax japonicus (SPJ) on acute myocardial ischemia injury rats induced by ligating of the left anterior descending branch (LAD), on the basis of this investigation, the possible mechanism of SPJ was elucidated. MATERIALS AND METHODS: SPJ was identified by high performance liquid chromatography-evaporative light scattering detection. Male Sprague-Dawley rats (200-220g) were randomly divided into four groups: sham-operated, LAD, LAD+l-SPJ (SPJ, 50mg/kg/day, orally) and LAD+h-SPJ (SPJ, 100mg/kg/day, orally). Before operation, the foregoing groups were pretreated with homologous drug once a day for 7 days, respectively. After twelve hours in LAD, the cardioprotective effects of SPJ were evaluated by infarct size, biochemical values, hemodynamic, and histopathological observations and the antioxidative and antiapoptotic relative gene expressions. RESULTS: SPJ significantly improved heart function and decreased infarct size; remarkably decreased levels of serum lactate dehydrogenase, creatine kinase, xanthine oxide and malondialdehyde content, increased contents of serum total antioxidant capacity, superoxide dismutase (SOD), glutathione peroxidase, catalase; quantitative real-time PCR results showed that SPJ might markedly reverse the down-regulated mRNA expressions of the SOD1, SOD2 and SOD3, ameliorate the increased Bax and caspase-3 mRNA expressions and decreased Bcl-2 mRNA expression and ratios of Bcl-2 to Bax. Histopathological observations provided supportive evidence for biochemical analyses, and with the dose of SPJ increasing, the aforesaid improvement became more and more strong. CONCLUSIONS: The studies demonstrated that in ischemic myocardium, oxidative stress caused the overgeneration and accumulation of reactive oxygen species (ROS), which was central of cardiac ischemic injury. SPJ exerted beneficially cardioprotective effects on myocardial ischemia injury rats, mainly scavenging oxidative stress-triggered overgeneration and accumulation of ROS, alleviating myocardial ischemia injury and cardiac cell death.

Application of liquid chromatography-tandem mass spectrometry method for the simultaneous quantitative analysis of propentofylline and its chiral metabolite M1 in rats.

A sensitive and selective liquid chromatographic-electrospray ionization mass spectrometric method for the simultaneous determination of propentofylline and enantiomers of its active metabolite M1 in rat serum, cortex and hippocampus was developed and validated according to GLP procedures. Sample preparations were carried out by liquid-liquid extraction using diethyl ether after the addition of the internal standard (pentoxifylline). The dried residue was reconstituted in mobile phase and injected onto a Chiralpak AD column (10 µm, 250 × 4.6 mm i.d.). The limit of quantification for propentofylline in serum, cortex and hippocampus was set at 0.25 ng/mL and for enantiomers of its metabolite M1 at 1.25 ng/mL. The established LC/ESI-MS/MS method has been successfully applied to an initial pharmacokinetic study of propentofylline and also to assessment of distribution of parent drug and enantiomers of its pharmacologically active metabolite M1 to cortex and hippocampus after intravenous administration of propentofylline to rats at a dose of 5 mg/kg.

Clinical pharmacokinetics of tonapofylline: evaluation of dose proportionality, oral bioavailability, and gender and food effects in healthy human subjects.

Tonapofylline is an antagonist of adenosine A1 receptor being developed for heart failure. In the present studies, pharmacokinetic characteristics, including dose proportionality, bioavailability, and effects of gender and food, were evaluated in healthy subjects receiving single-dose tonapofylline (0.2-375 mg) in a parallel or crossover design. Following oral administration, tonapofylline concentrations mostly peaked within 3 hours and declined over time in a multiple phasic manner. Based on a power model, dose proportionality of peak concentration (C(max)), area under the time-concentration curve for all values (AUC(all)), and area under the time-concentration curve to infinity (AUC(inf)) was concluded in a clinical setting. The bioavailability of tonapofylline was 81.2% (90% confidence interval, 70.6%-93.5%). Following intravenous administration, the steady-state volume of distribution of tonapofylline was estimated to be 756 mL/kg. The total clearance of tonapofylline was low (64.8 mL/h/kg), approximately 5% of hepatic blood flow. The terminal half-life was variable within groups and ranged from 11.2 to 24.2 hours across the dose range. Female subjects showed significantly higher C(max), AUC(all), and AUC(inf) than male subjects (P < .05). Food decreased C(max) by approximately 39%, whereas it did not appear to affect AUC(all) and AUC(inf). The intersubject variability of the pharmacokinetic parameters of tonapofylline was generally less than 30%. In these studies, a single dose of tonapofylline was safe and well tolerated.

Pharmacokinetic-pharmacodynamic modeling of methylxanthine derivatives in mice challenged with high-dose lipopolysaccharide.

AIMS: Pentoxifylline and lisofylline are methylxanthine derivatives that exhibit anti-inflammatory activity both in vitro and in vivo. This study was designed to develop a pharmacokinetic-pharmacodynamic (PK/PD) model to describe the inhibitory effect of these compounds on TNF-alpha production in mice challenged with bacterial lipopolysaccharide (LPS). METHODS: Male CD-1 mice received increasing intravenous doses of either compound simultaneously with high-dose LPS. A 2-compartment model with Michaelis-Menten-type elimination was used to describe the drug concentration versus time data. Serum TNF-alpha levels were fitted to an indirect response model. RESULTS: Pentoxifylline and lisofylline reduced LPS-induced TNF-alpha serum concentrations in a dose-dependent manner. PK/PD analysis revealed an almost 2-fold higher estimate of K(m) for pentoxifylline in comparison to lisofylline. The production and elimination rates of TNF-alpha were: k(in) = 2,167 pg/ml * h(-1) and k(out) = 1.65 h(-1), respectively. The drug concentration causing 50% of TNF inhibition (IC(50)) was markedly lower for pentoxifylline (0.47 vs. 1.61 microg/ml). CONCLUSIONS: It seems that pentoxifylline is more potent than lisofylline in inhibiting TNF-alpha production in vivo. The proposed PK/PD model allowed a better understanding of the pharmacological properties of both methylxanthine derivatives and may be helpful in appropriate dosage selection for further studies.