Global testing of a consensus solubility assessment to enhance robustness of the WHO biopharmaceutical classification system.

The WHO Biopharmaceutical Classification System (BCS) is a practical tool to identify active pharmaceutical ingredients (APIs) that scientifically qualify for a waiver of in vivo bioequivalence studies. The focus of this study was to engage a global network of laboratories to experimentally quantify the pH-dependent solubility of the highest therapeutic dose of 16 APIs using a harmonized protocol. Intra-laboratory variability was ≤5 %, and no apparent association of inter-laboratory variability with API solubility was discovered. Final classification "low solubility" vs "high solubility" was consistent among laboratories. In comparison to the literature-based provisional 2006 WHO BCS classification, three compounds were re-classified from "high" to "low-solubility". To estimate the consequences of these experimental solubility results on BCS classification, dose-adjusted in silico predictions of the fraction absorbed in humans were performed using GastroPlus®. Further expansion of these experimental efforts to qualified APIs from the WHO Essential Medicines List is anticipated to empower regulatory authorities across the globe to issue scientifically-supported guidance regarding the necessity of performing in vivo bioequivalence studies. Ultimately, this will improve access to affordable generic products, which is a critical prerequisite to reach Universal Health Coverage.

Biomimetic Artificial Membrane Permeability Assay over Franz Cell Apparatus Using BCS Model Drugs.

A major parameter controlling the extent and rate of oral drug absorption is permeability through the lipid bilayer of intestinal epithelial cells. Here, a biomimetic artificial membrane permeability assay (Franz-PAMPA Pampa) was validated using a Franz cells apparatus. Both high and low permeability drugs (metoprolol and mannitol, respectively) were used as external standards. Biomimetic properties of Franz-PAMPA were also characterized by electron paramagnetic resonance spectroscopy (EPR). Moreover, the permeation profile for eight Biopharmaceutic Classification System (BCS) model drugs cited in the FDA guidance and another six drugs (acyclovir, cimetidine, diclofenac, ibuprofen, piroxicam, and trimethoprim) were measured across Franz-PAMPA. Apparent permeability (Papp) Franz-PAMPA values were correlated with fraction of dose absorbed in humans (Fa%) from the literature. Papp in Caco-2 cells and Corti artificial membrane were likewise compared to Fa% to assess Franz-PAMPA performance. Mannitol and metoprolol Papp values across Franz-PAMPA were lower (3.20 × 10-7 and 1.61 × 10-5 cm/s, respectively) than those obtained across non-impregnated membrane (2.27 × 10-5 and 2.55 × 10-5 cm/s, respectively), confirming lipidic barrier resistivity. Performance of the Franz cell permeation apparatus using an artificial membrane showed acceptable log-linear correlation (R2 = 0.664) with Fa%, as seen for Papp in Caco-2 cells (R2 = 0.805). Data support the validation of the Franz-PAMPA method for use during the drug discovery process.

Crude plant extract versus single compounds for vitiligo treatment: Ex vivo intestinal permeability assessment on Brosimum gaudichaudii Trécul.

Roots´ bark extract of Brosimum gaudichaudii Trécul (EBGT) is traditionally used for photochemotherapy of vitiligo due to the presence of furanocoumarins psoralen (PSO) and 5-methoxypsoralen (5-MOP) as major compounds. Though plant extracts may provide additional highly permeable psoralens-like substances which may act synergically on vitiligo's therapy. Thus, the aim of this work was to develop an LC-MS/MS method for screening new highly permeable furanocoumarins from B. gaudichaudii and to compare biomarkers permeability and solubility provided as single compounds or as crude extract, according to BCS. An optimized LC-MS/MS method showed twelve permeable and bioactive compounds, among which 9 furanocoumarins, 2 pyranocoumarins and 1 dihydrocinnamic acid derivative were detected in EBGT samples. Solubility of PSO and 5-MOP was found to be, respectively, six- and eleven-fold higher in crude extract than as pure compounds. Permeability (Papp) of PSO and 5-MOP in EBGT were higher than metoprolol, the low/high BCS permeability class boundary reference compound. Hence, both biomarkers were considered as highly permeable (BCS2) compounds. Their permeability were concentration-dependent displaying values from 30.26 ± 5.13-8.21 ± 2.16 × 10-6 cm/s and 10.72 ± 1.73-6.07 ± 1.27 × 10-6 cm/s, respectively, over a wide range (2.3-200.0 mg mL-1). Thus, a carrier-mediated absorption process is suggested as the main mechanism. Accordingly, all additional permeated coumarins, identified by LC-MS/MS, showed to be at comparable amount of biomarkers in the permeated samples inferring similar high permeability rate. Moreover, biomarkers and other highly absorbable and bioactive linear furanocoumarins from EBGT may be used for vitiligo´s photochemotherapy. Taken together, these findings bring additional evidences for using crude plant extract when aiming synergistic effects of bioactive compounds on melanogenic therapies.

Effects of turmeric extract supplementation on inflammation and muscle damage after a half-marathon race: a randomized, double-blind, placebo-controlled trial.

PURPOSE: Strenuous exercise induces inflammation and muscle damage. Turmeric (Curcuma longa L.) is a widely used spice that exhibits potent anti-inflammatory response and appears to decrease indirect markers of muscle damage. A randomized, double-blind, placebo-controlled trial was conducted to evaluate the effects of Curcuma longa L. extract (CLE) on inflammation and muscle damage after a half-marathon race. METHODS: Twenty-eight healthy, normal-weight men were randomly assigned to one of two groups: (1) CLE (3 capsules per day, 500 mg each); or (2) placebo (PLA, 3 capsules per day, 500 mg of microcrystalline cellulose). Participants received the intervention for 4 weeks and immediately before and after the half-marathon race. Creatine kinase, lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, myoglobin, interleukins 6 and 10 were assessed at baseline, immediately before, after, and at 2, 24, and 48 h after the half-marathon race. RESULTS: The half-marathon race increased markers of inflammation and muscle damage. A greater increase in interleukin-10 was observed in the CLE group immediately after the competition compared to the PLA group (7.54 ± 1.45 vs 5.25 ± 0.59 pg/mL; p < 0.05; d = 0.55). Myoglobin concentration was lower 2 h after the race in participants from the CLE group compared to the PLA group (62.10 ± 8.26 vs 107.85 ± 18.45 ng/mL; p = 0.01; d = 0.86). CONCLUSION: Curcuma longa L. extract supplementation leads to an increase in IL-10 and decreased myoglobin in recreational male runners after a half-marathon race. TRIAL REGISTRATION NUMBER: U1111-1179-6335, February 13, 2016.

Bioanalytical method by HPLC-FLD for curcumin analysis in supplemented athletes.

In sports, curcumin, a substance derived from the rhizome of Curcuma longa (turmeric) plant with antioxidant effect 8 times greater than vitamin E, has attracted the attention of scientists because of its potent antioxidant action, since in athletes subjected to intense exercise the-endogenous mechanisms of neutralization of reactive species are saturated. However, the pharmacokinetic characteristics of curcumin do not favor its medicinal use due to its low absorption, accelerated metabolism and rapid systemic elimination. Thus, the determination of plasma levels in supplemented patients is a crucial step in their pharmacodynamic evaluation. Therefore, the objective of this work was to develop and validate an analytical method by HPLC-FLD for curcumin evaluation in plasma of supplemented athletes. Luna column (C18; 150 × 4 mm; 3 µm), acetonitrile: acetic acid pH 3.2 (45:55 to 60:40) as mobile phase, flow rate of 1 mL min-1, excitation at 429/285 nm and emission at 529 nm and injection of 10 µL were the chromatographic conditions used. Plasma samples were extracted using ethylacetate and methanol (95: 5, 500 µL) and estradiol (30 µg mL-1) as internal standard, with subsequent stirring (3 min) and centrifugation (8 min) (triple extraction). The organic fraction was evaporated under N2 (20 min) and the dried residue reconstituted in acetonitrile. The method was linear between 44 and 261 ng mL-1, showing intra-day (2.05.6%) and inter-day (4.0-5.1%) precision with accuracy and selectiveness (curcumin tR = 8.7 min and internal standard tR = 13.9 min with relative recovery of 83.2%). So, it can be successfully used for curcumin evaluation in plasma samples from supplemented athletes, as well as being an alternative and advantageous method to UV-Vis and MS/MS in bioavailability studies.

Electroanalysis Applied to Compatibility and Stability Assays of Drugs: Carvedilol Study Case.

Carvedilol (CRV) is a non-selective blocker of α and ß adrenergic receptors, which has been extensively used for the treatment of hypertension and congestive heart failure. Owing to its poor biopharmaceutical properties, CRV has been incorporated into different types of drug delivery systems and this necessitates the importance of investigating their compatibility and stability. In this sense, we have investigated the applicability of several electroanalytical tools to assess CRV compatibility with lipid excipients. Voltammetric and electrochemical impedance spectroscopy techniques were used to evaluate the redox behavior of CRV and lipid excipients. Results showed that Plurol® isostearic, liquid excipient, and stearic acid presented the greatest anode peak potential variation, and these were considered suitable excipients for CRV formulation. CRV showed the highest stability at room temperature and at 50 °C when mixed with stearic acid (7% w/w). The results also provided evidence that electrochemical methods might be feasible to complement standard stability/compatibility studies related to redox reactions.

4-Nerolidylcatechol: apoptosis by mitochondrial mechanisms with reduction in cyclin D1 at G0/G1 stage of the chronic myelogenous K562 cell line.

CONTEXT: 4-Nerolidylcatechol (4-NRC) has showed antitumor potential through apoptosis. However, its apoptotic mechanisms are still unclear, especially in leukemic cells. OBJECTIVES: To evaluate the cytotoxic potential of 4-NRC and its cell death pathways in p53-null K562 leukemic cells. MATERIALS AND METHODS: Cytotoxicity of 4-NRC (4.17-534.5 µM) over 24 h of exposure was evaluated by MTT assay. 4-NRC-induced apoptosis in K562 cells was investigated by phosphatidylserine (PS) externalization, cell cycle, sub-G1, mitochondrial evaluation, cytochrome c, cyclin D1 and intracellular reactive oxygen species (ROS) levels, and caspase activity analysis. RESULTS: IC50 values obtained were 11.40, 27.31, 15.93 and 15.70 µM for lymphocytes, K562, HL-60 and Jurkat cells, respectively. In K562 cells, 4-NRC (27 µM) promoted apoptosis as verified by cellular morphological changes, a significant increase in PS externalization and sub-G1 cells. Moreover, it significantly arrested the cells at the G0/G1 phase due to a reduction in cyclin D1 expression. These effects of 4-NRC also significantly promoted a reduction in mitochondrial activity and membrane depolarization, accumulation of cytosolic cytochrome c and ROS overproduction. Additionally, it triggered an increase in caspases -3/7, -8 and -9 activities. When the cells were pretreated with N-acetyl-l-cysteine ROS scavenger, 4-NRC-induced apoptosis was partially blocked, which suggests that it exerts cytotoxicity though not exclusively through ROS-mediated mechanisms. DISCUSSION AND CONCLUSION: 4-NRC has antileukemic properties, inducing apoptosis mediated by mitochondrial-dependent mechanisms with cyclin D1 inhibition. Given that emerging treatment concepts include novel combinations of well-known agents, 4-NRC could offer a promising alternative for chemotherapeutic combinations to maximize tumour suppression.

4-Nerolidylcatechol analogues as promising anticancer agents.

4-Nerolidylcatechol (1) is an isolated compound from Pothomorphe umbellata L. (Piperaceae) with promising antitumor cells properties. However it presents lability under light and room temperatures. Many efforts have been directed towards discovering anticancer agents endowed with cytotoxic activities. Here, we evaluated cytotoxic effects of 4-NRC analogues (LQFMs 2-6) and the cell death pathways induced by these compounds in multidrug-resistant K562 cells. Compounds (2-6) exhibited cytotoxic activities in a concentration-dependent manner against leukaemic cells, specially the compounds (3) and (5). Additionally, compounds (1), (3) and (5) promoted marked alterations on the cell morphology, including nuclear changes as demonstrated by Hoescht 33342 staining. Moreover, these compounds promoted apoptosis induction in K562 cells by phosphatidylserine exposure, increase of sub-G1 cells and modulation of the caspases-3/7, -8 and -9 activation. In addition, the pancaspase inhibitor z-VAD-fmk partially reduced the apoptosis induced by the compounds (1) and (5)-induced, suggesting caspase-dependent and caspase-independent cell death pathways. Compounds (1) and (5) also modified the cell cycle progression by G0/G1 and S arrest, respectively. Furthermore, compounds (1), (3) and (5) promoted mitochondrial dysfunction associated to accumulation of cytosolic cytochrome c and modulated the NF-ĸB activation. In addition, unlike their analogues, 4-NRC (1) also promoted a significant cyclin D1 inhibition. Together, these data suggest that the mechanism of cell death of 4-NRC and its analogues (3) and (5) occurs by apoptosis through mitochondrial mechanisms. Considering that LQFMs are biocompatible synthetic analogues produced by molecular simplification of (1) without the chiral centre, which is associated with the instability found in compound (1), we suggest that these compounds are promising candidates for further pre-clinical studies.

A Sensitive Medium-Throughput Method to Predict Intestinal Absorption in Humans Using Rat Intestinal Tissue Segments.

A range of in vitro, ex vivo, and in vivo approaches are currently used for drug development. Highly predictive human intestinal absorption models remain lagging behind the times because of numerous variables concerning permeability through gastrointestinal tract in humans. However, there is a clear need for a drug permeability model early in the drug development process that can balance the requirements for high throughput and effective predictive potential. The present study developed a medium throughput screening Snapwell (MTS-Snapwell) ex vivo model to provide an alternative method to classify drug permeability. Rat small intestine tissue segments were mounted in commercial Snapwell™ inserts. Unidirectional drug transport (A-B) was measured by collecting samples at different time points. Viability of intestinal tissue segments was measured by examining transepithelial electric resistance (TEER) and phenol red and caffeine transport. As a result, the apparent permeability (Papp; ×10(-6) cm/s) was determined for atenolol (10.7 ± 1.2), caffeine (17.6 ± 3.1), cimetidine (6.9 ± 0.1), metoprolol (12.6 ± 0.7), theophylline (15.3 ± 1.6) and, ranitidine (3.8 ± 0.4). All drugs were classified in high/low permeability according to Biopharmaceutics Classification System showing high correlation with human data (r = 0.89). These findings showed a high correlation with human data (r = 0.89), suggesting that this model has potential predictive capacity for paracellular and transcellular passively absorbed molecules.

4-Nerolidylcatechol and its synthetic analogues: antioxidant activity and toxicity evaluation.

4-Nerolidylcatechol (1) is a secondary metabolite of plants and is described as a promising anti-inflammatory, antimalarial, antiulcerogenic, analgesic and cytotoxic compound possibly due to its antioxidant profile. In this study, we evaluated the pharmacologic activity and the antioxidant and toxicological profiles of compound (1) and its synthetic analogues (2-6). The synthetic analogues were designed from the lead compound, (1), using a molecular-simplification strategy. Compound 5 showed, by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ß-carotene systems, similar antioxidant activity when compared to compound (1). The oxidative stress in erythrocyte membrane demonstrated the highly protective effect of compounds (4), (5) and (6) and high antioxidant/pro-oxidant activity in relation to the concentrations of compounds (1) and (3). Compounds (2), (4), (5) and (6) were haemobiocompatible. All compounds (1-6) showed cytotoxic effects in 3T3 cells, but compounds (2) and (6) were highly cytotoxic in this lineage when compared to compound (1). Compound (5) had a lower myelosuppressive effect in haematopoietic progenitor cells compared to (1). Both compounds, (1) and (5), showed low genotoxic effects in vitro, on human lymphocyte cells. In addition, these compounds also showed low-toxicity in vivo as defined a LD50 > 2000 mg/kg. In this assay, we did not observe death in the animals exposed to treatment with (1) and (5) compound. In conclusion, the structural design of the analogues as validated once compound (5) was found to have an antioxidant profile that was as potent as the lead compound (1). In addition, considering the safety profile, these compounds are promising as preventive and/or therapeutic agents against oxidative damage.

Anti-inflammatory effect of (E)-4-(3,7-dimethylocta-2,6-dienylamino)phenol, a new derivative of 4-nerolidylcatechol.

OBJECTIVES: We have investigated the anti-inflammatory and antinociceptive effects of (E)-4-(3,7-dimethylocta-2,6-dienylamino)phenol (LQFM-015), which was designed through molecular simplification strategy from 4-nerolidylcatechol. METHODS: The possible anti-inflammatory and antinociceptive effects were assayed on carrageenan-induced paw oedema and pleurisy, acetic acid-induced abdominal writhing and formalin tests in mice. KEY FINDINGS: LQFM-015 reduced the activity of PLA2 enzyme in vitro by 18%. Docking studies into the catalytic site of PLA2 were used to identify the binding mode of the LQFM-015. LQFM-015 showed a moderate antinociceptive effect, since this compound reduced the number of writhings by approximately up to 40% in the acetic acid-induced pain model; this antinociceptive activity also emerged in the second phase of the formalin-induced pain model (58% of inhibition). The anti-inflammatory action of LQFM-015 was confirmed in acute inflammation models, in which it reduced the formation of oedema to 52.78 ± 8.6 and 46.64 ± 5.2 at the second and third hour of carrageenan-induced paw oedema, respectively. Also in the carrageenan-induced pleurisy model, LQFM-015 reduced the migration of leucocytes by 26.0% and decrease myeloperoxidase activity by 50%. LQFM-015 showed different concentrations to inhibit 50% of isoenzyme cyclooxygenase activity (IC50); COX-1 IC50 = 36 µM) and COX-2 IC50 = 28 µM. CONCLUSIONS: LQFM-015 demonstrated inhibition of both PLA2 and COX enzymes; thus, the moderate antinociceptive effect of this compound could be attributed to its anti-inflammatory activity.

Quantitation of glucosamine sulfate in plasma by HPLC-MS/MS after administration of powder for oral solution formulation.

A rapid method for the quantification of glucosamine in human plasma using high-performance liquid chromatography coupled to tandem mass spectrometry was developed and validated. The sample preparation includes a simple deproteinization step, using D-[1-¹³C] glucosamine hydrochloride as an internal standard. Chromatographic separation was performed on an ACE Ciano column using isocratic elution with acetonitrile and aqueous 2 mM ammonium acetate containing 0.025% formic acid (80:20). Selected reaction monitoring was performed using the transitions m/z 180.1 → m/z 72.1 and m/z 181.0 → m/z 74.6 to quantify glucosamine and internal standard, respectively. The method was validated and proved to be linear, accurate and precise over the range 50-5000 ng/mL of glucosamine. Recovery rates higher than 90% were obtained for both glucosamine and internal standard. No matrix effect was detected in the samples. The validated method was successfully applied to a pharmacokinetic study after oral administration of a powder for oral solution formulation containing glucosamine sulfate.

Quantification of 4-nerolidylcatechol from Pothomorphe umbellata (Piperaceae) in rat plasma samples by HPLC-UV

The oral dosages formulations from Pothomorphe umbellata (Piperaceae) can only be therapeutically evaluated after establishing 4-nerolidylcatechol (4-NRC) pharmacokinetic profile in plasma. For that purpose, a unique analytical method validation using HPLC-UV detection was developed for analysis of rat plasma samples. The animals received 10 mg/kg b.w. of 4-NRC i.v.. Analytical conditions were set with C18 column, methanol: acetonitrile: water (62:20:18) as mobile phase, and a flow rate of 1.6 mL/min. The assay was linear from 1.0 - 80.0 mcg/mL. The recovery procedure was performed by liquid-liquid extraction showing quantitative extraction (106.4 ± 8.7 por cento)...

Biodisponibilidade oral do 4-nerolidilcatecol isolado e em extrato bruto de Pothomorphe umbellata (L) Miq administrado a ratos Sprague Dawley / 4-Nerolidylcatechol isolated and in Pothomorphe umbellata raw extract oral bioavailability administrated to Sprague Dawley rats

O 4-nerolidilcatecol é o metabólito secundário mais abundante do extrato hidroalcóolico liofilizado de raízes de Pothomorphe umbellata (Piperaceae), representando 21,5 porcento deste. Doses únicas de 10 mg/kg do composto isolado foram administradas via intravascular e oral a ratos Sprague Dawley, pesando entre 300-325 g. Paralelamente à obtenção dos parâmetros farmacocinéticos, realizou-se a avaliação da biodisponibilidade do extrato de raízes com a administração de dose oral única de 100 mg/kg. A quantificação das concentrações plasmáticas foi realizada por CLAE-EM/EM, com limite de quantificação de 2,5 ng/mL. O desvio padrão da precisão interensaio e intraensaio variou entre 2,9-10,1 porcento e 4,2-16,3 porcento...