Reversing the adverse biochemical effects in lead-intoxicated rats by N,N`- bis[(1,2-didehydro-1-hydroxy-2-thioxopyrid-4-yl)-carbonyl]- L-lysine.

N,N`-Bis[(1,2-didehydro-1-hydroxy-2-thioxopyrid-4-yl)-carbonyl]- L-lysine (HTPL) is a novel newly synthesized compound intended to be used for the chelation of lead in intoxicated animals. Subchronic lead intoxication experiments were carried out on Wistar male rats; these rats were intoxicated with lead and then treated with HTPL. Results were compared with those obtained with known compounds used for lead chelation therapy, such as disodium ethylnediaminetetraacetic acid (CaNa2EDTA) and meso-2,3-dimercaptosuccininc acid (DMSA), using different routes of administration. Biological samples of whole blood and urine were collected and analyzed for urinary proporphyrins, δ-aminolevulinic acid dehydratase, and zinc protoporphyrin. Results revealed that HTPL can remarkably reverse the toxic effects of lead intoxication at biochemical levels. Additionally, results showed that this agent is as good or even more potent than calcium disodium ethylnediaminetetraacetic acid (CaNa2EDTA) and meso-2,3-dimercaptosuccininc acid (DMSA) in reversing the toxic effect of lead. More importantly, HTPL was found effective when administrated intraperitoneally and orally.

Synthesis and characterization of new 1-hydroxy-2-pyridinethione derivatives: Their lead complexes and efficacy in the treatment of acute lead poisoning in rats.

A number of new mono- and dihydroxypyridinethione ligands have been synthesized via reaction of dimethylamine and amino acid esters with the active amide obtained from the reaction of 1-hydroxy-2-pyridinethione-4-carboxylic acid (1) and 1,1'-carbonyldiimidazole in DMF. Moreover, the lead complexes of these new ligands were also prepared. Structures of the newly synthesized compounds have been confirmed by different spectroscopic methods such as IR, 1H NMR, and 13C NMR, and by elemental analysis. The effect of these synthesized ligands on the excretion of lead, iron, and zinc, and their distribution in kidneys, liver, and bones in acutely intoxicated rats was investigated and results, for lead, were compared with those of the known drug meso-2,3-dimercaptosuccinic acid (DMSA). Results obtained revealed that compound 5 exhibits remarkable ability in total fecal and urinary excretion of lead and was superior to DMSA. In addition, results show that the concentration of lead in soft tissues and bones was lower in rats treated with HTPL than those treated with DMSA. Furthermore, the concentration of lead in liver tissues obtained from sub-chronic lead-intoxicated rats treated with HTPL was lower than those treated with DMSA and calcium disodium ethylenediaminetetraacetic acid (CaNa2EDTA).

Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma using HPLC coupled with tandem mass spectrometry: Application to bioequivalence studies.

A sensitive, specific and selective liquid chromatography/tandem mass spectrometric method has been developed and validated for the simultaneous determination of irbesartan and hydrochlorothiazide in human plasma. Plasma samples were prepared using protein precipitation with acetonitrile, the two analytes and the internal standard losartan were separated on a reverse phase C(18) column (50mmx4mm, 3microm) using water with 2.5% formic acid, methanol and acetonitrile (40:45:15, v/v/v (%)) as a mobile phase (flow rate of 0.70mL/min). Irbesartan and hydrochlorothiazide were ionized using ESI source in negative ion mode, prior to detection by multiple reaction monitoring (MRM) mode while monitoring at the following transitions: m/z 296-->269 and m/z 296-->205 for hydrochlorothiazide, 427-->175 for irbesartan. Linearity was demonstrated over the concentration range 0.06-6.00microg/mL for irbesartan and 1.00-112.00ng/mL for hydrochlorothiazide. The developed and validated method was successfully applied to a bioequivalence study of irbesartan (300mg) with hydrochlorothiazide (12.5mg) tablet in healthy volunteers (N=36).

Simultaneous determination of bisoprolol and hydrochlorothiazide in human plasma by HPLC coupled with tandem mass spectrometry.

A sensitive, specific and selective method has been developed for the simultaneous determination of bisoprolol and hydrochlorothiazide in human plasma. The method employed a state of the art LC-MS/MS operated in the positive and negative ionization switching modes. A simple sample preparation step involving protein precipitation with acetonitrile has been optimized; the analytes and the internal standard moxifloxacin were separated on a Purosphere STAR C8 column (125 mm x 4 mm, 5 microm). The mobile phase was an ammonium acetate solution (1 mM) with formic acid (0.2%): methanol and acetonitrile (65:17.5:17.5, v/v/v (%)), the flow rate was set at 0.65 mL/min. Bisoprolol and hydrochlorothiazide were ionized using ESI source prior to detection by Multiple Reaction Monitoring (MRM) mode while monitoring at the following transitions: positive m/z 326-->116 for bisoprolol, negative m/z 296-->269 and m/z 296-->205 for hydrochlorothiazide. Linearity was demonstrated over the concentration range 0.10-30.0 (ng/mL) for bisoprolol and 1.00-80.00 ng/mL for hydrochlorothiazide. The limits of detection were 0.100 (ng/mL) for bisoprolol and 1.00 (ng/mL) for hydrochlorothiazide. The validated method was successfully applied to a pharmacokinetic study of 5 mg bisoprolol fumarate with 12.5 mg hydrochlorothiazide tablet in healthy volunteers.

Reactions of sulfenic acid with 2-mercaptoethanol: a mechanism for the inhibition of gastric (H+-K+)-adenosine triphosphate by omeprazole.

The reactions of omeprazole, a potent proton pump inhibitor (PPI) were investigated in the presence of 2-mercapotoethanol. Reactions were monitored in solutions buffered to pH values ranging 2.0-8.0 using differential pulse polarography (DPP) at the static mercury drop electrode (SMDE). The fast, sensitive and selective electrochemical technique facilitated successive recordings of voltammograms (peak current (nA) vs. peak potential (volts vs. Ag/AgCl)) for all analytes in situe, including the 2-mercaptoethanol. In acidic solutions and in the presence of 2-mercaptoethanol, omeprazole undergoes degradation into three compounds, the first is a cyclic sulfenamide (D+), previously believed to be the active inhibitor of the H+, K+-ATPase, the second is the omeprazole dimer, and the third is the disulfide believed to be the product of reaction between 2-mercaptoethanol and D+. The cyclic sulfenamide (D+) solution was found to be stable in solutions containing 2-mercaptoethanol having pH values: 2.0, 4.0, and 6.0. This finding proved conclusively that the cyclic sulfenamide is not reactive toward the 2-mercaptoethanol. In contrast to previous reports, the conversion of the sulfenic acid intermediate into D+ was found to be irreversible. Due to this irreversibility, D+ and sulfenic acid were not rapidly interconvertable. The present work suggests that the active inhibitor is the sulfenic acid.

An in vitro investigation on acid catalyzed reactions of proton pump inhibitors in the absence of an electrophile.

An in vitro investigation of the complex system of acid catalyzed conversions of some proton pump inhibitors (PPI) have been carried out using differential pulse polarographhy (DPP) at the static mercury drop electrode (SMDE). Reactions were investigated in solutions buffered to pH values 2.0-8.0. The employed technique facilitated fast and timely monitoring of each PPI, in addition to its electroactive degradation products. Unlike previous techniques, which employed HPLC and UV spectroscopy, alone, or in combination, DPP facilitated fast and simultaneous recordings of all analytes in situ. This resulted in well-defined current-time profiles of individual electroactive degradation products, in addition to those of the starting materials. The results demonstrated that the rates of degradation of the investigated PPIs can be arranged in the following order: lansoprazole>omeprazole>pantoprazole. Furthermore, the rate of degradation of PPIs decreased with decreasing basicity of the corresponding benzimidazole nitrogen of PPIs, as predicted by the effect of individual substituents on each of the benzimidazole rings. The present study demonstrated that lansoprazole may have the fastest accumulation rate in the parietal cells, in addition to demonstrating the highest rate of conversion into the active inhibitor. At pH 6, however, pantoprazole was found to be the most stable, whereas lansoprazole and omeprazole underwent significant acid catalyzed decomposition. The cyclic sulfenamide of pantoprazole demonstrated highest stability among the three PPIs. Omeprazole, however, fell in the middle in terms of its stability and the stability of its corresponding cyclic sulfenamide. The present study may provide an insight for designing more potent new proton pump inhibitors.

Possible interethnic differences in omeprazole pharmacokinetics : comparison of Jordanian Arabs with other populations.

BACKGROUND AND OBJECTIVE: Considerable ethnic differences have been reported in the incidence of the poor metaboliser (PM) genotype of cytochrome P450 (CYP) 2C19. The frequency of this genotype was found to be much higher in Oriental persons (13-23%) than in American or European populations (3-5%). There are, however, no valid data published for Arabic subjects. The present study was conducted to evaluate pharmacokinetic parameters of omeprazole after a single dose in healthy Jordanian Arabic subjects and to compare the results with data published for other populations. METHODS: Seventy-four healthy male Jordanian Arabic volunteers contributed to the study, which was performed at Al Essra Hospital in Amman, Jordan. After an overnight fast, omeprazole was administered as a single Losec 20mg capsule. A total of 20 blood samples were collected over a 10-hour period after administration. Omeprazole pharmacokinetic parameters were determined from the plasma concentration-time profiles using the WinNonlin software. Kolmogorov-Smirnov's test and probit plots of omeprazole area under the plasma concentration-time curve (AUC) data were used to analyse the frequency distribution of phenotypic data. RESULTS: The mean pharmacokinetic parameters and their corresponding coefficient of variation (CV%) for peak plasma concentration (Cmax), AUC from time zero to infinity (AUCinfinity), time to reach Cmax (tmax), apparent oral clearance (CL/F) and elimination half-life (t(1/2)) were 314.96 ng/mL (56%), 923.2 ng . h/mL (108.6%), 2.1h (44%), 0.66 L/h/kg (92%) and 1.5 h (56.6%), respectively. Interindividual differences in the current study were high for all pharmacokinetic parameters, yet comparable to CVs reported in nonphenotyped subjects identified within other ethnic groups (40.3-159% for AUC and 39-48.2% for Cmax). The frequency distribution of all parameters, particularly the AUC, was shown to be trimodal. This has proposed the presence of three distinct phenotypes, designated as extensive metabolisers (EMs), slow-extensive metabolisers (SEMs), and PMs, with corresponding frequency of 36.5%, 39.2% and 24.3%, respectively. After stratification, the relative mean AUCs of omeprazole in EMs, SEMs and PMs were 1 : 2.7 : 9.3 (all p < 0.001). Accordingly, the CL/F of omeprazole showed a ratio of 9.8 : 3.6 : 1 for three phenotype groups, respectively. For other pharmacokinetic parameters including Cmax, t1/2, AUC normalised for bodyweight (AUCN), Cmax/dose and AUC/dose, there were also significant differences between the three groups. CONCLUSIONS: The current pharmacokinetic study revealed that the majority of the Jordanian Arabics seemed to be more properly classified within the EM phenotype. More specifically, the observed metabolic rates of heterozygous and homozygous Jordanian Arabic EMs were more comparable to those of Caucasian EMs than Oriental EMs. Consequently, higher dosage requirements can be expected among most of the Jordanian Arabics. Yet, the incidence of PMs is significant and they seemed to exhibit a similar pharmacokinetic pattern to Chinese PMs in terms of long-term exposure (clearance and AUC) as well as short-term exposure (Cmax) parameters, after adjustment for dose and bodyweight. Therefore, further clinical application of CYP2C19 polymorphism is anticipated in Jordanian Arabic mixed population, particularly if long-term use of omeprazole is intended.

Acid decomposition of omeprazole in the absence of thiol: a differential pulse polarographic study at the static mercury drop electrode (SMDE).

The reactions of omeprazole, a potent proton pump inhibitor (PPI), were investigated in the absence of a nucleophile. Reactions were monitored, using differential pulse polarography (DPP) at the static mercury drop electrode (SMDE), in solutions buffered to pH values ranging from 2.0 to 8.0. The fast, sensitive, and selective electrochemical technique facilitated to repeat recordings of successive voltammograms [peak current (nA) vs. peak potential (volts vs. Ag/AgCl saturated with 3.0 M KCl)]. The DPP signals of omeprazole and its degradation products, believed to be due to sulfur functional group (the principal site of electrode reaction), gave advantages over the previously employed UV detection technique. The latter primarily relied on pyridine and benzimidazole analytical signals, which are common reaction products of PPI in aqueous acidic solutions. After peak identification, the resulting current (nA)-time (s) profiles, demonstrated that omeprazole undergoes degradation to form two main stable compounds, the first is the cyclic sulfenamide (D(+)), previously believed to be the active inhibitor of the H(+), K(+)-ATPase, the second is omeprazole dimer. This degradation is highly dependant on pH. Unlike previous studies which reported that the lifetime of D(+) is few seconds, the cyclic sulfenamide (D(+)) was found to be stable for up to 5-20 min. The results further indicated that omeprazole converts into the cyclic sulfenamide in an irreversible reaction, consequently, D(+) and sulfenic acid (an intermediate which rapidly converts into D(+)) were not interconvertable. The present work suggested that the sulfenic acid is the active inhibitor in vivo. In addition, the omeprazole reactions, in the absence of the thiol, were not as complicated as were previously reported.

Measurement of protein cytokines in tissue extracts by enzyme-linked immunosorbent assays: application to lipopolysaccharide-induced differential milieu of cytokines.

OBJECTIVES: Determination of protein cytokines in local tissues would help to evaluate their local role in health, sickness behavior and immune-mediated diseases. Therefore, developing a simple quantitative method of protein cytokines in tissues/organs is highly important. METHODS: Mouse tissues were collected following intraperitoneal administration of endotoxin-free PBS or lipopolysaccharide. A mild detergent, 0.1% Igepal, was added in a buffer to enhance cytokines extraction. The tissues were then disrupted, homogenized, centrifuged and the supernatants were collected and assayed using solid-phase immunoassays. RESULTS: The presence of 0.1% Igepal extracted significantly more TNF-alpha from liver (322%: p<0.01), brain (358%: p<0.05), lungs (1600%: p<0.01), and more IL-10 from liver (220%: p<0.001), brain (4650%: p<0.001) than PBS alone. On the other hand, using 0.1% Igepal did not increase IFN-gamma extraction from liver, spleen, brain, lungs, skin and kidneys more than PBS alone. Furthermore, i.p. administration of LPS induced a differential milieu of cytokines. LPS increased significantly the production of TNF-alpha, IFN-gamma, and IL-10 from liver (521%, 123%, 72%: p<0.01, 0.04, 0.04), brain (470%, 122%, 280%: p< 0.01, 0.03, 0.01), peritoneal lavage (p<0.001) and blood (p<0.001). However, the pattern of increase was different for the above cytokines in spleen, skin, lungs and kidneys. CONCLUSIONS: The extraction of protein cytokines from tissues was superior with addition of mild detergent. Furthermore, our results showed a differential cytokines response to LPS with respect to tissue and cytokine type. This method should provide an important tool for studying local protein cytokines in behavioral pattern, sickness behavior, and immune-mediated diseases as well as to determine local therapeutic efficacy of immunomodulatory drugs.