Review of the pharmacokinetics of French maritime pine bark extract (Pycnogenol®) in humans.

The French maritime pine bark extract Pycnogenol® is a proprietary product from Pinus pinaster Aiton. It complies with the quality specifications in the United States Pharmacopeia monograph "Pine extract" in the section of dietary supplements. Pycnogenol® is standardized to contain 65-75% procyanidins which are a variety of biopolymers consisting of catechin and epicatechin monomeric units. The effects of Pycnogenol® have been researched in a multitude of human studies. The basis for any in vivo activity is the bioavailability of constituents and metabolites of the extract. General principles of compound absorption, distribution, metabolism and elimination as well as specific data from studies with Pycnogenol® are summarized and discussed in this review. Based on plasma concentration profiles it can be concluded that low molecular weight constituents of the extract, such as catechin, caffeic and ferulic acid, taxifolin are readily absorbed from the small intestine into systemic circulation. Procyanidin oligomers and polymers are subjected to gut microbial degradation in the large intestine yielding small bioavailable metabolites such as 5-(3',4'-dihydroxyphenyl)-γ-valerolactone. After intake of Pycnogenol®, constituents and metabolites have been also detected in blood cells, synovial fluid and saliva indicating a substantial distribution in compartments other than serum. In studies simultaneously investigating concentrations in different specimen, a preferential distribution of individual compounds has been observed, e.g., of ferulic acid and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone into synovial fluid compared to serum. The main route of elimination of constituents and metabolites of the French pine bark extract is the renal excretion. The broad knowledge accumulated regarding the pharmacokinetics of compounds and metabolites of Pycnogenol® constitute a rational basis for effects characterized on a cellular level and observed in human clinical studies.

Development and validation of a LC-MS/MS method for the quantification of phenolic compounds in human saliva after intake of a procyanidin-rich pine bark extract.

Plant-derived phenolic compounds are regularly ingested as food compounds or as food supplements. Concentrations of individual compounds and metabolites are typically measured in serum or urine samples. This, however, allows no conclusion on the distribution into organs and tissues. An easily accessible biofluid is saliva. At this point, it was not clear yet, whether polyphenols circulating in the blood would be secreted or diffuse into saliva. The purpose of the present study was to develop and validate a method using liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for analysis of phenolic compounds in human saliva. Method validation for the quantification of taxifolin, ferulic acid, caffeic acid, gallic acid, para-coumaric acid, and protocatechuic acid and the gut microbial catechin metabolite δ-(3,4-dihydroxyphenyl)-γ-valerolactone (M1) in human saliva was performed according to current guidelines for bioanalytical method validation. The lower limit of quantification ranged from 0.82 ng/ml for M1 to 8.20 ng/ml for protocatechuic acid. The method was successfully applied to an authentic saliva sample of a volunteer after swallowing of procyanidin-rich pine bark extract capsules (dietary supplement Pycnogenol®). All polyphenols except ferulic acid were quantified at concentrations ranging from 1.20 ng/ml (M1) to 10.34 ng/ml (gallic acid). Notably, in contrast to serum samples, all phenolic compounds were present without sulfate or glucuronic acid conjugation in saliva, suggesting an enzymatic deconjugation, e.g., by a ß-glucuronidase activity, during compound transfer from serum to saliva. Since M1 is only produced in the gut, its presence in saliva ruled out the possibility of sample contamination by phenolic compounds residing in the oral cavity after food intake. To the best of our knowledge, this is the first time that the gut microbiota-derived metabolite M1 has been detected in saliva. To further investigate the role of phenolic compounds in saliva, the described analytical method can be applied in clinical studies investigating the biodistribution of polyphenols and their metabolites.

Determination of Guanfacine in Oral Fluid and Serum of Children and Adolescents with Attention-Deficit/Hyperactivity Disorder: A Short Communication.

BACKGROUND: Guanfacine, a selective α2A-adrenoreceptor agonist, is a second-line medication for treating children and adolescents with attention-deficit/hyperkinetic disorder. The dosage administered as milligram per body weight to balance the potential benefits and risks of treatment. Therapeutic drug monitoring (TDM) is useful for identifying a patient's therapeutic window to optimize individual drug dosing and reduce the risk of adverse drug reactions. However, in children and adolescents, intravenous sample collection is especially stressful and thus remains a primary challenge, restricting the use of TDM. Therefore, evaluating alternative specimens to facilitate TDM is a worthwhile task. The aim of this study was to assess the feasibility of using oral fluid for TDM of guanfacine in children and adolescents. METHODS: In this article, 9 patients (median age 8.1 years; 6 boys and 3 girls) undergoing treatment with guanfacine were included. Simultaneously collected oral fluid and serum samples were deproteinized using methanol containing a stable isotope-labeled internal standard before the determination of guanfacine by liquid chromatography-tandem mass spectrometry. Pearson correlation and paired t test were used for statistical analysis. RESULTS: The mean serum guanfacine concentration was 3 times higher than that detected in oral fluid (7.47 ng/mL versus 2.36 ng/mL; t (8) = 5.94; P < 0.001). A strong positive linear correlation (r = 0.758, P = 0.018) was identified between oral fluid and serum concentrations. A strong but nonsignificant negative correlation (r = -0.574, P = 0.106) was detected between the oral fluid pH and oral fluid-to-serum concentration ratio. CONCLUSIONS: The strong correlation between oral fluid and serum concentration and the probable small effect of oral fluid pH on oral fluid-to-serum concentration ratio supports guanfacine as a suitable candidate for TDM in oral fluid.

Stability and antioxidant capacity of epigallocatechin gallate in Dulbecco's modified eagle medium.

Though the instability of polyphenols in cell culture experiment has been investigated previously, the underlying mechanism is not completely clear yet. Therefore, in this study, the stability of epigallocatechin gallate (EGCG) in cell culture medium DMEM was investigated at 4 °C and 37 °C via UPLC-MS-MS analysis followed by determination of the antioxidant capacity of EGCG. EGCG was instable in DMEM and formed various degradation products derived from its dimer with increasing incubation time with many isomers being formed at both temperatures. The dimer products were more stable at 4 °C than at 37 °C. The structure and formation mechanism of five products were analyzed with four unidentified. Ascorbic acid significantly improved the stability of EGCG by protecting EGCG from auto-oxidation in DMEM, particularly at 4 °C. The antioxidative activity of EGCG in DMEM was determined by DPPH, ABTS and FRAP assay. The antioxidative properties of EGCG continuously decreased over 8 h in DMEM, which was consistent with its course of degradation.

Relationship Between Amphetamine Concentrations in Saliva and Serum in Children and Adolescents With Attention-Deficit/Hyperactivity Disorder.

BACKGROUND: Therapeutic drug monitoring (TDM) is a valid tool for the optimization of psychopharmacotherapy; however, in child and adolescent psychiatry, uncomfortable intravenous sample collection is the main challenge and restricts the use of TDM. Therefore, it is important to evaluate alternate specimens to facilitate TDM. The aim of this study was to evaluate the feasibility of using saliva for the TDM of amphetamine in children and adolescents with attention-deficit/hyperactivity disorder. METHODS: In this study, 28 patient samples (mean age, 11.3 years; boys, 23; and girls, 5) treated with lisdexamfetamine were included. The active compound amphetamine was extracted and derivatized before quantification by high-performance liquid chromatography with fluorescence detection. Nonparametric Spearman rank correlations were used for correlation analyses; for clinical validation, Bland-Altman analysis was applied. RESULTS: The median amphetamine concentrations in saliva were 2.7 times higher (range 0.7-23.6) than those in serum (257.8 ng/mL versus 77.2 ng/mL; z = -4.51, P < 0.001). A strong positive linear correlation was observed between saliva and serum concentrations (ρ = 0.628, P < 0.001). The ratio of saliva-to-serum concentration was strongly pH dependent (ρ = -0.712, P < 0.001). Therefore, a transformation formula, factoring in salivary pH, to calculate serum concentrations from the measured saliva concentrations was applied. Theoretical and measured serum amphetamine concentrations were subjected to Bland-Altman analysis. Using an acceptance limit of 20%, only 21% (n = 6) of samples fulfilled this criterion. CONCLUSIONS: Amphetamine paired saliva-to-serum concentrations were highly variable and strongly affected by salivary pH, indicating that saliva is an inappropriate sampling matrix for TDM of amphetamine. Furthermore, Bland-Altman analysis did not support saliva as a suitable matrix for TDM.

Constituents and Metabolites of a French Oak Wood Extract (Robuvit®) in Serum and Blood Cell Samples of Women Undergoing Hysterectomy.

Ellagitannins are signature constituents of oak wood and their consumption has been associated with various health benefits. In vivo, they undergo metabolic degradation including gut microbial metabolism yielding urolithins. Only limited data is available about compounds being present in blood after intake of an extract from French oak wood, Robuvit®. In the course of a randomized, double-blind, controlled clinical investigation, 66 patients undergoing hysterectomy received placebo or 300 mg Robuvit® per day before and over 8 weeks after surgery. Serum and blood cell samples were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The number of urolithin producers and the urolithin levels increased after intake of Robuvit®. In serum samples, the median concentration of urolithin A was 14.0 ng/ml [interquartile range (IQR) 57.4] after 8 weeks. Urolithin B was determined at 22.3 ng/ml (IQR 12.6), urolithin C at 2.66 ng/ml (IQR 2.08). In blood cells, lower concentrations and only urolithins A and B were detected. A statistically significant association of lower post-surgical pain scores with metabotype A was detected (p < 0.05). To conclude, supplementation with French oak wood extract raised urolithin generation in patients and suggested health advantages for urolithin-producers.

Flavonols with a catechol or pyrogallol substitution pattern on ring B readily form stable dimers in phosphate buffered saline at four degrees celsius.

Phosphate buffered saline (PBS) is a commonly used buffer in biological research. Herein, the stability of a series of flavonoids, i.e. myricetin, kaempferol, baicalein, luteolin and quercetin, were assessed in PBS within 5 s. Apigenin proved very stable in PBS and was therefore used as a control. Kaempferol and baicalein were less stable with small amounts of oxidized and hydroxylated products being detected. The other flavonoids were unstable and their dimers were identified in PBS at 4 °C under normal atmospheric conditions. Flavonols with a catechol or pyrogallol substitution pattern on ring B readily formed stable dimers and oxidized products in PBS (pH = 7.4) at 4 °C within 5 s. The chosen experimental conditions improved the stability of dimers and allowed their detection.

Screening of Inhibitory Effects of Polyphenols on Akt-Phosphorylation in Endothelial Cells and Determination of Structure-Activity Features.

Polyphenols exert beneficial effects in type 2 diabetes mellitus (T2DM). However, their mechanism of action remains largely unknown. Endothelial Akt-kinase plays a key role in the pathogenesis of cardiovascular complications in T2DM and therefore the modulation of its activity is of interest. This work aimed to characterize effects of structurally different polyphenols on Akt-phosphorylation (pAkt) in endothelial cells (Ea.hy926) and to describe structure-activity features. A comprehensive screening via ELISA quantified the effects of 44 polyphenols (10 µM) on pAkt Ser473. The most pronounced inhibitors were luteolin (44 ± 18%), quercetin (36 ± 8%), urolithin A (35 ± 12%), apigenin, fisetin, and resveratrol; (p < 0.01). The results were confirmed by Western blotting and complemented with corresponding experiments in HUVEC cells. A strong positive and statistically significant correlation between the mean inhibitory effects of the tested polyphenols on both Akt-residues Ser473 and Thr308 (r = 0.9478, p = 0.0003) was determined by immunoblotting. Interestingly, the structural characteristics favoring pAkt inhibition partially differed from structural features enhancing the compounds' antioxidant activity. The present study is the first to quantitatively compare the influence of polyphenols from nine different structural subclasses on pAkt in endothelial cells. These effects might be advantageous in certain T2DM-complications involving over-activation of the Akt-pathway. The suggested molecular mode of action of polyphenols involving Akt-inhibition contributes to understanding their effects on the cellular level.

Dried blood spot testing for estimation of renal function and analysis of metformin and sitagliptin concentrations in diabetic patients: a cross-sectional study.

PURPOSE: Dried blot spot (DBS) analysis of drugs or clinical parameters offers many advantages. We investigated the feasibility of using DBS for analysis of anti-diabetic drugs concomitantly with the estimated creatinine clearance (Clcrea). METHODS: The cross-sectional study involved physicians in an enabling analysis with 70 diabetic patients and community pharmacists in a field investigation with 84 participants. All 154 DBS samples were analyzed for creatinine, metformin, and sitagliptin. RESULTS: The diabetic patients revealed of a wide range of age (32-88 years), BMI values (19.8-54.7 kg/m2), and extent of polypharmacotherapy (1-21 drugs). A correlation factor to convert capillary blood creatinine from DBS into plasma concentrations was determined. Patients' Clcrea ranged from 21.6-155.9 mL/min. The results indicated statistically significant correlations (p < 0.05) between the use of two or three particular drug classes (diuretics, NSAIDs, renin-angiotensin system blockers) and a decreased renal function. DBS concentrations of metformin ranged between 0.23-4.99 µg/mL. The estimated elimination half-life (t ½) of metformin was 11.9 h in patients with a ClCrea higher than 60 mL/min and 18.5 h for diabetics with lower ClCrea. Sitagliptin capillary blood concentrations ranged between 11.12-995.6 ng/mL. Calculated t ½ of sitagliptin were 8.4 h and 13.0 h in patients with a ClCrea above and below 60 mL/min, respectively. CONCLUSIONS: DBS allow for the analysis of concentrations of predominantly renally eliminated drugs and community pharmacists can provide a valuable contribution to DBS sampling.

Investigations into neuroprotectivity, stability, and water solubility of 7-O-cinnamoylsilibinin, its hemisuccinate and dehydro derivatives.

Derivatives of the recently described potent neuroprotective 7-O-cinnamoylsilibinin ester were prepared: its hemisuccinate to improve water solubility and the dehydrosilibinin ester that was shown to form in assay media to investigate its role in overall neuroprotective effects. 7-O-Cinnamoyl-2,3-dehydrosilibinin is less neuroprotective than 7-O-cinnamoylsilibinin in a murine hippocampal cell line (HT-22) and we conclude that the dehydrosilibinin derivatives are not the actual carriers of neuroprotective properties, at least in the assay applied. Solubility of the test compounds was determined in shake-flask experiments and the ester's solubility was greatly improved by introduction of a hemisuccinate at the 23-position of silibinin. Time-stability curves in assay media were recorded. The hemisuccinate ester did not act as a prodrug to release 7-O-cinnamoylsilibinin but is the second ester bond to be cleaved. Nevertheless, it still exhibits significant neuroprotection. Therefore, its greatly increased solubility might effectively counterbalance lower in vitro neuroprotection.

Regioselective synthesis of 7-O-esters of the flavonolignan silibinin and SARs lead to compounds with overadditive neuroprotective effects.

A series of neuroprotective hybrid compounds was synthesized by conjugation of the flavonolignan silibinin with natural phenolic acids, such as ferulic, cinnamic and syringic acid. Selective 7-O-esterfication without protection groups was achieved by applying the respective acyl chlorides. Sixteen compounds were obtained and SARs were established by evaluating antioxidative properties in the physicochemical FRAP assay, as well as in a cell-based neuroprotection assay using murine hippocampal HT-22 cells. Despite weak activities in the FRAP assay, esters of the α,ß-unsaturated acids showed pronounced overadditive effects at low concentrations greatly exceeding the effects of equimolar mixtures of silibinin and the respective acids in the neuroprotection assay. Cinnamic and ferulic acid esters (5a and 6a) also showed overadditive effects regarding inhibition of microglial activation, PC12 cell differentiation, in vitro ischemia as well as anti-aggregating abilities against Aß42 peptide and τ protein. Remarkably, the esters of ferulic acid with silybin A and silybin B (11a and 11b) showed a moderate but significant difference in both neuroprotection and in their anti-aggregating capacities. The results demonstrate that non-toxic natural antioxidants can be regioselectively connected as esters with medium-term stability exhibiting very pronounced overadditive effects in a portfolio of biological assays.

Cellular pharmacodynamic effects of Pycnogenol® in patients with severe osteoarthritis: a randomized controlled pilot study.

BACKGROUND: The standardized maritime pine bark extract (Pycnogenol®) has previously shown symptom alleviating effects in patients suffering from moderate forms of knee osteoarthritis (OA). The cellular mechanisms for this positive impact are so far unknown. The purpose of the present randomized pilot controlled study was to span the knowledge gap between the reported clinical effects of Pycnogenol® and its in vivo mechanism of action in OA patients. METHODS: Thirty three patients with severe OA scheduled for a knee arthroplasty either received 100 mg of Pycnogenol® twice daily or no treatment (control group) three weeks before surgery. Cartilage, synovial fluid and serum samples were collected during surgical intervention. Relative gene expression of cartilage homeostasis markers were analyzed in the patients' chondrocytes. Inflammatory and cartilage metabolism mediators were investigated in serum and synovial fluid samples. RESULTS: The oral intake of Pycnogenol® downregulated the gene expression of various cartilage degradation markers in the patients' chondrocytes, the decrease of MMP3, MMP13 and the pro-inflammatory cytokine IL1B were statistically significant (p ≤ 0.05). Additionally, protein concentrations of ADAMTS-5 in serum were reduced significantly (p ≤ 0.05) after three weeks intake of the pine bark extract. CONCLUSIONS: This is the first report about positive cellular effects of a dietary supplement on key catabolic and inflammatory markers in patients with severe OA. The results provide a rational basis for understanding previously reported clinical effects of Pycnogenol® on symptom scores of patients suffering from OA. TRIAL REGISTRATION: ISRCTN10754119 . Retrospectively registered 08/10/2015.

Distribution of Constituents and Metabolites of Maritime Pine Bark Extract (Pycnogenol®) into Serum, Blood Cells, and Synovial Fluid of Patients with Severe Osteoarthritis: A Randomized Controlled Trial.

The present randomized controlled study aimed to investigate the in vivo distribution of constituents or metabolites of the standardized maritime pine bark extract Pycnogenol®. Thirty-three patients with severe osteoarthritis scheduled for a knee arthroplasty were randomized to receive either 200 mg per day Pycnogenol® (P+) or no treatment (Co) over three weeks before surgery. Serum, blood cells, and synovial fluid samples were analyzed using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization (LC-ESI/MS/MS). Considerable interindividual differences were observed indicating pronounced variability of the polyphenol pharmacokinetics. Notably, the highest polyphenol concentrations were not detected in serum. Catechin and taxifolin primarily resided within the blood cells while the microbial catechin metabolite δ-(3,4-dihydroxy-phenyl)-γ-valerolactone, ferulic, and caffeic acid were mainly present in synovial fluid samples. Taxifolin was detected in serum and synovial fluid exclusively in the P+ group. Likewise, no ferulic acid was found in serum samples of the Co group. Calculating ratios of analyte distribution in individual patients revealed a simultaneous presence of some polyphenols in serum, blood cells, and/or synovial fluid only in the P+ group. This is the first evidence that polyphenols distribute into the synovial fluid of patients with osteoarthritis which supports rationalizing the results of clinical efficacy studies.

Analysis of metformin, sitagliptin and creatinine in human dried blood spots.

For analysis of the anti-diabetic drugs metformin and sitagliptin and the renal clearance marker creatinine in the same human dried blood spot (DBS) extract two liquid chromatography methods employing HPLC/UV and LC-ESI-MS/MS have been developed and validated. An accurate volume of 40µL blood was spotted on a sampling paper which was extracted using 90% acetonitrile with 10% formic acid. The new methods were shown to be selective, accurate and precise. The validated ranges were 0.2-5µg/mL for metformin, 1.5-15µg/mL for creatinine and 3-500ng/mL for sitagliptin. Since drug analysis in DBS determines whole blood concentrations as opposed to the typically used plasma levels the partition ratios between human plasma and blood cells, c(P)/c(BC), were elucidated in vitro to gain insight into the significance of blood cells as compartment of distribution for both compounds. The c(P)/c(BC) was found to be 4.65±0.73 for metformin and 5.58±0.98 for sitagliptin. While an accumulation of metformin in erythrocytes was already known we now present the first evidence that sitagliptin distributes into human blood cells. The analytical methods were also successfully applied to authentic capillary blood samples from two diabetic patients regularly taking a combination of metformin and sitagliptin. Both samples revealed analyte trough concentrations well above the lower limit of quantification of the respective compounds. Therefore, the present study offers a methodological basis for the DBS analysis of metformin and sitagliptin in relation to the patients' creatinine concentration.

Profiling a gut microbiota-generated catechin metabolite's fate in human blood cells using a metabolomic approach.

The microbial catechin metabolite δ-(3,4-dihydroxy-phenyl)-γ-valerolactone (M1) has been found in human plasma samples after intake of maritime pine bark extract (Pycnogenol). M1 has been previously shown to accumulate in endothelial and blood cells in vitro after facilitated uptake and to exhibit anti-inflammatory activity. The purpose of the present research approach was to systematically and comprehensively analyze the metabolism of M1 in human blood cells in vitro and in vivo. A metabolomic approach that had been successfully applied for drug metabolite profiling was chosen to detect 19 metabolite peaks of M1 which were subsequently further analyzed and validated. The metabolites were categorized into three levels of identification according to the Metabolomics Standards Initiative with six compounds each confirmed at levels 1 and 2 and seven putative metabolites at level 3. The predominant metabolites were glutathione conjugates which were rapidly formed and revealed prolonged presence within the cells. Although a formation of an intracellular conjugate of M1 and glutathione (M1-GSH) was already known two GSH conjugate isomers, M1-S-GSH and M1-N-GSH were observed in the current study. Additionally detected organosulfur metabolites were conjugates with oxidized glutathione and cysteine. Other biotransformation products constituted the open-chained ester form of M1 and a methylated M1. Six of the metabolites determined in in vitro assays were also detected in blood cells in vivo after ingestion of the pine bark extract by two volunteers. The present study provides the first evidence that multiple and structurally heterogeneous polyphenol metabolites can be generated in human blood cells. The bioactivity of the M1 metabolites and their contribution to the previously determined anti-inflammatory effects of M1 now need to be elucidated.

Stability of dietary polyphenols under the cell culture conditions: avoiding erroneous conclusions.

Most data of bioactivity from dietary polyphenols have been derived from in vitro cell culture experiments. In this context, little attention is paid to potential artifacts due to chemical instability of these natural antioxidants. An early degradation time ((C)T10) and half-degradation time ((C)T50) were defined to characterize the stability of 53 natural antioxidants incubated in Dulbecco's modified Eagle's medium (DMEM) at 37 °C. The degree of hydroxylation of flavones and flavonols significantly influenced the stability in order resorcinol-type > catechol-type > pyrogallol-type, with the pyrogallol-type being least stable. In contrast, any glycosylation of polyphenols obviously enhanced their stability. However, the glycosylation was less important compared to the substitution pattern of the nucleus rings. Methoxylation of flavonoids with more than three hydroxyl groups typically improved their stability as did the hydrogenation of the C2═C3 double bond of flavonoids to corresponding flavanoids. There was no significant correlation between the antioxidant potential of polyphenols and their stability. Notably, the polyphenols were clearly more stable in human plasma than in DMEM, which may be caused by polyphenol-protein interactions. It is strongly suggested to carry out stability tests in parallel with cell culture experiments for dietary antioxidants with catechol or pyrogallol structures and pyrogallol-type glycosides in order to avoid artifacts.

Highly sensitive analysis of polyphenols and their metabolites in human blood cells using dispersive SPE extraction and LC-MS/MS.

Blood cells, particularly erythrocytes, present a significant compartment for distribution of drugs and endogenous compounds and have been suggested to be factored in pharmacokinetic and pharmacodynamic evaluations. We previously detected the binding of polyphenols to red blood cells and found indications for a facilitated uptake of the bioactive procyanidin metabolite δ-(3,4-dihydroxy-phenyl)-γ-valerolactone (M1) into human erythrocytes. The purpose of the present investigation was to develop an effective, sensitive and robust liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify low concentrations of polyphenols in human blood cells. Various sample preparation methods including classic sample clean-up techniques and variations of the QuEChERS (quick, easy, cheap, effective, rugged and safe) approach were compared regarding compound recovery, matrix effects and overall process efficiency. The QuEChERS technique which involves a liquid-liquid extraction and clean-up by dispersive solid-phase extraction yielded best results. The method was fully validated for the six analytes: (+)-catechin, ferulic acid, M1, taxifolin, caffeic acid and δ-3-methoxy-4-hydroxy-phenyl- γ-valerolactone (M2) in human blood cells with an optimised QuEChERS sample preparation and prior enzymatic hydrolysis of analyte conjugates. The lower limits of quantification for the analytes ranged from 0.12 ng/mL for M1, M2 and taxifolin to 48.40 ng/mL for caffeic acid. The application of the method to a blood cell sample of a volunteer ingesting 100 mg/day of the standardised pine bark extract Pycnogenol(®) over the course of 3 weeks revealed measurable steady-state concentrations of catechin, M1, taxifolin, ferulic acid and M2. To our knowledge, this is the first report of using the QuEChERS approach for detection and quantification of plant-derived compounds in human blood cells. The method can be applied in pharmacokinetic studies to determine the distribution of polyphenols and their metabolites in human whole blood, blood cells or erythrocytes. This might contribute in gaining deeper insights into the in vivo distribution of polyphenols and their metabolites.

Suppression of cytokine release by fluticasone furoate vs. mometasone furoate in human nasal tissue ex-vivo.

BACKGROUND: Topical glucocorticosteroids are the first line therapy for airway inflammation. Modern compounds with higher efficacy have been developed, but head-to-head comparison studies are sparse. OBJECTIVE: To compare the activity of two intranasal glucocorticoids, fluticasone furoate (FF) and mometasone furoate (MF) with respect to the inhibition of T helper (Th)1, Th2 and Th17 cytokine release in airway mucosa. METHODS: We used an ex-vivo human nasal mucosal tissue model and employed pre- and post- Staphylococcus aureus enterotoxin B (SEB)-challenge incubations with various time intervals and drug concentrations to mimic typical clinical situations of preventive or therapeutic use. RESULTS: At a fixed concentration of 10-10 M, FF had significantly higher suppressive effects on interferon (IFN)-γ, interleukin (IL)-2 and IL-17 release, but not IL-5 or tumor necrosis factor (TNF)-α, vs. MF. While the maximal suppressive activity was maintained when FF was added before or after tissue stimulation, the cytokine suppression capacity of MF appeared to be compromised when SEB-induced cell activation preceded the addition of the drug. In a pre-challenge incubation setting with removal of excess drug concentrations, MF approached inhibition of IL-5 and TNF-α after 6 and 24 hours while FF maximally blocked the release of these cytokines right after pre-incubation. Furthermore, FF suppressed a wider range of T helper cytokines compared to MF. CONCLUSION: The study demonstrates the potential of our human mucosal model and shows marked differences in the ability to suppress the release of various cytokines in pre- and post-challenge settings between FF and MF mimicking typical clinical situations of preventive or therapeutic use.

Influence of diabetes on the pharmacokinetic behavior of natural polyphenols.

The development of food fortified with polyphenols and polyphenol-rich foods represents a novel approach to prevent or attenuate type 2 diabetes. It has been reported that type 2 diabetes may affect the pharmacokinetics of various drugs in several animal models. There is powerful evidence linking dietary polyphenols consumption with the risk factors defining type 2 diabetes, even if some opposite results occurred. This mini-review summarizes important advances on diabetes-associated changes in pharmacokinetics of natural polyphenols. The pharmacokinetic behavior between drugs and dietary polyphenols probably may be different due to (i) Ingested dose/amount per day. The dietary polyphenol intake per day is much higher than that of clinical drugs; (ii) Complexity of the components. Clinical drugs are well-characterized and typically small molecules. However, the polyphenols in diet are unimaginably complex; (iii) Interaction with food proteins. Although the effects of food proteins on the bioavailability of polyphenols are still not examined in much detail, direct binding interactions of polyphenols to proteins always occur; (iv) The most common polyphenols in the human diet have a low intrinsic activity and are poorly absorbed from the intestine, highly metabolized, or rapidly eliminated. Although there is very limited information available so far, it is proposed that type 2 diabetes influences the pharmacokinetic behavior of dietary polyphenols including: i) competition of glucose with polyphenols regarding binding to plasma proteins; ii) weakened non-covalent interaction affinities of plasma proteins for natural polyphenols due to protein glycation in type II diabetes; iii) the enhanced clearance of polyphenols in type 2 diabetes. An understanding of diabetes-associated changes in absorption, distribution, metabolism, elimination and bioactivities of natural polyphenols as well as the mechanism of the variability should lead to the improvement of the benefits of these polyphenols and clinical outcomes for diabetics.