Targeting ERS-mitophagy in hippocampal neurons to explore the improvement of memory by tea polyphenols in aged type 2 diabetic rats.

Increasing evidence demonstrated that mitophagy and endoplasmic reticulum stress (ERS) was closely associated with memory decline in elderly type 2 diabetes mellitus (T2DM). Tea polyphenols (TP), an excellent natural antioxidant, has been reported to have neuroprotective properties in aging and diabetes, but the underlying mechanisms are still not fully understood. This study targets ERS-mitophagy in hippocampal neurons to investigate the improvement effect of memory in aged T2DM rats by TP. Rats were randomly divided into the control group, the aged group, the aged T2DM model group, the TP 75, 150, 300 mg/kg groups. TP 300 mg/kg ameliorated mitophagy by decreasing the levels of p-mTOR (S2448), P62 and HSP60 and increasing the levels of PINK1 and Parkin, the ratio of LC3Ⅱ/LC3Ⅰ, co-localization of LC3 and HSP60 and the number of autophagosomes and autolysosomes. TP 300 mg/kg attenuated ERS by downregulating the levels of p-PERK, p-eIF2α, ATF4, GRP78 and restoring the ER structure. To further verify epigallocatechin gallate (EGCG), which is the main active component of TP, enhanced mitophagy by inhibiting ERS, PC12 cells were pretreated with ERS activator tunicamycin (TM) or ERS inhibitor 4-phenylbutyric acid (4-PBA). The results showed that the improvement of mitophagy by EGCG was inhibited by TM and promoted by 4-PBA. Collectively, ERS-mitophagy in hippocampal neurons plays a key role in the improvement of memory by TP in aged T2DM rats. This study will provide a new perspective and strategy for the prevention of memory decline in elderly with T2DM.

Early elimination of uremic toxin ameliorates AKI-to-CKD transition.

Acute kidney injury (AKI)-related fibrosis is emerging as a major driver of chronic kidney disease (CKD) development. Aberrant kidney recovery after AKI is multifactorial and still poorly understood. The accumulation of indoxyl sulfate (IS), a protein-bound uremic toxin, has been identified as a detrimental factor of renal fibrosis. However, the mechanisms underlying IS-related aberrant kidney recovery after AKI is still unknown. The present study aims to elucidate the effects of IS on tubular damage and its involvement in the pathogenesis of AKI-to-CKD transition. Our results showed that serum IS started to accumulate associated with the downregulation of tubular organic anion transporter but not observed in the small-molecule uremic toxins of the unilateral ischemia-reperfusion injury (UIRI) without a contralateral nephrectomy model. Serum IS is positively correlated with renal fibrosis and binding immunoglobulin protein (BiP) and CAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) expression induction in the UIRI with a contralateral nephrectomy model (UIRI+Nx). To evaluate the effects of IS in the AKI-to-CKD transition, we administered indole, a precursor of IS, at the early stage of UIRI. Our results demonstrated IS potentiates renal fibrosis, senescence-associated secretory phenotype (SASP), and activation of endoplasmic reticulum (ER) stress, which is attenuated by synergistic AST-120 administration. Furthermore, we clearly demonstrated that IS exposure potentiated hypoxia-reperfusion (H/R) induced G2/M cell cycle arrest, epithelial-mesenchymal transition (EMT) and aggravated ER stress induction in vitro. Finally, the ER chemical chaperon, 4-phenylbutyric acid (4-PBA), successfully reversed the above-mentioned AKI-to-CKD transition. Taken together, early IS elimination in the early stage of AKI is likely to be a useful strategy in the prevention and/or treatment of the AKI-to-CKD transition.

AMPA induced cognitive impairment in rats: Establishing the role of endoplasmic reticulum stress inhibitor, 4-PBA.

Glutamate excitotoxicity and endoplasmic reticulum (ER) recently have been found to be instrumental in the pathogenesis of various neurodegenerative diseases. However, the paucity of literature deciphering the inter-linkage among glutamate receptors, behavioral alterations, and ER demands thorough exploration. Reckoning the aforesaid concerns, a prospective study was outlined to delineate the influence of ER stress inhibition via 4-phenylbutyric acid (PBA) on α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) excitotoxicity-induced behavioral aspects and possible ER stress-glutamate linkage. Male SD rats were randomly divided into four groups namely sham (surgical control+vehicle, group 1), AMPA-induced excitotoxic group 2 receive a single intra-hippocampal injection of 10 mM AMPA, group 3 received AMPA along with PBA (i.p, 100 mg/kg body weight) for 15 days, and group 4 received PBA alone. Behavioral analyses were performed prior to the sacrifice of animals and hippocampus was extracted thereafter for further analysis. AMPA-induced excitotoxicity exhibited significant impairment of locomotion as well as cognitive functions. The levels of neurotransmitters such as dopamine, homo vanillic acid (HVA), norepinephrine, and serotonin were reduced accompanied by reduced expression of GLUR1 and GLUR4 (glutamate receptor) as well as loss of neurons in different layers of hippocampus. ER stress markers were upregulated upon AMPA excitotoxicity. However, chemical chaperone PBA supplementation remarkably mitigated the behavioral alterations along with expression of glutamate and ER stress intermediates/markers in AMPA excitotoxic animals. Therefore, the present exploration convincingly emphasizes the significance of ER stress and its inhibition via PBA in combating cognitive impairment as well as improving locomotion in excitotoxic animals.

Amphetamine-like Neurochemical and Cardiovascular Effects of α-Ethylphenethylamine Analogs Found in Dietary Supplements.

Dietary supplements often contain additives not listed on the label, including α-ethyl homologs of amphetamine such as N,α-diethylphenethylamine (DEPEA). Here, we examined the neurochemical and cardiovascular effects of α-ethylphenethylamine (AEPEA), N-methyl-α-ethylphenethylamine (MEPEA), and DEPEA as compared with the effects of amphetamine. All drugs were tested in vitro using uptake inhibition and release assays for monoamine transporters. As expected, amphetamine acted as a potent and efficacious releasing agent at dopamine transporters (DAT) and norepinephrine transporters (NET) in vitro. AEPEA and MEPEA were also releasers at catecholamine transporters, with greater potency at NET than DAT. DEPEA displayed fully efficacious release at NET but weak partial release at DAT (i.e., 40% of maximal effect). In freely moving, conscious male rats fitted with biotelemetry transmitters for physiologic monitoring, amphetamine (0.1-3.0 mg/kg, s.c.) produced robust dose-related increases in blood pressure (BP), heart rate (HR), and motor activity. AEPEA (1-10 mg/kg, s.c.) produced significant increases in BP but not HR or activity, whereas DEPEA and MEPEA (1-10 mg/kg, s.c.) increased BP, HR, and activity. In general, the phenethylamine analogs were approximately 10-fold less potent than amphetamine. Our results show that α-ethylphenethylamine analogs are biologically active. Although less potent than amphetamine, they produce cardiovascular effects that could pose risks to humans. Given that MEPEA and DEPEA increased locomotor activity, these substances may also have significant abuse potential. SIGNIFICANCE STATEMENT: The α-ethyl homologs of amphetamine have significant cardiovascular, behavioral, and neurochemical effects in rats. Given that these compounds are often not listed on the ingredient labels of dietary supplements, these compounds could pose a risk to humans using these products.

Intravenous Administration of Butaphosphan and Cyanocobalamin Combination to Late-Pregnant Dairy Cows Reduces Their Insulin Resistance After Calving.

Dairy cows suffer insulin resistance following parturition and lactogenesis. Several researchers attempted to reduce insulin resistance via dietary and parenteral supplementations of different substances to promote metabolic performance of dairy cows. Due to mechanisms of actions of butaphosphan in combination with cyanocobalamin, we hypothesized that this compound may reduce insulin resistance of dairy cows following parturition; hence, the effects of the intravenous administration of butaphosphan and cyanocobalamin to prepartum dairy cows on their insulin resistance after calving were evaluated. Twenty-four multiparous Holstein dairy cows were enrolled 3 weeks prior to parturition and divided into four equal groups, including control (Ctrl) and butaphosphan and cyanocobalamin (B+C) 1, 2, and 3. Ctrl cows received 15 mL of 0.9% NaCl solution and B+C 1, 2, and 3 groups intravenously received 2, 4, and 6 mL/100 kg BW of 10% butaphosphan and 0.005% cyanocobalamin combination over three periods of 3 consecutive days, including 21-19, 12-10, and 3-1 days before calving, respectively. Intravenous glucose tolerance test was performed weekly 1, 2, and 3 weeks after parturition to evaluate the insulin resistance phenomenon. Circulating levels of glucose, insulin, non-esterified fatty acids (NEFA), and beta-hydroxybutyric acid (BHBA) were assessed 1, 2, and 3 weeks after calving. Ctrl cows were the most insulin-resistant group, and B+C1 group was the most insulin-sensitive, followed by B+C2 and B+C3 groups. The NEFA and BHBA levels in the B+C3 group were significantly lower than those in the other groups. In conclusion, intravenous administration of butaphosphan and cyanocobalamin to the late-pregnant dairy cows may reduce their insulin resistance after calving.

Selenium Attenuates Chronic Heat Stress-Induced Apoptosis via the Inhibition of Endoplasmic Reticulum Stress in Mouse Granulosa Cells.

Heat stress induces apoptosis in various cells. Selenium, an essential micronutrient, has beneficial effects in maintaining the cellular physiological functions. However, its potential protective action against chronic heat stress (CHS)-induced apoptosis in granulosa cells and the related molecular mechanisms are not fully elucidated. In this study, we investigated the roles of selenium in CHS-induced apoptosis in mouse granulosa cells and explored its underlying mechanism. The heat treatment for 6-48 h induced apoptosis, potentiated caspase 3 activity, increased the expression levels of apoptosis-related gene BAX and ER stress markers, glucose-regulated protein 78 (GRP78), and CCAAT/enhancer binding protein homologous protein (CHOP) in mouse granulosa cells. The treatment with ER stress inhibitor 4-PBA significantly attenuated the adverse effects caused by CHS. Selenium treatment significantly attenuated the CHS- or thapsigargin (Tg, an ER stress activator)-induced apoptosis, potentiation of caspase 3 activity, and the increased protein expression levels of BAX, GRP78, and CHOP. Additionally, treatment of the cells with 5 ng/mL selenium significantly ameliorated the levels of estradiol, which were decreased in response to heat exposure. Consistently, administering selenium supplement alleviated the hyperthermia-caused reduction in the serum estradiol levels in vivo. Together, our findings indicate that selenium has protective effects on CHS-induced apoptosis via inhibition of the ER stress pathway. The current study provides new insights in understanding the role of selenium during the process of heat-induced cell apoptosis.

Primary Care Management of Upper Respiratory Infections in the Women's Health Care Setting.

Upper respiratory infections (URIs) are the most common reason for which individuals seek health care services in the outpatient clinical setting. This case report describes the clinical presentation and management of a woman with a URI. The signs and symptoms of URIs, physical examination, differential diagnoses, and treatment plan options available in the ambulatory care setting are reviewed. Current evidence-based guidelines are discussed, and recommendations for clinical practice are reviewed. Discussion of the incidental treatment of URIs in the specialty care setting is also addressed.

Facile synthesis of amine-functional reduced graphene oxides as modified quick, easy, cheap, effective, rugged and safe adsorbent for multi-pesticide residues analysis of tea.

Amine-functional reduced graphene oxide (amine-rGO) with different carbon chain length amino groups were successfully synthesized. The graphene oxides (GO) reduction as well as amino grafting were achieved simultaneously in one step via a facile solvothermal synthetic strategy. The obtained materials were characterized by X-ray diffraction, Raman spectroscopy, Fourier-transform infrared spectrometry and X-ray photoelectron spectroscopy to confirm the modification of GO with different amino groups. The adsorption performance of catechins and caffeine from tea acetonitrile extracts on different amine functional rGO samples were evaluated. It was found that tributylamine-functional rGO (tri-BuA-rGO) exhibited the highest adsorption ability for catechins and caffeine compared to GO and other amino group functional rGO samples. It was worth to note that the adsorption capacity of catechins on tri-BuA-rGO was 11 times higher than that of GO (203.7mgg-1 vs 18.7mgg-1). Electrostatic interaction, π-π interaction and surface hydrophilic-hydrophobic properties of tri-BuA-rGO played important roles in the adsorption of catechins as well as caffeine. The gravimetric analysis confirmed that the tri-BuA-rGO achieved the highest efficient cleanup preformance compared with traditional dispersive solid phase extraction (dSPE) adsorbents like primary-secondary amine (PSA), graphitized carbon black (GCB) or C18. A multi-pesticides analysis method based on tri-BuA-rGO is validated on 33 representative pesticides in tea using gas chromatography coupled to tandem mass spectrometry or high-performance liquid chromatography coupled with tandem mass spectrometry. The analysis method gave a high coefficient of determination (r2>0.99) for each pesticide and satisfactory recoveries in a range of 72.1-120.5%. Our study demonstrated that amine functional rGO as a new type of QuEChERS adsorbent is expected to be widely applied for analysis of pesticides at trace levels.

Endoplasmic reticulum stress and dysregulation of calcium homeostasis mediate Cu-induced alteration in hepatic lipid metabolism of javelin goby Synechogobius hasta.

The present study was conducted to investigate the effect of Cu exposure on endoplasmic reticulum (ER) stress and Ca(2+) homeostasis, and also explore the underlying mechanism of the ER stress and Ca(2+) homeostasis in the Cu-induced change of hepatic lipid metabolism in javelin goby Synechogobius hasta. To this end, four experiments were conducted. In experiment 1, the full-length cDNA sequences of two ER molecular chaperones [glucose-regulated protein 78 (GRP78) and calreticulin (CRT)] and three ER stress sensors [PKR-like ER kinase (PERK), inositol requiring enzyme (IRE)-1α, and activating transcription factor (ATF)-6α] cDNAs were firstly characterized from S. hasta. The predicted amino acid sequences for the S. hasta GRP78, CRT, PERK, IRE-1α and ATF-6α revealed that the proteins contained all of the structural features characteristic in other species. mRNAs of the five genes were expressed in various tissues, but their mRNA levels varied among tissues. In experiment 2, S. hasta were exposed to four waterborne Cu concentrations (control, 19µg/l, 38µg/l, and 57µg/l, respectively) for 60days. Cu exposure evoked ER stress in liver of S. hasta in a time- and concentration-course change. In experiment 3, specific inhibitors, 2-aminoethyldiphenyl borate (2-APB) and dantrolene, were used to explore whether Ca(2+) release from ER was involved in the Cu-induced ER stress change. Dantrolene and 2-APB prevented Cu-induced intracellular Ca(2+) elevation, which demonstrated the release of Ca(2+) from the ER was mediated by both RyR and IP3R. In experiment 4, a chemical chaperone, 4-phenyl butyric acid (4-PBA), was used to demonstrate whether Cu-induced alteration in lipid metabolism was suppressed through the attenuation of ER stress. Cu exposure evoked ER stress and sterol-regulator element-binding protein-1c (SREBP-1c) activation in hepatocytes of S. hasta, resulting in dysregulation of hepatic lipid metabolism. 4-PBA attenuated the Cu-induced elevation of mRNA expression of ER stress-related genes. For the first time, our study cloned GRP78, CRT, PERK, IRE-1α and ATF-6α genes in S. hasta and demonstrated their differential expression among tissues. Moreover, the study demonstrated the molecular mechanism by which ER stress might underlie the change of lipid metabolism induced by Cu in S. hasta.

Determination of designer doping agent--2-ethylamino-1-phenylbutane--in dietary supplements and excretion study following single oral supplement dose.

The quantitative analysis of a new designer doping agent, 2-ethylamino-1-phenylbutane (EAPB) and its metabolite, 2-amino-1-phenylbutane (APB) in urine samples, and the determination of EAPB in dietary supplement samples, have been presented. The main purpose of the present study was to develop simple and reliable gas chromatography-mass spectrometry method (GC-MS) for excretion study following a single oral administration of dietary supplements containing EAPB. Three analytical methods for the determination of EAPB in urine and supplement samples, and APB in urine samples using the GC-MS system, have been validated. The method of the determination of EAPB in supplement samples was applied to analyze seventeen dietary supplements, CRAZE and DETONATE. Two other methods were used to determine the urinary excretion profile of EAPB and APB in the case of three healthy volunteers and, on further investigation, it was applied to the anti-doping control in sport. Quantification was obtained on the basis of the ions at m/z 86, 58 and 169, monitored for EAPB, APB and diphenylamine (used as an internal standard), respectively. The limits of detection and quantification were 2.4 and 7.3µg/g for EAPB in the case of supplement analysis, 2.9 and 8.8ng/mL for EAPB in the case of urine analysis, and 3.2 and 9.7ng/mL for APB. The other validation parameters as linearity, precision and trueness have been also investigated with the acceptable results. The extraction yield of all presented methods was above 69%. EAPB was detected in fourteen analyzed supplements (not included EAPB in their labels) and its content varied between 1.8 and 16.1mg/g. Following oral administration of three supplements with EAPB to one male and two female volunteers, the parent compound of EAPB and its metabolite were monitored and the excretion parameters as the maximum concentration of the analyte in urine (2.2-4.2µg/mL for EAPB; 1.1-5.1µg/mL for APB) and the time for the maximum height of the excretion peak (2-8h and 22h in one case for EAPB; 20-22h and 4h in one case for APB) have been indicated. EAPB and APB were detected at the level above 50ng/mL (50% of the minimum required performance level for stimulants in the anti-doping control in-competition in sport) in the urine up to 46-106h and 58-120h, respectively. Additionally, the result of the anti-doping control during swimming competition of one athlete, whose urine sample was analyzed for stimulants and narcotics, has been presented. The qualitative and quantitative analyses of new designer agents in urine samples and the excretion studies of these substances are of a great importance in the anti-doping control in sport. Moreover, the presentation of detection examples of these agents in supplements that haven't got included an information about them in the labeling, make athletes (and other supplement customers) more and more aware of the risk of the supplement use and possible health and doping consequences.

Osage Orange (Maclura pomifera L.) Seed Oil Poly(α-hydroxydibutylamine) Triglycerides: Synthesis and Characterization.

Milled Osage orange seeds (Maclura pomifera (Raf.) Schneid) were Soxhlet extracted with hexane, and portions of the extract were treated with activated carbon before solvent removal. The crude oil was winterized and degummed by centrifugation at low temperature. Decantation of the centrifugate gave an admixture of the triglycerides and free fatty acids. The free fatty acid content of the oil was removed when portions of the admixture were diluted with hexane and shaken with cold aqueous ammonium hydroxide (0.1 M) solution. The desiccant-dried organic phase was concentrated under reduced pressure to give the cleaned Osage orange triglyceride after solvent removal by rotary evaporation at 67 °C. Epoxidation of the resulting cleaned triglyceride was effected by reaction with in situ generated peroxy performic acid in H2O2. The oxirane rings of the derivatized oil were then opened using N,N-dibutylamine catalyzed by anhydrous ZnCl2 to afford the poly(α-hydroxydibutylamine) triglyceride. The purpose of this work was to derivatize and thereby stabilize this highly unsaturated tree oil for its eventual use in lubrication applications.

Inhibition of monoamine oxidase (MAO) by α-ethylphenethylamine and N,α-diethylphenethylamine, two compounds related to dietary supplements.

Phenethylamines can interact with the metabolic enzyme monoamine oxidase (MAO), which can cause neurochemical dysfunction or changes in drug potency. A methamphetamine analog, N,α-diethylphenethylamine (N,α-DEPEA), was recently discovered in athletic performance-enhancing supplements, along with discovery of its metabolite, α-ethylphenethylamine (AEPEA). In vitro inhibition of human recombinant MAO by AEPEA and N,α-DEPEA was evaluated by measuring the fluorescence of 4-hydroxyquinoline produced from MAO substrate, kynuramine. AEPEA competitively inhibited human recombinant MAO A (Ki = 14.0 µM), which was 17-fold stronger compared to MAO B (Ki = 234 µM). Furthermore, N,α-DEPEA was a weak inhibitor of both MAO A (Ki = 251 µM) and MAO B (Ki = 159 µM). Trends regarding MAO A inhibition were explored among structural analogs, yielding the following ranking: amphetamine (Ki = 5.3 µM), AEPEA (Ki = 14.0 µM), methamphetamine (Ki = 17.2 µM), phentermine (Ki = 196 µM), and N,α-DEPEA (Ki = 251 µM). This study provides important data relating chemical structures and biochemical effects for two emerging compounds associated with dietary supplements.

Amphiphilic cellulose nanocrystals from acid-free oxidative treatment: physicochemical characteristics and use as an oil-water stabilizer.

A chemical pretreatment for producing cellulose nanocrystals (CNCs) with periodate oxidation and reductive amination is reported. This new functionalization of cellulose fibers dispenses an alternative method for fabricating individual CNCs without the widely used acid hydrolysis process. CNCs can be directly modified during the pretreatment step, and no additional post-treatments are required to tune the surface properties. Three butylamine isomers were tested to fabricate CNCs with amphiphilic features. After mechanical homogenization, CNCs occurred as individual crystallinities without aggregation where high uniformity in terms of shape and size was obtained. The elemental analysis and (1)H NMR measurement show that iso- and n-butylamine attach the highest number of butylamino groups to the cellulose fibers. Linking the alkyl groups increases the hydrophobic nature of the CNCs, where water contact angles from self-standing films up to 110.5° are reported. Since these butylamino-functionalized CNCs have hydrophobic characteristics in addition to the hydrophilic backbone of cellulose, the stabilization impact on oil/water emulsions is demonstrated as a potential application.

Designer phenethylamines routinely found in human urine: 2-ethylamino-1-phenylbutane and 2-amino-1-phenylbutane.

2-Ethylamino-1-phenylbutane (EAPB) and 2-amino-1-phenylbutane (APB) were identified by gas chromatography-mass spectrometry in multiple urine samples submitted for stimulant drug testing and screened positive for amphetamines by enzyme immunoassay. Forty-two samples from all over the USA were found, containing both analytes during a 3-month period May-July 2013. A sports dietary supplement 'CRAZE' has been determined to be one of the sources of EAPB supply. EAPB along with its suggested metabolite APB were detected in a urine sample, obtained from a person known to use 'CRAZE'.

LC-MS-MS analysis of dietary supplements for N-ethyl-α-ethyl-phenethylamine (ETH), N, N-diethylphenethylamine and phenethylamine.

There has been a recent rise in the number of cases of athletes being banned from competition because of positive tests for prohibited substances in their biological specimens. Most of these substances are on the World Anti-Doping Agency (WADA) prohibited list, while others are not specifically named on the list. N-Ethyl-α-ethyl-phenethylamine (ETH), a derivative of phenethylamine (PEA), is one of these unlisted substances and shares chemical and biological effects to the amphetamines, which are listed on the WADA prohibited substances list. It is classified as Category 6B stimulant on the list. This study was directed toward the development of an liquid chromatography tandem mass spectrometry (LC-MS-MS) method for the analysis of ETH in performance-enhancing dietary supplement. A standard was prepared and confirmed by spectroscopic analysis, which was then used to develop the analytical procedure. The procedure was validated and found to have an limit of detection of 2.5 ng/mL, limit of quantification of 5 ng/mL and upper limit of linearity of 500 ng/mL, with within-day variability at the 10-ng/mL level range of 3.88-7.89% (n = 6) and 1.39-3.36% (n = 6) for the 100-ng/mL level. The day-to-day variability was 9.8% for the low control and 3.1% for the high control. The method was used to analyze a variety of dietary supplements for ETH as well as PEA and its N, N-diethyl derivative (NDP).

Influence of short chain organic acids and bases on the wetting properties and surface energy of submicrometer ceramic powders.

The effect of short chained organic acids and bases on the surface energy and wetting properties of submicrometer alumina powder was assessed. The surface chemistry of treated powders was determined by means of Diffuse Reflectance Infrared Fourier Transform spectroscopy and compared to untreated powder. The wetting of powders was measured using a modified Washburn method, based on the use of precompacted powder samples. The geometric factor needed to calculate the contact angle was derived from measurements of the porous properties of the powder compacts. Contact angle measurements with several probe liquids before and after modification allowed a theoretical estimation of the surface energy based on the surface tension component theory. Trends in the surface energy components were linked to observations in infrared spectra. The results showed that the hydrophobic character of the precompacted powder depends on both the chain length and polar group of the modifying agent.

Inhibition of cough-reflex sensitivity by benzonatate and guaifenesin in acute viral cough.

Acute cough due to viral upper respiratory tract infection (URI) is the most common form of cough and accounts for tremendous expenditure on prescription and non-prescription cough products worldwide. However, few agents have been shown in properly conducted clinical trials to be effective for cough due to URI. The present study evaluated the effect of benzonatate 200mg (B), guaifenesin 600 mg (G), their combination (B+G), and placebo (P) on capsaicin-induced cough in 30 adult nonsmokers with acute URI. On 3 separate days within a 7-day period, 1h after ingesting randomly assigned study drug in a double-blind fashion, subjects underwent capsaicin cough challenge testing, which involved inhalation of incremental doubling concentrations of capsaicin until the concentration of capsaicin inducing 5 or more coughs (C(5)) was attained. Each subject received 3 of 4 possible study drugs. G (p=0.01) but not B (p=NS) inhibited cough-reflex sensitivity (log C(5)) relative to P. The combination of B+G suppressed capsaicin-induced cough to a greater degree than B alone (p<0.001) or G alone (p=0.008). The mechanism by which the combination of B+G causes a potentiation of antitussive effect remains to be elucidated. Our results suggest that B+G may be an effective therapy for acute cough due to the common cold (URI).

Towards automation of a valuable preclinical cardiac safety pharmacology assay: Evaluation of the effects of cardiac ion channel blockers on cardiac repolarisation in vitro.

INTRODUCTION: Purkinje fibre repolarisation assays are valuable tools for identifying compounds which affect cardiac ion channels. The throughput of compound testing in this assay is low therefore we designed a novel recording system to improve screening and animal tissue usage efficiencies. METHODS: The system was used to evaluate compounds using standard sharp microelectrode techniques. Animal tissue usage efficiencies were quantified by adding up the total number of Purkinje fibres from which recordings were attempted and dividing this by the number of experimental data sets generated, to arrive at a 'fibres per data set' ratio. Test compounds were dofetilide (3 x 10(-10) to 10(-8) M), cisapride (10(-8) to 3 x 10(-7) M), terodiline (10(-6) to 3 x 10(-5) M) and verapamil (3 x 10(-7) to 10(-5) M). RESULTS: Using the novel modified system, 21 data sets were generated from 29 fibres, compared to 24 data sets from 41 fibres using the conventional manual recording system, demonstrating a 24% improvement in the efficiency of animal tissue usage. Comparing data from the manual and modified systems revealed differences in absolute values for all parameters including APD90 (308.73 +/- 9.97 ms, n = 24, compared to 275.27 +/- 8.25 ms, n = 21, respectively; P < 0.05). Differences in the magnitude of changes in action potential parameters between the systems were also evident for all compounds including terodiline (1 x 10(-5) M) which caused a -27.1 +/- 16.5% reduction in APD50 in the manual system, compared to a - 55.2 +/- 2.2% reduction in the modified system. DISCUSSION: Although the value of the present study is limited by the small sample sizes, it has demonstrated utility of the modified system in improving efficiency of animal tissue usage. It offers potential utility in a higher throughput screening environment for examining the electrophysiological properties of novel compounds in native cardiac tissues, particularly where functional patch clamp data are limited.

In vitro preclinical cardiac assessment of tolterodine and terodiline: multiple factors predict the clinical experience.

Terodiline and tolterodine are drugs used to treat urinary incontinence. Terodiline was removed from the market in 1991 for proarrhythmia, whereas tolterodine has a generally benign clinical cardiac profile. To assess differences in the electrophysiologic actions of these drugs, we evaluated their effects on hERG current (HEK cells) and cardiac Purkinje fiber repolarization. The IC50 for hERG block (37 degrees C) by tolterodine was 9.6 nM and by terodiline was 375 nM, values near or below clinical concentrations. Tolterodine elicited concentration-dependent prolongation of the action potential duration (APD90). In contrast, terodiline depressed the action potential plateau and induced triangulation without affecting APD90. The triangulation ratios (normalized ratio of APD50 over APD90) for terodiline were 0.94 and 0.59 for 1.0 and 10 microM and for tolterodine, were 0.99 and 0.97 at 7 and 70 nM. In summary, tolterodine, a potent hERG blocker, has a benign clinical cardiac profile at therapeutic concentrations that may be due to its lack of triangulation, as well as extensive plasma protein binding. However, at supratherapeutic concentrations, preclinical data predict risk of QT prolongation. These data suggest that hERG block and triangulation are among multiple factors that must be considered in preclinical cardiac safety assessments.

Cytochrome P450 enzyme-mediated degradation of Echinacea alkylamides in human liver microsomes.

Echinacea preparations are widely used herbal remedies for the prevention and treatment of colds. In this study we have investigated the metabolism by human liver microsomes of the alkylamide components from an Echinacea preparation as well as that of pure synthetic alkylamides. No significant degradation of alkylamides was evident in cytosolic fractions. Time- and NADPH-dependent degradation of alkylamides was observed in microsomal fractions suggesting they are metabolised by cytochrome P450 (P450) enzymes in human liver. There was a difference in the susceptibility of 2-ene and 2,4-diene pure synthetic alkylamides to microsomal degradation with (2E)-N-isobutylundeca-2-ene-8,10-diynamide (1) metabolised to only a tenth the extent of (2E,4E,8Z,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamide (3) under identical incubation conditions. Markedly less degradation of 3 was evident in the mixture of alkylamides present in an ethanolic Echinacea extract, suggesting that metabolism by liver P450s was dependent both on their chemistry and the combination present in the incubation. Co-incubation of 1 with 3 at equimolar concentrations resulted in a significant decrease in the metabolism of 3 by liver microsomes. This inhibition by 1, which has a terminal alkyne moiety, was found to be time- and concentration-dependent, and due to a mechanism-based inactivation of the P450s. Alkylamide metabolites were detected and found to be the predicted epoxidation, hydroxylation and dealkylation products. These findings suggest that Echinacea may effect the P450-mediated metabolism of other concurrently ingested pharmaceuticals.