Opioid-Induced Reductions in Gait Variability in Healthy Volunteers and Individuals with Knee Osteoarthritis.

OBJECTIVE: To investigate differences in gait variability induced by two different single-dose opioid formulations and an inert placebo in healthy volunteers and knee osteoarthritis patients. DESIGN: Experimental, randomized, double-blinded, crossover study of inert placebo (calcium tablets), 50 mg of tapentadol, and 100 mg of tramadol. SETTING: Laboratory setting. SUBJECTS: Healthy volunteers and knee osteoarthritis patients. METHODS: At three visits, separated by seven days, one tablet was administered per visit according to the randomization code. At each visit, a baseline measurement was done before tablet administration, after which hourly measurements were performed for six hours, yielding a total of seven measurements per visit. Gait variability was measured by three-dimensional gait analysis, recorded during six minutes of continuous treadmill walking at self-selected speed. One hundred seventy gait cycles were identified from detection of clear events of the knee joint angle trajectories. Gait variability was assessed as average standard deviations over a gait cycle of the sacrum displacements and accelerations; sagittal plane ankle, knee, and hip joint angles; step widths; and stride times. RESULTS: Twenty-four opioid-naïve and neurologically intact participants (12 healthy volunteers and 12 knee osteoarthritis patients) were included and completed the experiment. Tapentadol reduced the variability of sacrum displacements and accelerations compared with placebo and tramadol. There were no differences between experimental conditions regarding the variability in lower-extremity joint angle variability, step widths, or stride times. CONCLUSIONS: In opioid-naïve and neurologically intact individuals, tapentadol seems to reduce movement variability during treadmill walking, compared with placebo and tramadol. This can be interpreted as a loss of adaptability that might increase the risk of falling if the system is perturbed.

Investigation of factors affecting the stability of lysozyme spray dried from ethanol-water solutions.

Formulation composition and processing conditions can be adjusted to enhance the structural integrity as well as the bioactivity of proteins in the spray drying process. In this study, lysozyme was chosen as a model pharmaceutical protein to study these aspects when spray drying from water-ethanol mixtures. The effect of formulation additives (trehalose, Tween 20 and phosphate-buffered saline) and processing conditions (inlet temperature and storage time of lysozyme in the feed solution before the spray drying process) on the protein bioactivity was investigated. The results showed that the bioactivities of spray dried lysozyme with these additives were about 5-10% higher than that without additives. The bioactivity of the spray dried lysozyme was found to increase with a decrease in the inlet temperature from 130°C to 80°C, with similar findings when shortening the storage time of the feed solutions prior to spray drying. Fourier Transform Infrared (FTIR) and Circular Dichroism (CD) results showed that the native structures of lysozyme were largely restored upon reconstitution of the spray dried powder in water after the spray drying process. This suggests that the bioactivity of lysozyme could be preserved adequately by optimization of both the formulation composition and process conditions even when spray drying from a water-ethanol mixture.

An Optimised Method for Routine Separation and Quantification of Major Alkaloids in Cortex Cinchona by HPLC Coupled with UV and Fluorescence Detection.

INTRODUCTION: Authentication of herbal products to ensure efficacy and safety require efficient separation and quantification of constituents. Standard assays for Cinchona bark used for the treatment of malaria and production of quinine, either use only spectrophotometry to detect two pairs of diastereoisomers of quinine and cinchonine type alkaloids (European Pharmacopoeia, Ph.Eur.) or liquid chromatography primarily optimised for detection of the four major alkaloids. However, numerous minor alkaloids occur in Cinchona and related species and efficient separation including gradient elution is necessary in order to obtain the full pattern of constituents in bark samples. OBJECTIVE: To develop an optimised HPLC method for separation and quantitative analysis of the four major alkaloids in Cinchona bark using UV detection. METHODOLOGY: Dimethyl sulphoxide (DMSO) extracts of 50 mg of pulverised barks were prepared using ultrasonication. The chromatographic separation was performed on an XB-C18 column packed with 2.6 µm particles. Gradient elution using an ammonium formate buffer and methanol as organic modifier over 26 min was based on non-chiral separation of the diastereoisomers and the high solvent selectivity of methanol. Post column UV detection was performed at 250 nm and 330 nm. Fluorescence detection was performed using 330 nm for excitation and 420 nm for emission. RESULTS: The optimised HPLC method facilitates efficient separation and quantification of the four major alkaloids in 26 min with a limit of quantification of 5 µg/g from 50 mg bark sample. CONCLUSION: The optimised HPLC method offers a simple and efficient quantification of the four major alkaloids. Copyright © 2017 John Wiley & Sons, Ltd.

Fractional laser-assisted topical delivery leads to enhanced, accelerated and deeper cutaneous 5-fluorouracil uptake.

BACKGROUND: Topical 5-Fluorouracil (5-FU) exhibits suboptimal efficacy for non-melanoma skin cancer, attributed to insufficient intracutaneous penetration. This study investigates the impact of ablative fractional laser (AFXL) at different laser-channel depths on cutaneous 5-FU pharmacokinetics and biodistribution. METHODS: In vitro porcine skin underwent AFXL-exposure using a fractional 10,600 nm CO2-laser, generating microscopic ablation zones (MAZ) reaching the dermoepidermal junction (MAZ-ED), superficial-(MAZ-DS), or mid-dermis(MAZ-DM). 5-FU in AFXL-exposed and control skin was measured in Franz diffusion cells at 4 and 24 hours (n = 55). HPLC quantified 5-FU in full-thickness skin, specific skin depths of 100µm-1500µm, and transcutaneous receiver-compartments. Qualitative matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) visualized 5-FU in selected samples. RESULTS: Overall, AFXL enhanced and accelerated 5-FU uptake versus unexposed controls, with increased accumulation in deep skin layers (p < 0.01). While total, 24-hour 5-FU uptake in control skin was 0.096 mg/cm3 (0.19% of applied concentration), AFXL delivered up to 4.707 mg/cm3 (MAZ-DM; 9.41% uptake, 49-fold enhancement) (p = 0.002; 24 hours). Indicating accelerated delivery, 5-FU in laser-exposed samples at 4 hours was at least 10-fold that of 24-hour controls (p = 0.002). Deeper laser-channels increased delivery throughout the skin (MAZ-ED vs. MAZ-DM; p<0.01). MALDI-MSI confirmed enhanced, accelerated, deeper and more uniform 5-FU distribution after AFXL versus controls. CONCLUSIONS: AFXL offers laser-channel depth-dependent, enhanced and accelerated 5-FU uptake, with increased deposition in deep skin layers.

Effect of ethanol as a co-solvent on the aerosol performance and stability of spray-dried lysozyme.

In the spray drying process, organic solvents can be added to facilitate drying, accommodate certain functional excipients, and modify the final particle characteristics. In this study, lysozyme was used as a model pharmaceutical protein to study the effect of ethanol as a co-solvent on the stability and aerosol performance of spray-dried protein. Lysozyme was dissolved in solutions with various ratios of ethanol and water, and subsequently spray-dried. A change from spherical particles into wrinkled and folded particles was observed upon increasing the ratio of ethanol in the feed. The aerosol performance of the spray-dried lysozyme from ethanol-water solution was improved compared to that from pure water. The conformation of lysozyme in the ethanol-water solution and spray dried powder was altered, but the native structure of lysozyme was restored upon reconstitution in water after the spray drying process. The enzymatic activities of the spray-dried lysozyme showed no significant impact of ethanol; however, the lysozyme enzymatic activity was ca. 25% lower compared to the starting material. In conclusion, the addition of ethanol as a co-solvent in the spray drying feed for lysozyme did not compromise the conformation of the protein after drying, while it improved the inhaled aerosol performance.

Direct coupling of a flow-flow electromembrane extraction probe to LC-MS.

A fully integrated and automated electromembrane extraction LC-MS (EME-LC-MS) system has been developed and characterized. Hyphenation of a flow-flow EME probe to LC-MS was accomplished by using an in-built 10-port switching valve of the LC-MS system. The 10-port switching valve decoupled the high pressure of the UHPLC-system from the low pressure required for operation of the EME-probe by automated switching between a sample extraction/analysis and a sample load position. In the sample load position the extracted analytes were loaded into a HPLC sample loop. By switching the valve to the sample extraction/analysis position the setup allowed simultaneous analysis of previously loaded analytes while extracting a new sample. Performance of the system was characterized with respect to precision and linearity (RSD < 2.5%, R(2): 0.998) and the setup was applied for studying the in-vitro metabolism of methadone by rat liver microsomes. As the metabolic reaction proceeded, methadone and its metabolites were extracted and analyzed in parallel by LC-MS using either isocratic or gradient elution. Compared to a conventional in-vitro metabolism analysis based on protein precipitation followed by LC-MS analysis the fully automated EME-LC-MS system offers a significant time saving and in addition demonstrates increased sensitivity as the analytes were automatically enriched during the extraction process. The experiment revealed 6 to 16 times higher S/N ratios of the EME-LC-MS method compared to protein precipitation followed by LC-MS and thus concomitantly lower LOD and LOQ. The setup integrates a complete analytical workflow of rapid extraction, enrichment, separation and detection of analytes in a fully automated manner. These attributes make the developed system a powerful alternative approach for a wide range of analytical applications.

Inhibition of ATP synthesis by fenbufen and its conjugated metabolites in rat liver mitochondria.

Fenbufen is an arylpropionic acid derivative belonging to the group of non-steroidal anti-inflammatory drugs (NSAIDs). Even though fenbufen is considered a safe drug, some adverse reactions including hepatic events have been reported. To investigate whether mitochondrial damage could be involved in the drug induced liver injury (DILI) by fenbufen, the inhibitory effect of fenbufen and its conjugated metabolites on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria was investigated. Fenbufen glucuronide (F-GlcA), fenbufen-N-acetyl cysteine-thioester (F-NAC) and fenbufen-S-glutathione thioester (F-SG) were found to be more potent inhibitors compared to parent fenbufen (F), whereas fenbufen-O-carnitine (F-carn), fenbufen-glycine (F-gly) and fenbufen-N-acetyl lysine amide (F-NAL) were less potent compared to fenbufen. Fenbufen-CoA thioester (F-CoA) was equally potent as fenbufen in inhibiting ATP synthesis. Fenbufen showed time and concentration dependent inhibition of ATP synthesis with Kinact of 4.4 min(-1) and KI of 0.88 µM and Kinact/KI ratio of 5.01 min(-1) µM(-1). Data show that fenbufen did not act through opening MPT pore, nor did incubation of mitochondria with reduced GSH and fenbufen show any protective effect on fenbufen mediated inhibition of oxidative phosphorylation. Inclusion of NADPH in mitochondrial preparations with fenbufen did not modulate the inhibitory effects, suggesting no role of CYP mediated oxidative metabolites on the ATP synthesis in isolated mitochondria. The results from the present experiments provide evidence that fenbufen and its metabolites could be involved in mitochondrial toxicity through inhibition of ATP synthesis.

Mitochondrial toxicity of diclofenac and its metabolites via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria: Possible role in drug induced liver injury (DILI).

Diclofenac is a widely prescribed NSAID, which by itself and its reactive metabolites (Phase-I and Phase-II) may be involved in serious idiosyncratic hepatotoxicity. Mitochondrial injury is one of the mechanisms of drug induced liver injury (DILI). In the present work, an investigation of the inhibitory effects of diclofenac (Dic) and its phase I [4-hydroxy diclofenac (4'-OH-Dic) and 5-hydroxy diclofenac (5-OH-dic)] and Phase-II [diclofenac acyl glucuronide (DicGluA) and diclofenac glutathione thioester (DicSG)] metabolites, on ATP synthesis in rat liver mitochondria was carried out. A mechanism based inhibition of ATP synthesis is exerted by diclofenac and its metabolites. Phase-I metabolite (4'-OH-Dic) and Phase-II metabolites (DicGluA and DicSG) showed potent inhibition (2-5 fold) of ATP synthesis, where as 5-OH-Dic, one of the Phase-I metabolite, was a less potent inhibitor as compared to Dic. The calculated kinetic constants of mechanism based inhibition of ATP synthesis by Dic showed maximal rate of inactivation (Kinact) of 2.64 ± 0.15 min(-1) and half maximal rate of inactivation (KI) of 7.69 ± 2.48 µM with Kinact/KI ratio of 0.343 min(-1) µM(-1). Co-incubation of mitochondria with Dic and reduced GSH exhibited a protective effect on Dic mediated inhibition of ATP synthesis. Our data from this study strongly indicate that Dic as well as its metabolites could be involved in the hepato-toxic action through inhibition of ATP synthesis.

Mitochondrial toxicity of selective COX-2 inhibitors via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria.

Cyclooxygenase-2 (COX-2) inhibitors (coxibs) are non-steroidal anti-inflammatory drugs (NSAIDs) designed to selectively inhibit COX-2. However, drugs of this therapeutic class are associated with drug induced liver injury (DILI) and mitochondrial injury is likely to play a role. The effects of selective COX-2 inhibitors on inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria were investigated. The order of potency of inhibition of ATP synthesis was: lumiracoxib (IC50: 6.48 ± 2.74 µM)>celecoxib (IC50: 14.92 ± 6.40 µM)>valdecoxib (IC50: 161.4 ± 28.6 µM)>rofecoxib (IC50: 238.4 ± 79.2 µM)>etoricoxib (IC50: 405.1 ± 116.3 µM). Mechanism based inhibition of ATP synthesis (Kinact 0.078 min(-1) and KI 21.46 µM and Kinact/KI ratio 0.0036 min(-1)µM(-1)) was shown by lumiracoxib and data suggest that the opening of the MPT pore may not be the mechanism of toxicity. A positive correlation (with r(2)=0.921) was observed between the potency of inhibition of ATP synthesis and the log P values. The in vitro metabolism of coxibs in rat liver mitochondria yielded for each drug substance a major single metabolite and identified a hydroxy metabolite with each of the coxibs and these metabolites did not alter the inhibition profile of ATP synthesis of the parent compound. The results suggest that coxibs themselves could be involved in the hepatotoxic action through inhibition of ATP synthesis.

Characterization of midazolam metabolism in locusts: the role of a CYP3A4-like enzyme in the formation of 1'-OH and 4-OH midazolam.

1. The metabolism of midazolam was investigated in vivo in locusts in order to evaluate the presence of an enzyme with functionality similar to human CYP3A4/5. 2. Hydroxylated metabolites of midazolam identical to human metabolites were detected in locusts and the apparent affinities (Km values) were in the same range as reported in humans (in locusts: 7-23 and 33-85 µM for the formation of the 1'-OH and 4-OH metabolites, respectively). 3. The formation of hydroxylated metabolites could successfully be inhibited by co-administration of ketoconazole, a known CYP3A4/5 inhibitor. 4. Besides phase I metabolites, a number of conjugated metabolites were detected using high-resolution mass spectrometry. The most abundant metabolites detected were structurally identified by (1)H NMR as two N-glucosides. NMR analysis strongly suggested that the glycosylation occurred at the two nitrogens (either one in each case) of the imidazole ring. 5. Distribution of midazolam and the glucose conjugates were successfully measured using desorption electrospray mass spectrometry imaging revealing time-dependent changes in distribution over time. 6. In conclusion, it appears that an enzyme with functionality similar to human CYP3A4/5 is present in locusts. However, it appears that conjugation with glucose is the main detoxification pathway of midazolam in locusts.

An invertebrate model for CNS drug discovery: Transcriptomic and functional analysis of a mammalian P-glycoprotein ortholog.

BACKGROUND: ABC efflux transporters at the blood brain barrier (BBB), namely the P-glycoprotein (P-gp), restrain the development of central nervous system (CNS) drugs. Consequently, early screening of CNS drug candidates is pivotal to identify those affected by efflux activity. Therefore, simple, high-throughput and predictive screening models are required. The grasshopper (locust) has been developed as an invertebrate in situ model for BBB permeability assessment, as it has shown similarities to vertebrate models. METHODS: Transcriptome profiling of ABC efflux transporters in the locust brain was performed. Subsequently, identified transcripts were matched with their counterparts in human, rat, mouse and Drosophila melanogaster, based on amino acid sequence similarity, and phylogenetic trees were constructed to reveal the most likely evolutionary history of the proteins. Further, functional characterization of a P-gp ortholog was achieved through transport studies, using a selective P-gp substrate and locust brain in situ, followed by kinetic analyses. RESULTS: A protein with high sequence similarity to the ABCB1 gene of vertebrates was found in the locust brain, which encodes P-gp in human and is considered the most vital efflux pump. Functionally, this model showed transport kinetic behaviors comparable to those obtained from in vitro models. Particularly, substrate affinity of the putative P-gp was observed as in P-gp expressing cells lines, used for predicting drug penetration across biological barriers. CONCLUSION: Findings suggest a conserved mechanism of brain efflux activity between insects and vertebrates, confirming that this model holds promise for inexpensive and high-throughput screening relative to in vivo models, for CNS drug discovery.

Topically applied methotrexate is rapidly delivered into skin by fractional laser ablation.

OBJECTIVES: Methotrexate (MTX) is a chemotherapeutic and anti-inflammatory drug that may cause systemic adverse effects. This study investigated kinetics and biodistribution of MTX delivered topically by ablative fractional laser (AFXL). METHODS: In vitro passive diffusion of 10 mg/ml MTX (1 w/v%) was measured from 0.25 to 24 h through AFXL-processed and intact porcine skin in Franz Cells (n = 46). A 2,940 nm fractional Erbium Yttrium Aluminium Garnet laser generated mid-dermal microchannels at 2.4% density, and 256 mJ/microchannel. HPLC quantified MTX-concentrations in extracts from mid-dermal skin sections, donor and receiver compartments. Fluorescence microscopy of UVC-activated MTX-fluorescence and desorption electro-spray ionization mass spectrometry imaging (DESI-MSI) evaluated MTX biodistribution. RESULTS: AFXL-processed skin facilitated rapid MTX delivery through cone-shaped microchannels of 690 µm ablation depth, lined by the 47 µm thermal coagulation zone (CZ). Quantitatively, MTX was detectable by HPLC in mid-dermis after 15 min, significantly exceeded deposition in intact skin after 1.5 h, and saturated skin after 7 h at a 10-fold increased MTX-deposition versus intact skin (3.08 vs 0.30 mg/cm(3), p = 0.002). Transdermal permeation was < 1.5% of applied MTX before skin saturation, and increased up to 8.0% after 24 h. Qualitatively, MTX distributed into CZ within 15 min (p = 0.015) and further into surrounding dermal tissue after 1.5 h (p = 0.004). After skin saturation at 7 h, MTX fluorescence intensities in CZ and tissue were similar and DESI-MSI confirmed MTX biodistribution throughout the mid-dermal skin section. CONCLUSIONS: MTX absorbs rapidly into mid-dermis of AFXL-processed skin with minimal transdermal permeation until skin saturation, suggesting a possible alternative to systemic MTX for some skin disorders.

Real Time Extraction Kinetics of Electro Membrane Extraction Verified by Comparing Drug Metabolism Profiles Obtained from a Flow-Flow Electro Membrane Extraction-Mass Spectrometry System with LC-MS.

A simple to construct and operate, "dip-in" electromembrane extraction (EME) probe directly coupled to electrospray ionization-mass spectrometry (ESI-MS) for rapid extraction and real time analysis of various analytes was developed. The setup demonstrated that EME-MS can be used as a viable alternative to conventional protein precipitation followed by liquid chromatography-mass spectrometry (LC-MS) for studying drug metabolism. Comparison of EME-MS with LC-MS for drug metabolism analysis demonstrated for the first time that real time extraction of analytes by EME is possible. Metabolism kinetics were investigated for three different drugs: amitriptyline, promethazine, and methadone. By comparing the EME-MS extraction profiles of the drug substances and formed drug metabolites with the metabolism profiles obtained by conventional protein precipitation followed by LC-MS good correlation was obtained with only very limited time delay in the extraction. The results indicate that, by tuning the electromembrane properties, for example, by optimizing the extraction voltage, extremely fast extraction kinetics can be obtained. A metabolic profile could be generated while the drug was metabolized offering a significant time saving as compared to conventional LC-MS where laborious protein precipitation or other sample pretreatments are required before analysis. This makes the developed EME-MS setup a highly promising sample preparation method for various kinds of applications where fast and real-time analysis of analytes is of interest.

Evaluation of microwave oven heating for prediction of drug-excipient compatibilities and accelerated stability studies.

Microwave ovens have been used extensively in organic synthesis in order to accelerate reaction rates. Here, a set up comprising a microwave oven combined with silicon carbide (SiC) plates for the controlled microwave heating of model formulations has been applied in order to investigate, if a microwave oven is applicable for accelerated drug stability testing. Chemical interactions were investigated in three selected model formulations of drug and excipients regarding the formation of ester and amide reaction products. In the accelerated stability studies, a design of experiments (DoE) approach was applied in order to be able to rank excipients regarding reactivity: Study A: cetirizine with PEG 400, sorbitol, glycerol and propylene glycol. Study B: 6-aminocaproic acid with citrate, acetate, tartrate and gluconate. Study C: atenolol with citric, tartaric, malic, glutaric, and sorbic acid. The model formulations were representative for oral solutions (co-solvents), parenteral solutions (buffer species) and solid dosage forms (organic acids applicable for solubility enhancement). The DoE studies showed overall that the same impurities were generated by microwave oven heating leading to temperatures between 150°C and 180°C as compared to accelerated stability studies performed at 40°C and 80°C using a conventional oven. Ranking of the reactivity of the excipients could be made in the DoE studies performed at 150-180°C, which was representative for the ranking obtained after storage at 40°C and 80°C. It was possible to reduce the time needed for drug-excipient compatibility testing of the three model formulations from weeks to less than an hour in the three case studies. The microwave oven is therefore considered to be an interesting alternative to conventional thermal techniques for the investigation of drug-excipient interactions during preformulation.

Structure elucidation and quantification of impurities formed between 6-aminocaproic acid and the excipients citric acid and sorbitol in an oral solution using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy.

Concentrated solutions containing 6-aminocaproic acid and the excipients citric acid and sorbitol have been studied at temperatures of 50°C, 60°C, 70°C and 80°C as well as at 20°C. It has previously been reported that the commonly employed citric acid is a reactive excipient, and it is therefore important to thoroughly investigate a possible reaction between 6-aminocaproic acid and citric acid. The current study revealed the formation of 3-hydroxy-3,4-dicarboxy-butanamide-N-hexanoic acid between 6-aminocaproic acid and citric acid by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy (NMR). Less than 0.03% of 6-aminocaproic acid was converted to 3-hydroxy-3,4-dicarboxy-butanamide-N-hexanoic acid after 30 days of storage at 80°C. Degradation products of 6-aminocaproic acid were also observed after storage at the applied temperatures, e.g., dimer, trimer and cyclized 6-aminocaproic acid, i.e., caprolactam. No reaction products between D-sorbitol and 6-aminocaproic acid could be observed. 3-Hydroxy-3,4-dicarboxy-butanamide-N-hexanoic acid, dimer and caprolactam were also observed after storage at 20°C for 3 months. The findings imply that an oral solution of 6-aminocaproic acid is relatively stable at 20°C at the pH values 4.00 and 5.00 as suggested in the USP for oral formulations. Compliance with the ICH guideline Q3B is expected.

Distribution of terfenadine and its metabolites in locusts studied by desorption electrospray ionization mass spectrometry imaging.

Desorption electrospray ionization (DESI) mass spectrometry (MS) imaging was used to image locusts dosed with the antihistamine drug terfenadine. The study was conducted in order to elucidate a relatively high elimination rate of terfenadine from the locust hemolymph. In this one of the few MS imaging studies on insects, a method for cryosectioning of whole locusts was developed, and the distributions of a number of endogenous compounds are reported, including betaine and a number of amino acids and phospholipids. Terfenadine was detected in the stomach region and the intestine walls, whereas three different metabolites-terfenadine acid (fexofenadine), terfenadine glucoside, and terfenadine phosphate-were detected in significantly smaller amounts and only in the unexcreted feces in the lower part of the intestine. The use of MS/MS imaging was necessary in order to detect the metabolites. With use of DESI-MS imaging, no colocalization of the drug and the metabolites was observed, suggesting a very rapid excretion of metabolites into the feces. Additional liquid chromatography-MS investigations were performed on hemolymph and feces and showed some abundance of terfenadine and the three metabolites, although at low levels, in both the hemolymph and the feces.

An Efficient, Robust, and Inexpensive Grinding Device for Herbal Samples like Cinchona Bark.

An effective, robust, and inexpensive grinding device for the grinding of herb samples like bark and roots was developed by rebuilding a commercially available coffee grinder. The grinder was constructed to be able to provide various particle sizes, to be easy to clean, and to have a minimum of dead volume. The recovery of the sample when grinding as little as 50 mg of crude Cinchona bark was about 60%. Grinding is performed in seconds with no rise in temperature, and the grinder is easily disassembled to be cleaned. The influence of the particle size of the obtained powders on the recovery of analytes in extracts of Cinchona bark was investigated using HPLC.

Characterization of a novel brain barrier ex vivo insect-based P-glycoprotein screening model.

In earlier studies insects were proposed as suitable models for vertebrate blood-brain barrier (BBB) permeability prediction and useful in early drug discovery. Here we provide transcriptome and functional data demonstrating the presence of a P-glycoprotein (Pgp) efflux transporter in the brain barrier of the desert locust (Schistocerca gregaria). In an in vivo study on the locust, we found an increased uptake of the two well-known Pgp substrates, rhodamine 123 and loperamide after co-administration with the Pgp inhibitors cyclosporine A or verapamil. Furthermore, ex vivo studies on isolated locust brains demonstrated differences in permeation of high and low permeability compounds. The vertebrate Pgp inhibitor verapamil did not affect the uptake of passively diffusing compounds but significantly increased the brain uptake of Pgp substrates in the ex vivo model. In addition, studies at 2°C and 30°C showed differences in brain uptake between Pgp-effluxed and passively diffusing compounds. The transcriptome data show a high degree of sequence identity of the locust Pgp transporter protein sequences to the human Pgp sequence (37%), as well as the presence of conserved domains. As in vertebrates, the locust brain-barrier function is morphologically confined to one specific cell layer and by using a whole-brain ex vivo drug exposure technique our locust model may retain the major cues that maintain and modulate the physiological function of the brain barrier. We show that the locust model has the potential to act as a robust and convenient model for assessing BBB permeability in early drug discovery.

Application of temperature-correlated mobility theory for optimizing the MEKC separation of the main lignans from Schisandra Chinensis Fructus and its prescription Yuye Decoction.

The present work shows the application of the temperature-correlated mobility theory for the optimization of the separation and peak alignment of the main lignans from water extracts of traditional Chinese medicine Schisandra Chinensis Fructus as well as its prescription Yuye Decoction (Jade Fluid Decoction; YYD). This is the first application of this theory for MEKC separations, and the data presentation allows a much easier peak tracking and thereby identification of the analytes. Most interestingly, the data obtained and presented in the mobility scale at 298 K, show that Schisantherin A, which is easily mistaken as one of the analytes using traditional time scale, was actually not detected in Schisandra Chinensis Fructus and Yuye Decoction (Jade Fluid Decoction) water extracts. This proves the value of the temperature-correlated mobility scale for method optimization of complex samples. Thus, in the temperature-correlated mobility scale, the optimization of the system conditions for the MEKC separations can easily be achieved by correcting for viscosity changes. Also, the influence of the operating temperature can be monitored in a more distinct way.

Kinetics of the esterification of active pharmaceutical ingredients containing carboxylic acid functionality in polyethylene glycol: formulation implications.

Polyethylene glycols (PEGs) are attractive as excipients in the manufacture of drug products because they are water soluble and poorly immunogenic. They are used in various pharmaceutical preparations. However, because of their terminal hydroxyl groups, PEGs can participate in esterification reactions. In this study, kinetics of two active pharmaceutical ingredients, cetirizine and indomethacin possessing carboxylic acid functionality, has been studied in PEG 400 and PEG 1000 at 50 °C, 60 °C, 70 °C, and 80 °C. HPLC-UV was applied for the determination of concentrations in the kinetic studies, whereas HPLC-MS was used to identify reaction products. The esterification reactions were observed to be reversible. A second-order reversible kinetic model was applied and rate constants were determined. The rate constants demonstrated that cetirizine was esterified about 240 times faster than indomethacin at 80 °C. The shelf-life for cetirizine in a PEG 400 formulation at 25 °C expressed as t(95%) was predicted to be only 30 h. Further, rate constants for esterification of cetirizine in PEG 1000 in relation to PEG 400 decreased by a factor of 10, probably related to increased viscosity. However, it is important to be aware of this drug-excipient interaction, as it can reduce the shelf-life of a low-average molecular weight PEG formulation considerably.