Dried Blood Spots as Matrix for Evaluation of Valproate Levels and the Immediate and Delayed Metabolomic Changes Induced by Single Valproate Dose Treatment.

The immediate and delayed metabolic changes in rats treated with valproate (VPA), a drug used for the treatment of epilepsy, were profiled. An established approach using dried blood spots (DBS) as sample matrices for gas chromatography/mass spectrometry-based metabolomics profiling was modified using double solvents in the extraction of analytes. With the modified method, some of the previously undetectable metabolites were recovered and subtle differences in the metabolic changes upon exposure to a single dose of VPA between males and female rats were identified. In male rats, changes in 2-hydroxybutyric acid, pipecolic acid, tetratriacontane and stearic acid were found between the control and treatment groups at various time points from 2.5 h up to 24 h. In contrast, such differences were not observed in female rats, which could be caused by the vast inter-individual variations in metabolite levels within the female group. Based on the measured DBS drug concentrations, clearance and apparent volume of distribution of VPA were estimated and the values were found to be comparable to those estimated previously from full blood drug concentrations. The current study indicated that DBS is a powerful tool to monitor drug levels and metabolic changes in response to drug treatment.

Investigating PEGDA and GelMA Microgel Models for Sustained 3D Heterotypic Dermal Papilla and Keratinocyte Co-Cultures.

Hair follicle morphogenesis is heavily dependent on reciprocal, sequential, and epithelial-mesenchymal interaction (EMI) between epidermal stem cells and the specialized cells of the underlying mesenchyme, which aggregate to form the dermal condensate (DC) and will later become the dermal papilla (DP). Similar models were developed with a co-culture of keratinocytes and DP cells. Previous studies have demonstrated that co-culture with keratinocytes maintains the in vivo characteristics of the DP. However, it is often challenging to develop three-dimensional (3D) DP and keratinocyte co-culture models for long term in vitro studies, due to the poor intercellular adherence between keratinocytes. Keratinocytes exhibit exfoliative behavior, and the integrity of the DP and keratinocyte co-cultured spheroids cannot be maintained over prolonged culture. Short durations of culture are unable to sufficiently allow the differentiation and re-programming of the keratinocytes into hair follicular fate by the DP. In this study, we explored a microgel array approach fabricated with two different hydrogel systems. Using poly (ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), we compare their effects on maintaining the integrity of the cultures and their expression of important genes responsible for hair follicle morphogenesis, namely Wnt10A, Wnt10B, and Shh, over prolonged duration. We discovered that low attachment surfaces such as PEGDA result in the exfoliation of keratinocytes and were not suitable for long-term culture. GelMA, on the hand, was able to sustain the integrity of co-cultures and showed higher expression of the morphogens overtime.

Keratinocytes maintain compartmentalization between dermal papilla and fibroblasts in 3D heterotypic tri-cultures.

OBJECTIVES: Reproducing human hair follicles in vitro is often limited by various reasons such as the lack of a systematic approach to culture distinct hair follicle cell types to reproduce their spatial relationship. Here, we reproduce hair follicle-like constructs resembling the spatial orientation of different cells in vivo, to study the role of keratinocytes in maintaining cellular compartmentalization among hair follicle-related cells. MATERIALS AND METHODS: Dermal papilla (DP) cells, HaCaT keratinocytes and human dermal fibroblast (HDF) cells were seeded sequentially into three-dimensional (3D) microwells fabricated from polyethylene glycol diacrylate hydrogels. Quantitative polymerase chain reaction was used to compare inductive gene expression of 3D and two-dimensional (2D) DP. DP and HaCaT cells were transfected with green fluorescent protein and red fluorescent protein lentivirus, respectively, to enable cell visualization using confocal microscopy. RESULTS: The 3D DP cultures showed significantly enhanced expression of essential DP genes as compared 2D cultures. Core-shell configurations containing keratinocytes forming the outer shell and DP forming the core were observed. Migratory polarization was mediated by cell-cell interaction between the keratinocytes and HDF cells, while preserving the aggregated state of the DP cells. CONCLUSIONS: Keratinocytes may play a role in maintaining compartmentalization between the DP and the surrounding HDF residing in the dermis, and therefore maintains the aggregative state of the DP cells, necessary for hair follicle development and function.

Impact of substrate stiffness on dermal papilla aggregates in microgels.

Interaction between cells and the extracellular environment plays a vital role in cellular development. The mechanical property of a 3-dimensional (3D) culture can be modified to mimic in vivo conditions. Dermal papilla (DP) cells are shown to gradually lose their inductivity in hair cycle development in a 2-dimensional culture. They are shown to partially restore their inductivity when transferred into a 3D microenvironment. In this study, a microarray fabricated from three different concentrations of poly-ethylene-glycol-diacrylate 3500, namely 5%, 10% and 15% w/v, yielded increasing substrate stiffness. The impact of varying substrate stiffness was tested for DP cell viability, attachment, and selected hair inductive markers. DP aggregates were shown to be viable and exhibited greater spreading with increasing substrate stiffness. Moreover, DP aggregates cultured on a softer substrate showed a greater fold change of gene and protein expressions than those cultured on a harder substrate.

Role of serum albumin as a nanoparticulate carrier for nose-to-brain delivery of R-flurbiprofen: implications for the treatment of Alzheimer's disease.

OBJECTIVES: R-flurbiprofen (R-FP) was found to offer neuroprotective effects by inhibiting mitochondrial calcium overload induced by ß-amyloid peptide toxicity in Alzheimer's disease (AD). However, poor brain penetration after oral administration posed a challenge to its further development for AD treatment. In this study, we investigated the potential of serum albumin as nanoparticulate carriers for nose-to-brain delivery of R-FP to improve its brain accumulation. METHODS: Mice were subjected to three treatment groups: (1) intranasal R-FP solution, (2) oral R-FP solution and (3) intranasal R-FP albumin nanoparticles. We also investigated whether the in-vivo R-FP level achieved in the brain afforded by intranasal administration of R-FP nanoparticles had any effect on mitochondrial respiratory activity in an in-vitro AD model. KEY FINDINGS: Our in-vivo experiments demonstrate that the intranasal administration of serum albumin-based R-FP nanoparticles achieved higher brain-to-plasma ratio profile as compared to intranasal and oral administration of a simple R-FP solution. We observed significantly improved basal and maximal mitochondrial respiration in cells treated with R-FP albumin nanoparticles at in-vivo brain concentration. CONCLUSIONS: Serum albumin-based nanoparticles administered via the nasal route may be a viable approach in delivering therapeutic agents to the brain to alleviate mitochondrial dysfunction in AD.

Intranasal administration of carbamazepine-loaded carboxymethyl chitosan nanoparticles for drug delivery to the brain.

Epilepsy is considered as a common and diverse set of chronic neurological disorders and its symptoms can be controlled by antiepileptic drugs (AEDs). The presence of p-glycoprotein and multi-drug resistance transporters in the blood-brain barrier could prevent the entry of AEDs into the brain, causing drug resistant epilepsy. To overcome this problem, we propose using carboxymethyl chitosan nanoparticles as a carrier to deliver carbamazepine (CBZ) intra-nasally with the purpose to bypass the blood-brain barrier thus to enhance the brain drug concentration and the treatment efficacy. Results so far indicate that the developed CBZ-NPs have small particle size (218.76 ± 2.41 nm) with high drug loading (around 35%) and high entrapment efficiency (around 80%). The in vitro release profiles of CBZ from the NPs are in accordance with the Korsmeyer-peppas model. The in vivo results show that both encapsulation of CBZ in nanoparticles and the nasal route determined the enhancement of the drug bioavailability and brain targeting characteristics.

Intranasal administration of brain-targeted HP-ß-CD/chitosan nanoparticles for delivery of scutellarin, a compound with protective effect in cerebral ischaemia.

OBJECTIVES: Scutellarin (SCU) is a traditional Chinese medicine used for the treatment of ischaemic cerebrovascular disease, but its clinic applications have been limited due to its poor water solubility, poor bioavailability and short half-life. In comparison with the conventional oral and intravenous administration, nasal administration may help targeting the drug more directly to brain. Thus, we proposed to employ a novel SCU-loaded HP-ß-CD/chitosan nanoparticles (CD/CS-SCU-NPs) to deliver SCU to brain through the nasal route. METHODS: CD/CS-SCU-NPs were prepared by an ionic cross-linking method. The NPs formulation was tested in vivo in C57BL mice. The concentrations of SCU in brain and plasma after intranasal and oral administration of the CD/CS-SCU-NPs and after intranasal administration of SCU solution (SCU-SL) were determined and brain targeting parameters were calculated. KEY FINDINGS: Compared to the intranasal administration of SCU-SL, intranasal and oral administration of the CD/CS-SCU-NPs increased accumulation of SCU in brain, indicating that CD/CS-SCU-NPs have obvious brain targeting advantage, although the advantage is more evident after intranasal administration. CONCLUSIONS: Findings from in-vivo study indicated that much higher SCU brain exposure was observed after intranasal administration of the CD/CS-SCU-NPs. Administration of CD/CS-SCU-NPs through the nasal route would have potential to treat ischemic cerebrovascular disease.

Testing of a novel percutaneous temporary aortic valve in a rabbit model of acute severe aortic regurgitation: an experimental study.

PURPOSE: Device management of hemodynamic instability due to acute aortic regurgitation is not available. A novel, catheter-based, temporary aortic valve (TAV) has been in development. Early prototypes (balloon-based TAV) have undergone proof-of-concept studies in mathematical, bench and animal models. The redesigned membrane-based TAV prototype is evaluated in a rabbit model of acute severe aortic regurgitation. METHODS: Acute aortic regurgitation was simulated by deploying a self-expanding endovascular stent across the aortic annulus. Eight rabbits of body weights ranging 4.9-5.4 kg were randomly assigned to two groups: those received additional hemodynamic support with the TAV prototype immediately after aortic regurgitation was induced versus no TAV support. The survival times of the two groups were compared. RESULTS: Comparing the groups with TAV versus without TAV, the mean body weights were similar: 4.99 ± 0.06 vs. 5.10 ± 0.22 kg (p = 0.71). The mean stent sizes used to create acute aortic regurgitation were similar: 6.25 ± 0.50 vs. 6.75 ± 0.50 mm, respectively (p = 0.53). The mean survival times also did not differ significantly: 21.00 ± 15.41 vs. 8.25 ± 2.75 minutes, respectively (p = 0.45). A slight trend appeared to be in favor of longer survival in the TAV supported group. CONCLUSIONS: In a rabbit model of acute massive aortic regurgitation, the use of the TAV support prototype did not hasten the animals' death, but rather survival may be enhanced by the use of the device. Future studies specifically designed to evaluate the efficacy of the TAV catheter can be valuable in this new technology.

Formulation optimization of scutellarin-loaded HP-ß-CD/chitosan nanoparticles using response surface methodology with Box-Behnken design.

The aim of this paper is to investigate and optimize the preparation of scutellarin (SCU)-loaded HP-ß-CD/chitosan (CS) nanoparticles (CD/CS-SCU-NPs). CD/CS-SCU-NPs were prepared by ionic cross-linking method and the process and formulation variables were optimized using response surface methodology (RSM) with a three-level, three factor Box-Behnken design (BBD). The independent variables were the added amounts of CS, sodium tripolyphosphate (TPP) and Pluronic F-68 during the preparation. Dependent variables (responses) were particle size and entrapment efficiency. Mathematical equations and respond surface plots were used to correlate independent and dependent variables. The preparation process and formulation variables were optimized to achieve minimum particle size and maximum entrapment efficiency by calculating the overall desirability value (OD). The optimized NP formulation was characterized for particle size, PDI, zeta potential, entrapment efficiency and in vitro drug release. According to the results, an optimized CD/CS-SCU-NP formulation was prepared. Results for particle size, PDI, zeta potential and entrapment efficiency were found to be around 200 nm, 0.5, 25 mV, and 70% respectively. For in vitro study, the release of SCU from the NPs exhibited a biphasic release and was in accordance with Higuchi equation. The optimized preparation was simple with the probability for industrialization. The combination use of RSM, BBD and overall desirability values could provide a promising application for incorporating CD into CS nanoparticles as drug delivery carrier and help develop lab-scale procedures.

Qualitative coronary flow evaluation of a pre-clinical percutaneous temporary aortic valve.

A published balloon-based percutaneous temporary aortic valve (TAV), with a specific fixed gap-to-aorta cross-sectional area ratio, was shown to provide haemodynamic support in acute aortic regurgitation (AR). The fixed gap of the balloon-TAV, however, limits the ability to optimize the gap size balancing coronary flow vs AR protection. Hence, a reduced diastolic gap may improve AR protection, but could reduce coronary flow and increase systolic TAV flow resistance. A new membrane-based TAV, which avoids these design limitations, could guide gap size optimization and advance the development into a pre-clinical tool. The re-designed TAV prototype has a membrane-cone collapsible in systole to reduce flow resistance and expands in diastole with a gap-to-aorta cross-sectional area ratio that can be tailored to optimize AR protection and coronary flow. Without the concern for systolic TAV flow resistance, a lower limit of the gap:aorta cross-sectional area ratio could be determined. The ability of the membrane-TAV design in determining an optimal gap:aorta ratio is tested in an in vitro flow chamber. Three prototypes with reducing gap:aorta cross-sectional area ratios (35%, 15%, 0%) were tested in a flow chamber of simulated acute severe AR. Correspondingly, increasing in forward cardiac output volumes, coronary flow:aortic regurgitant volume ratios and reduction in aortic regurgitant volumes were observed (p < 0.001) in the three models. The membrane-TAV concept contains a design feature for optimization of LV protection from acute AR and coronary perfusion by defining an optimal gap:aorta ratio. Along with the results from the balloon-TAV, a clinically useful percutaneous device for the management of acute severe aortic regurgitation is becoming possible.

Influence of drug transporters and stereoselectivity on the brain penetration of pioglitazone as a potential medicine against Alzheimer's disease.

Pioglitazone is currently undergoing clinical trials for treatment of Alzheimer's disease (AD). However, poor brain penetration remains an obstacle to development of the drug for such intended clinical uses. In this study, we demonstrate that the inhibition of P-glycoprotein (P-gp) significantly increases brain penetration of pioglitazone, whereas inhibition of breast cancer resistance protein (BCRP) has little effect. We also investigate the stereoselectivity of pioglitazone uptake in the brain. When mice were dosed with racemic pioglitazone, the concentration of (+)-pioglitazone was 46.6% higher than that of (-)-pioglitazone in brain tissue and 67.7% lower than that of (-)-pioglitazone in plasma. Dosing mice with pure (+)-pioglitazone led to a 76% increase in brain exposure levels compared to those from an equivalent dose of racemic pioglitazone. Pure (+)-pioglitazone was also shown to have comparable amyloid-lowering capabilities to the racemic pioglitazone in an in vitro AD model. These results suggest that P-gp may act as a stereoselective barrier to prevent pioglitazone entry into the brain. Dosing with (+)-pioglitazone instead of the racemic mixture may result in higher levels of brain exposure to pioglitazone, thus potentially improving the development of pioglitazone treatment of AD.

Validation of a rapid and sensitive LC-MS/MS method for determination of exemestane and its metabolites, 17ß-hydroxyexemestane and 17ß-hydroxyexemestane-17-O-ß-D-glucuronide: application to human pharmacokinetics study.

A novel, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the evaluation of exemestane pharmacokinetics and its metabolites, 17ß-dihydroexemestane (active metabolite) and 17ß-dihydroexemestane-17-O-ß-D-glucuronide (inactive metabolite) in human plasma. Their respective D3 isotopes were used as internal standards. Chromatographic separation of analytes was achieved using Thermo Fisher BDS Hypersil C18 analytic HPLC column (100 × 2.1 mm, 5 µm). The mobile phase was delivered at a rate of 0.5 mL/min by gradient elution with 0.1% aqueous formic acid and acetonitrile. The column effluents were detected by API 4000 triple quadrupole mass spectrometer using electrospray ionisation (ESI) and monitored by multiple reaction monitoring (MRM) in positive mode. Mass transitions 297 > 121 m/z, 300 > 121 m/z, 299 > 135 m/z, 302 > 135 m/z, 475 > 281 m/z, and 478 > 284 m/z were monitored for exemestane, exemestane-d3, 17ß-dihydroexemestane, 17ß-dihydroexemestane-d3, 17ß-dihydroexemestane-17-O-ß-D-glucuronide, and 17ß-dihydroexemestane-17-O-ß-D-glucuronide-d3 respectively. The assay demonstrated linear ranges of 0.4-40.0 ng/mL, for exemestane; and 0.2-15.0 ng/mL, for 17ß-dihydroexemestane and 17ß-dihydroexemestane-17-O-ß-D-glucuronide, with coefficient of determination (r2) of > 0.998. The precision (coefficient of variation) were ≤10.7%, 7.7% and 9.5% and the accuracies ranged from 88.8 to 103.1% for exemestane, 98.5 to 106.1% for 17ß-dihydroexemestane and 92.0 to 103.2% for 17ß-dihydroexemestane-17-O-ß-D-glucuronide. The method was successfully applied to a pharmacokinetics/dynamics study in breast cancer patients receiving exemestane 25 mg daily orally. For a representative patient, 20.7% of exemestane in plasma was converted into 17ß-dihydroexemestane and 29.0% of 17ß-dihydroexemestane was inactivated as 17ß-dihydroexemestane-17-O-ß-D-glucuronide 24 hours after ingestion of exemestane, suggesting that altered 17-dihydroexemestane glucuronidation may play an important role in determining effect of exemestane against breast cancer cells.

Comprehensive physicochemical, pharmacokinetic and activity profiling of anti-TB agents.

OBJECTIVES: The discovery and development of TB drugs has met limited success, with two new drugs approved over the last 40 years. Part of the difficulty resides in the lack of well-established in vitro or in vivo targets of potency and physicochemical and pharmacokinetic parameters. In an attempt to benchmark and compare such properties for anti-TB agents, we have experimentally determined and compiled these parameters for 36 anti-TB compounds, using standardized and centralized assays, thus ensuring direct comparability across drugs and drug classes. METHODS: Potency parameters included growth inhibition, cidal activity against growing and non-growing bacteria and activity against intracellular mycobacteria. Pharmacokinetic parameters included basic physicochemical properties, solubility, permeability and metabolic stability. We then attempted to establish correlations between physicochemical, in vitro and in vivo pharmacokinetic and pharmacodynamic indices to tentatively inform future drug discovery efforts. RESULTS: Two-thirds of the compounds tested showed bactericidal and intramacrophage activity. Most compounds exhibited favourable solubility, permeability and metabolic stability in standard in vitro pharmacokinetic assays. An analysis of human pharmacokinetic parameters revealed associations between lipophilicity and volume of distribution, clearance, plasma protein binding and oral bioavailability. Not surprisingly, most compounds with favourable pharmacokinetic properties complied with Lipinski's rule of five. CONCLUSIONS: However, most attempts to detect in vitro-in vivo correlations were unsuccessful, emphasizing the challenges of anti-TB drug discovery. The objective of this work is to provide a reference dataset for the TB drug discovery community with a focus on comparative in vitro potency and pharmacokinetics.

Metabolic profiling of CHO-AßPP695 cells revealed mitochondrial dysfunction prior to amyloid-ß pathology and potential therapeutic effects of both PPARγ and PPARα Agonisms for Alzheimer's disease.

In this study, we performed gas chromatography time-of-flight mass spectrometry (GC-TOFMS)-based extracellular metabolic profiling on AßPP-transfected CHO cells (CHO-AßPP695) and its wildtype. Orthogonal partial least squares discriminant analysis (OPLS-DA) was then used to identify discriminant metabolites, which gave clues on the effects of AßPP transgene on cellular processes. To confirm the hypotheses generated based on the metabolic data, we performed biochemical assays to gather further evidence to support our findings. The OPLS-DA showed a robust differentiation following 24 h of incubation (Q2(cum) = 0.884) and 15 discriminant metabolites were identified. In contrast, extracellular Aß42 was identified to increase significantly in CHO-AßPP695 only after incubation for 48 h. The observed 24-h metabolic fluxes were associated with increased mitochondrial AßPP and reduced mitochondrial viabilities, which occurred before extracellular Aß accumulation. We also investigated the therapeutic potential of peroxisome proliferator-activated receptor gamma (PPARγ) agonists, namely rosiglitazone (RSG) and pioglitazone (PIO), by employing the same approach to characterize the metabolic profiles of CHO-AßPP695 treated with RSG and PIO, with or without their respective receptor blockers. Treatment with PIO was found to reduce the perturbation of the discriminant metabolites in CHO-AßPP695 to a larger extent than treatment with RSG. We also attributed the PIO effects on the lowering of Aß42, and restoration of mitochondrial activity to PPARγ and PPARα agonism, respectively. Taken together, PIO was demonstrated to be therapeutically superior to RSG. Our findings provide further insights into early disease stages in this AßPP model, and support the advancement of PIO in AD therapy.

Pharmacokinetic-pharmacodynamic analysis of spiroindolone analogs and KAE609 in a murine malaria model.

Limited information is available on the pharmacokinetic (PK) and pharmacodynamic (PD) parameters driving the efficacy of antimalarial drugs. Our objective in this study was to determine dose-response relationships of a panel of related spiroindolone analogs and identify the PK-PD index that correlates best with the efficacy of KAE609, a selected class representative. The dose-response efficacy studies were conducted in the Plasmodium berghei murine malaria model, and the relationship between dose and efficacy (i.e., reduction in parasitemia) was examined. All spiroindolone analogs studied displayed a maximum reduction in parasitemia, with 90% effective dose (ED90) values ranging between 6 and 38 mg/kg of body weight. Further, dose fractionation studies were conducted for KAE609, and the relationship between PK-PD indices and efficacy was analyzed. The PK-PD indices were calculated using the in vitro potency against P. berghei (2× the 99% inhibitory concentration [IC99]) as a threshold (TRE). The percentage of the time in which KAE609 plasma concentrations remained at >2× the IC99 within 48 h (%T>TRE) and the area under the concentration-time curve from 0 to 48 h (AUC0-48)/TRE ratio correlated well with parasite reduction (R2=0.97 and 0.95, respectively) but less so for the maximum concentration of drug in serum (Cmax)/TRE ratio (R2=0.88). The present results suggest that for KAE609 and, supposedly, for its analogs, the dosing regimens covering a T>TRE of 100%, AUC0-48/TRE ratio of 587, and a Cmax/TRE ratio of 30 are likely to result in the maximum reduction in parasitemia in the P. berghei malaria mouse model. This information could be used to prioritize analogs within the same class of compounds and contribute to the design of efficacy studies, thereby facilitating early drug discovery and lead optimization programs.

Metabonomic profiling of bladder cancer.

Early diagnosis and life-long surveillance are clinically important to improve the long-term survival of bladder cancer patients. Currently, a noninvasive biomarker that is as sensitive and specific as cystoscopy in detecting bladder tumors is lacking. Metabonomics is a complementary approach for identifying perturbed metabolic pathways in bladder cancer. Significant progress has been made using modern metabonomic techniques to characterize and distinguish bladder cancer patients from control subjects, identify marker metabolites, and shed insights on the disease biology and potential therapeutic targets. With its rapid development, metabonomics has the potential to impact the clinical management of bladder cancer patients in the future by revolutionizing the diagnosis and life-long surveillance strategies and stratifying patients for diagnostic, surgical, and therapeutic clinical trials. An introduction to metabonomics, typical metabonomic workflow, and critical evaluation of metabonomic investigations in identifying biomarkers for the diagnosis of bladder cancer are presented.

Clinical validation and implications of dried blood spot sampling of carbamazepine, valproic acid and phenytoin in patients with epilepsy.

To facilitate therapeutic monitoring of antiepileptic drugs (AEDs) by healthcare professionals for patients with epilepsy (PWE), we applied a GC-MS assay to measure three AEDs: carbamazepine (CBZ), phenytoin (PHT) and valproic acid (VPA) levels concurrently in one dried blood spot (DBS), and validated the DBS-measured levels to their plasma levels. 169 PWE on either mono- or polytherapy of CBZ, PHT or/and VPA were included. One DBS, containing ∼15 µL of blood, was acquired for the simultaneous measurement of the drug levels using GC-MS. Simple Deming regressions were performed to correlate the DBS levels with the plasma levels determined by the conventional immunoturbimetric assay in clinical practice. Statistical analyses of the results were done using MedCalc Version 12.6.1.0 and SPSS 21. DBS concentrations (Cdbs) were well-correlated to the plasma concentrations (Cplasma): r=0.8381, 0.9305 and 0.8531 for CBZ, PHT and VPA respectively, The conversion formulas from Cdbs to plasma concentrations were [0.89×CdbsCBZ+1.00]µg/mL, [1.11×CdbsPHT-1.00]µg/mL and [0.92×CdbsVPA+12.48]µg/mL respectively. Inclusion of the red blood cells (RBC)/plasma partition ratio (K) and the individual hematocrit levels in the estimation of the theoretical Cplasma from Cdbs of PHT and VPA further improved the identity between the observed and the estimated theoretical Cplasma. Bland-Altman plots indicated that the theoretical and observed Cplasma of PHT and VPA agreed well, and >93.0% of concentrations was within 95% CI (±2SD); and similar agreement (1∶1) was also found between the observed Cdbs and Cplasma of CBZ. As the Cplasma of CBZ, PHT and VPA can be accurately estimated from their Cdbs, DBS can therefore be used for drug monitoring in PWE on any of these AEDs.

Pharmacokinetics-pharmacodynamics analysis of bicyclic 4-nitroimidazole analogs in a murine model of tuberculosis.

PA-824 is a bicyclic 4-nitroimidazole, currently in phase II clinical trials for the treatment of tuberculosis. Dose fractionation pharmacokinetic-pharmacodynamic studies in mice indicated that the driver of PA-824 in vivo efficacy is the time during which the free drug concentrations in plasma are above the MIC (fT>MIC). In this study, a panel of closely related potent bicyclic 4-nitroimidazoles was profiled in both in vivo PK and efficacy studies. In an established murine TB model, the efficacy of diverse nitroimidazole analogs ranged between 0.5 and 2.3 log CFU reduction compared to untreated controls. Further, a retrospective analysis was performed for a set of seven nitroimidazole analogs to identify the PK parameters that correlate with in vivo efficacy. Our findings show that the in vivo efficacy of bicyclic 4-nitroimidazoles correlated better with lung PK than with plasma PK. Further, nitroimidazole analogs with moderate-to-high volume of distribution and Lung to plasma ratios of >2 showed good efficacy. Among all the PK-PD indices, total lung T>MIC correlated the best with in vivo efficacy (rs = 0.88) followed by lung Cmax/MIC and AUC/MIC. Thus, lung drug distribution studies could potentially be exploited to guide the selection of compounds for efficacy studies, thereby accelerating the drug discovery efforts in finding new nitroimidazole analogs.

Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS)-based metabolomics for comparison of caffeinated and decaffeinated coffee and its implications for Alzheimer's disease.

Findings from epidemiology, preclinical and clinical studies indicate that consumption of coffee could have beneficial effects against dementia and Alzheimer's disease (AD). The benefits appear to come from caffeinated coffee, but not decaffeinated coffee or pure caffeine itself. Therefore, the objective of this study was to use metabolomics approach to delineate the discriminant metabolites between caffeinated and decaffeinated coffee, which could have contributed to the observed therapeutic benefits. Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS)-based metabolomics approach was employed to characterize the metabolic differences between caffeinated and decaffeinated coffee. Orthogonal partial least squares discriminant analysis (OPLS-DA) showed distinct separation between the two types of coffee (cumulative Q(2) = 0.998). A total of 69 discriminant metabolites were identified based on the OPLS-DA model, with 37 and 32 metabolites detected to be higher in caffeinated and decaffeinated coffee, respectively. These metabolites include several benzoate and cinnamate-derived phenolic compounds, organic acids, sugar, fatty acids, and amino acids. Our study successfully established GC-TOF-MS based metabolomics approach as a highly robust tool in discriminant analysis between caffeinated and decaffeinated coffee samples. Discriminant metabolites identified in this study are biologically relevant and provide valuable insights into therapeutic research of coffee against AD. Our data also hint at possible involvement of gut microbial metabolism to enhance therapeutic potential of coffee components, which represents an interesting area for future research.

Evaluation of meisoindigo, an indirubin derivative: in vitro antileukemic activity and in vivo pharmacokinetics.

Meisoindigo has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia (CML) in China since the 1980s. In the present study, the in vitro antileukemic activity of meisoindigo was investigated in acute promyelocytic leukemia (APL) cells, acute myeloid leukemia (AML) cells, and myelomonocytic leukemia cells (NB4, NB4.007/6, HL-60 and U937) comprising both retinoic acid-sensitive and retinoic acid-resistant cells. We found that meisoindigo effectively inhibited the growth and/or proliferation of these four cell types at µM levels. The effects of meisoindigo in these cells are related to its proliferation inhibition and apoptosis induction, and are independent of cell cycle arrest, indicating that meisoindigo could be possible in the treatment of APL, AML and retinoic acid resistant APL. The in vivo pharmacokinetics of meisoindigo and its major circulatory metabolites in rat plasma were then investigated by a newly developed and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The profiles of plasma concentration versus time were plotted and the relevant pharmacokinetic parameters were calculated for meisoindigo and its reductive metabolites. The plasma concentrations of meisoindigo after oral administration were much lower than the in vitro IC50s determined in the leukemic cells. The contradicting poor pharmacokinetic characteristics and the established clinical efficacy of meisoindigo could indicate the presence of active metabolites in vivo.