High-pressure Liquid Chromatographic Assay Research Articles

A Mass Balance and Disposition Study of the DNA Methyltransferase Inhibitor Zebularine (NSC 309132) and Three of Its Metabolites in Mice

To elucidate the in vivo metabolic fate of zebularine (NSC 309132), a DNA methyltransferase inhibitor proposed for clinical evaluation in the treatment of cancer. Male, CD(2)F(1) mice were dosed i.v. with 100 mg/kg 2-[(14)C]zebularine. At specified times between 5 and 1,440 minutes, mice were euthanized. Plasma, organs, carcass, urine, and feces were collected and assayed for total radioactivity. Plasma and urine were also analyzed for zebularine and its metabolites with a previously validated high-pressure liquid chromatography assay. A similar experiment was done with 2-[(14)C]uridine, the proposed primary metabolite of zebularine. Maximum plasma concentrations were 462, 306, 33.6, 21.7, and 11.5 mumol/L for total radioactivity, zebularine, uridine, uracil (each at 5 minutes), and dihydrouracil (at 15 minutes), respectively. Total radioactivity, zebularine, uridine, uracil, and dihydrouracil were rapidly eliminated from plasma, and after 45 minutes, none of the individual compounds could be quantitated by high-pressure liquid chromatography. Plasma data were consistent with sequential conversion of zebularine to uridine, uracil, and dihydrouracil. 2-Pyrimidinone was not observed. Prolonged retention of radioactivity, at concentrations higher than in plasma, was observed in tissues. Recovery of given radioactivity in urine (30.3% of dose), feces (0.4% of dose), cage wash (7.9% of dose), and tissues and carcass (6.1% of dose) after 24 hours implied that up to 55% of radioactivity was expired as (14)CO(2). Comparison of zebularine and uridine pharmacokinetic data indicated that approximately 40% of the zebularine dose was converted to uridine. Zebularine is extensively and rapidly metabolized into endogenous compounds that are unlikely to have effects at the concentrations observed.

Safety of Voriconazole in a Patient with CYP2C9*2/CYP2C9*2 Genotype

Voriconazole, a broad-spectrum triazole antifungal agent, is well absorbed, with a high oral bioavailability of 96% (6). Maximal plasma concentrations are observed 1 to 2 h after drug administration. The volume of distribution is estimated to be 4.6 liters/kg, and the plasma protein binding is 58%. A terminal elimination half-life (t1/2) of 8 h in CYP2C19 extensive metabolizers (EMs) and a t1/2 of 15 h in CYP2C19 poor metabolizers (PMs) were reported (4). Due to a possible saturation of metabolism, voriconazole exhibits nonlinear pharmacokinetics (6). The main metabolite is voriconazole N oxide, which is formed by CYP2C19, CYP3A4, and to a lesser extent CYP2C9 (2). CYP2C19 is a polymorphically expressed enzyme, with 2.2% of the Caucasian population being PMs (9) with a genetically determined absence of active enzyme. It is known that voriconazole pharmacokinetics are substantially influenced by the CYP2C19 genotype (4, 7). A reduction of voriconazole metabolic clearance in PMs of CYP2C19 is expected; data published so far indicate approximately threefold higher voriconazole area-under-the-concentration-time-curve or maximum-concentration-of-drug-in-serum values in CYP2C19 PMs than in homozygous EMs (4, 7). Two common allelic variants of CYP2C9 have a markedly reduced catalytic activity (about 20% for CYP2C9*2 and less than 10% for CYP2C9*3) compared with that of the wild-type enzyme (CYP2C9*1) (3). Accordingly, PMs have an impaired metabolism of phenytoin, tolbutamide, glipizide, and warfarin, although PMs are very uncommon (0.2 to 1.0% of Caucasians but essentially 0% of Southeast Asians) (10). So far no in vivo data are available on the influence of CYP2C9 genetic polymorphism on voriconazole pharmacokinetics. An ongoing study on the pharmacokinetics of voriconazole patients included those who had received their oral loading dose of voriconazole (400 mg) as a regular therapeutic drug treatment during hospitalization and a 12-h (dosing interval) pharmacokinetic profile after the first dose was obtained. We identified one Caucasian patient as a homozygous carrier of the CYP2C9*2 allele, using an established genotyping method using real-time fluorescence PCR on a LightCycler (Roche, Mannheim, Germany) (1). Additionally, the CYP2C19 genotype was also determined, which was homozygous for the wild-type allele (CYP2C19*1/CYP2C19*1). Plasma voriconazole concentrations were determined in the reference laboratory at Heidelberg University using a fully validated high-pressure liquid chromatography (HPLC) assay as described elsewhere (5, 8). Noncompartmental pharmacokinetic parameters were calculated using WinNonlin 4.1 (Pharsight, Mountain View, CA). We compared the obtained pharmacokinetic parameters with those from an earlier study with volunteers whose CYP2C9 and CYP2C19 genotypes were characterized (4). In our patient, the apparent oral clearance (Cl/F), area under the concentration-time curve from zero hours to infinity (AUC0-∞), volume of distribution (Vz), and t1/2 values for voriconazole were not different from those of CYP2C9 EMs who were also CYP2C19 EMs after a single oral dose of 400 mg voriconazole (4) (shown in Table ​Table11). TABLE 1. Pharmacokinetic parameters after administration of 400 mg voriconazole as a single oral dose in a CYP2C9*2/CYP2C9*2 patient (CYP2C19 homozygous EM) compared to results for eight CYP2C9 EM healthy volunteers (CYP2C19 homozygous EM) A conclusion could be that a reduced-metabolizer status of CYP2C9, and therefore low catalytic activity of the enzyme, does not alter the pharmacokinetics of voriconazole. From our data it can be supported that CYP2C9 plays only a minor role in the elimination (metabolism) of voriconazole.

Comparison of Three Valved Holding Chambers for the Delivery of Fluticasone Propionate–HFA to an Infant Face Model

The purpose of this study was to compare three valved holding chambers (VHC) with facemasks attached. One VHC (AeroChamber Max[TM] with medium mask) was made with materials that dissipate surface electrostatic charge, and the others (OptiChamber Advantage and ProChamber[TM] with pediatric facemask) were made from non-conducting materials. The OptiChamber Advantage and ProChamber VHCs were each washed with an ionic detergent and drip dried before testing to minimize surface electrostatic charge. The AeroChamber Max VHCs were tested "out of the package" and also after wash, rinse, and drying. An infant face model incorporating an electrostatic filter in the oral cavity was connected to a breath simulator using a standard waveform for a small child. The fit of each VHC with facemask was demonstrated by agreement of inspiratory flow measurements between a pneumotachograph connected to the system with those set on the simulator. An HFA-fluticasone propionate metered dose inhaler (MDI; 125 microg/dose) was inserted into the VHC, two actuations were delivered, and the filters were subsequently assayed using high-pressure liquid chromatography (HPLC). Testing and sample assay order was randomized, and HPLC assays were undertaken blinded. Drug delivery efficiency expressed as a percentage of the total dose of fluticasone propionate (250 microg) for the AeroChamber Max VHC "out-of-the-package" was 22.0(0.7)% (mean [99% CI]) and 21.2(1.5)% when pre-washed/rinsed. Results for the pre-washed ProChamber and OptiChamber Advantage VHCs were 10.2(0.55)% and 8.8(1.9)%, respectively. The more efficient delivery of medication via VHCs made from electrostatic charge dissipative materials should be considered when choosing doses for small children.

Estimation of microsomal CYP1A2 activity by high performance liquid chromatography

Phenacetin has been reported to be a reliable probe for the estimation of microsomal CYP1A2 activity. Phenacetin is converted to paracetamol by CYP1A2. A high pressure liquid chromatographic assay for the estimation of CYP1A2 activity in microsomes, was evaluated with caffeine as the internal standard. An RP C-18 supelcosil (150 × 4.6 mm, 5 µm) column was used for the assay. The mobile phase composition was acetonitrile:water (15:85), with a flow rate of 0.7 ml/min, injection volume of 100 µl, and detection at 240 nm. The retention times for paracetamol, caffeine and phenacetin were found to be 4.9, 8.6 and 15.5 min, respectively. The limit of detection was 0.1 µM for phenacetin, and the limit of quantitation was 0.2 µM for paracetamol. The percent coefficient of variation associated with paracetamol determination after duplicate estimation, was found to be in the range 0.02 to 7.7% for buffer matrix, and 0.8 to 9.7% for microsomal matrix. The mean rate of paracetamol formation in rat liver and guinea pig liver microsomes was 0.027 and 0.161 nmoles/min/nmole of P450, respectively. No interference was observed from incubation mixture components.

GTP Cyclohydrolase II Structure and Mechanism

GTP cyclohydrolase II converts GTP to 2,5-diamino-6-beta-ribosyl-4(3H)-pyrimidinone 5'-phosphate, formate and pyrophosphate, the first step in riboflavin biosynthesis. The essential role of riboflavin in metabolism and the absence of GTP cyclohydrolase II in higher eukaryotes makes it a potential novel selective antimicrobial drug target. GTP cyclohydrolase II catalyzes a distinctive overall reaction from GTP cyclohydrolase I; the latter converts GTP to dihydroneopterin triphosphate, utilized in folate and tetrahydrobiopterin biosynthesis. The structure of GTP cyclohydrolase II determined at 1.54-A resolution reveals both a different protein fold to GTP cyclohydrolase I and distinctive molecular recognition determinants for GTP; although in both enzymes there is a bound catalytic zinc. The GTP cyclohydrolase II.GMPCPP complex structure shows Arg(128) interacting with the alpha-phosphonate, and thus in the case of GTP, Arg(128) is positioned to act as the nucleophile for pyrophosphate release and formation of the proposed covalent guanylyl-GTP cyclohydrolase II intermediate. Tyr(105) is identified as playing a key role in GTP ring opening; it is hydrogen-bonded to the zinc-activated water molecule, the latter being positioned for nucleophilic attack on the guanine C-8 atom. Although GTP cyclohydrolase I and GTP cyclohydrolase II both use a zinc ion for the GTP ring opening and formate release, different residues are utilized in each case to catalyze this reaction step.

Study on early diagnosis of invasive pulmonary aspergillosis in a rat model

目的 建立高效液相色谱技术(HPLC)检测(1-3)-β-D-葡聚糖的方法, 并与巢式聚合酶链反应(PCR)方法进行比较,评价其在实验大鼠侵袭性肺曲霉病(IPA)早期诊断中的意义.方法将烟曲霉孢子注入实验大鼠左肺以制作IPA模型;采集各组实验大鼠血样及脏器标本,进行巢式PCR、HPLC及培养方法的检测.结果 HPLC检测(1-3)-β-D-葡聚糖最低检测限度为1～2 pg/ml,临界值为15 pg/ml.模型组检测值和阳性率均较对照组高,差异有统计学意义(P＜0.01);随感染时间延长检测值和阳性率逐渐上升,各组差异有统计学意义(P＜0.01);感染死亡大鼠血液标本的检测值与死亡时间呈负相关关系,其相关系数r为-0.949(P＜0.01).1周内HPLC方法敏感度(77.8%)和特异度(91.7%)均较巢式PCR高,但差异无统计学意义(P＞0.05),两者敏感度均高于血培养,差异均有统计学意义(P＜0.01).结论 HPLC 检测大鼠血中(1～3)-β-D-葡聚糖浓度,在早期预测大鼠肺曲霉感染的发生、发展及预后判断方面明显优于传统的血培养,也要优于巢式PCR方法。

In vitro metabolism of a thrombin inhibitor and quantitation of metabolically generated cyanide

During the metabolic characterization of compound I, 2-{6-cyano-3-[(2,2-difluoro-2-pyridin-2-ylethyl)amino]-2-oxopyrazin-1(2H)-yl]- N-[(3-fluoropyridin-2-yl)methyl]acetamide, evidence was obtained for extensive oxidative bioactivation of the pyrazinone ring system and some of the resulting metabolites were apparently devoid of the cyano moiety. Two assays, a spectrophotometric and a high-pressure liquid chromatography (HPLC) pre-column derivatization method, were evaluated for their ability to detect and quantify cyanide that is metabolically generated from liver microsomal incubations. When I was incubated (45 μM) in the presence of NADPH-fortified human liver microsomes for 2 h, 7.5 μM of cyanide was detected using the spectrophotometric assay and 8.9 μM was measured using the HPLC methodology. Overall, the results from the two assays appeared to agree reasonably well with each other. However, the HPLC assay was the preferred method for the evaluation of cyanide formation in vitro due to its sensitivity, reliability, and ease of use.

Neurochemical characterization of nervous elements innervating the body wall of earthworms (Lumbricus, Eisenia): immunohistochemical and pharmacological studies

The distribution and chemical neuroanatomy of nervous elements and certain pharmacological-physiological characteristics of the innervation of the body wall in earthworms are described. Solitary sensory bipolar cells can be found among the epithelial cells. These bipolar cells contain serotonin, tyrosine hydroxylase, histamine, gamma-amino-butyric acid (GABA), Eisenia tetradecapeptide, proctolin or rhodopsin in various combinations. In the body wall, the plexus sub-muscularis is composed of nerve fibres only, whereas the plexus sub-epithelialis and muscularis also contain solitary nerve cells. These cells display histamine, GABA or neuropeptide Y immunoreactivity. The fibres of the three plexuses are reactive to serotonin, histamine, Eisenia tetradecapeptide, proctolin, GABA and neuropeptide Y antibodies. FMRFamide-immunoreactive fibres of the plexus muscularis originate from the central nervous system, whereas axons containing the other studied molecules are derived from both peripheral and central structures. High pressure liquid chromatography assays have revealed serotonin, dopamine and histamine in the body wall. Contractions of the body wall musculature can be elicited with serotonin and FMRFamide. Serotonin-evoked contractions are suppressed by the application of GABA. Serotonin acts both directly on the muscle cell receptors and indirectly through initiating transmitter release from the nervous elements, whereas the FMRFamide-induced contractions seem to be mediated through the muscle cell receptors only. The pharmacological profiles of the serotonin and GABA receptors resemble those of the vertebrate 5-HT(3) and GABA(B) receptor types. Our findings indicate that both the sensory and efferent system of the annelid body wall operate by means of a variety of neuroactive compounds, suggesting a complex role of signalling systems in the regulation of this organ.

Penetration of topically applied gatifloxacin 0.3%, moxifloxacin 0.5%, and ciprofloxacin 0.3% into the aqueous humor

To investigate the aqueous penetration of 3 commercially available ophthalmic fluoroquinolones. Prospective, double-masked, clinical study. Fifty-two eyes of 52 patients. Fifty-two patients undergoing cataract surgery were given preoperative topical gatifloxacin 0.3% (Zymar), moxifloxacin 0.5% (Vigamox), or ciprofloxacin 0.3% (Ciloxan). The patients were instructed to use their antibiotic drops 4 times a day for 3 days before surgery. On the day of surgery, patients were given their assigned antibiotic every 15 minutes for 3 doses, 1 hour before their procedure. At the time of surgery, 0.1 ml of aqueous fluid was aspirated from the anterior chamber with an air cannula needle attached to a tuberculin syringe. The aspirate was immediately stored at -70 degrees C. Fluoroquinolone concentrations were determined by reverse-phase high-pressure liquid chromatography assay technique with ultraviolet detection at a wavelength of 275 nm. Mean aqueous concentration of gatifloxacin in 16 eyes was 0.63 microg/ml (standard deviation [SD], 0.30), moxifloxacin in 14 eyes was 1.31 microg/ml (SD, 0.46), and the mean concentration of ciprofloxacin in 22 eyes was 0.15 microg/ml (SD, 0.11). Both moxifloxacin (P<0.001) and gatifloxacin (P<0.005) penetrated the aqueous humor at significantly higher levels than ciprofloxacin. Moxifloxacin penetrated into the aqueous humor at significantly higher levels than gatifloxacin (P<0.05). The anterior chamber levels of moxifloxacin and gatifloxacin may be due to the difference in antibiotic concentration.

Corneal pharmacokinetics of topically applied azithromycin and clarithromycin

To determine corneal levels of topically administered azithromycin and clarithromycin in a rabbit model. Experimental animal study. Corneas of New Zealand albino rabbits were treated with topical azithromycin (2 mg/ml or 4 mg/ml) or clarithromycin (10 mg/ml). Topical azithromycin was prepared from an intravenous solution and topical clarithromycin from a suspension for oral use. All rabbits received one drop every 2 hours on the right eye. Groups of rabbits were treated for the following intervals: 6, 12, 24, and 48 hours (four rabbits for each combination of time point, drug, and dose). Corneal tissue was removed 1 hour after the last application. To investigate stability of tissue azithromycin levels, an additional group of four rabbits was treated for 24 hours, but corneal tissue was not removed until 24 hours later. Samples were homogenized, and drug concentrations were measured using high-pressure liquid chromatography (HPLC) analysis and bioactivity assay. Corneal concentrations of azithromycin increased with drug dosage and duration of application. Rabbits treated with azithromycin tolerated the drug well without signs of irritation. Clarithromycin was undetectable in corneal tissue by HPLC and bioactivity assay for all rabbits. Some rabbits treated with clarithromycin had signs of ocular surface irritation. Measurable concentrations of azithromycin are achieved in corneal tissue after topical application in a rabbit model, and the drug is well tolerated. Azithromycin may be a useful antibiotic for the topical treatment of human corneal infections, but clarithromycin, in currently available formulations, may not be effective because of poor tissue penetration.

PHASE I PHARMACOKINETIC STUDY OF A SINGLE INTRAVESICAL INSTILLATION OF GEMCITABINE ADMINISTERED IMMEDIATELY AFTER TRANSURETHRAL RESECTION PLUS MULTIPLE RANDOM BIOPSIES IN PATIENTS WITH SUPERFICIAL BLADDER CANCER

In this phase I study we determined the pharmacokinetic and toxicity profiles of a single intravesical instillation of gemcitabine administered immediately after complete transurethral resection (TUR) plus multiple random biopsies. Ten patients with superficial bladder cancer clinically staged as Ta/T1 with no carcinoma in situ were included. A single dose of gemcitabine was administered intra-vesically immediately after TUR plus 6 random biopsies. Five patients received 1,500 mg and 5 received 2,000 mg diluted in 100 ml saline. Retention time in the bladder was 60 minutes. Concentrations of gemcitabine and dFdU (2',2'-difluoro-2'-deoxyuridine) were determined by high pressure liquid chromatography assay. Treatment was clinically well tolerated in all patients. Two patients in the 1,500 mg group had minimal hipogastric discomfort and 1 in the 2,000 mg group had grade 1 bladder spasms. There was no remarkable systemic toxicity on hematology or biochemistry at any dose level on day 12 or 30. One patient per dose level showed tumor recurrence on 3-month repeat cystourethroscopy. Mean maximum gemcitabine concentration was 1.8 microg/ml and the mean last AUC was 158 microg/ml*minute. There was large interpatient variability but no significant differences between the 2 dose levels. Single intravesical instillation of gemcitabine immediately after TUR and multiple random biopsies for superficial bladder cancer are a safe and well tolerated treatment. The favorable toxicity and pharmacokinetic profiles of intravesical gemcitabine support future phase II studies with this agent.

Spread of novel aminoglycoside resistance gene aac(6')-Iad among Acinetobacter clinical isolates in Japan.

A novel aminoglycoside resistance gene, aac(6')-Iad, encoding aminoglycoside 6'-N-acetyltransferase, was identified in Acinetobacter genospecies 3 strain A-51. The gene encoded a 144-amino-acid protein, which shared modest identity (up to 36.7%) with some of the aminoglycoside 6'-N-acetyltransferases. The results of high-pressure liquid chromatography assays confirmed that the protein is a functional aminoglycoside 6'-N-acetyltransferase. The enzyme conferred resistance to amikacin, tobramycin, sisomicin, and isepamicin but not to gentamicin. The prevalence of this gene among Acinetobacter clinical isolates in Japan was then investigated. Of 264 Acinetobacter sp. strains isolated from geographically diverse areas in Japan in 2002, 16 were not susceptible to amikacin, and aac(6')-Iad was detected in 7. Five of the producers of aminoglycoside 6'-N-acetyltransferase type Iad were identified as Acinetobacter baumannii, and two were identified as Acinetobacter genospecies 3. These results suggest that aac(6')-Iad plays a substantial role in amikacin resistance among Acinetobacter spp. in Japan.

The K+-induced increases in noradrenaline and dopamine release are accompanied by reductions in the release of their intraneuronal metabolites from the rat anterior hypothalamus. An in vivo brain microdialysis study.

The novel technique of microdialysis has been used to examine the basal and K+-induced release of catecholamines and metabolites from the anterior hypothalamus of the urethane-anaesthetized rat in vivo. A high pressure liquid chromatographic assay was developed to simultaneously measure endogenous noradrenaline, dopamine and their intraneuronal metabolites 3,4-dihydroxyphenylglycol (DOPEG) and 3,4-dihydroxyphenylacetic acid (DOPAC) respectively, in each 60 microliters dialysate sample. The effect of replacing Ca2+ in the perfusion fluid with a low concentration of Cd2+, which blocks Ca2+ effects, was also studied. Increasing the K+ concentration in the perfusion fluid elicited a concentration-dependent increase in noradrenaline and dopamine release. In contrast, there were marked reductions in DOPEG and DOPAC which were not concentration-dependent. In the Ca2+-depleted conditions, the K+-induced increase in amine release was significantly attenuated, but the reductions in the metabolites were not affected. We suggest that the mechanisms contributing to the observed reductions in the metabolites may be inhibition of neuronal reuptake, an increase in neuronal efflux, an enhancement of vesicular uptake and a decrease in vesicular efflux.

Biosynthesis, Purification, and Substrate Specificity of Severe Acute Respiratory Syndrome Coronavirus 3C-like Proteinase

The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. In order to understand the active form and the substrate specificity of the enzyme, we have cloned, expressed, and purified SARS 3C-like proteinase. Analytic gel filtration shows a mixture of monomer and dimer at a protein concentration of 4 mg/ml and mostly monomer at 0.2 mg/ml, which correspond to the concentration used in the enzyme assays. The linear decrease of the enzymatic-specific activity with the decrease of enzyme concentration revealed that only the dimeric form is active and the dimeric interface could be targeted for structural-based drug design against SARS 3C-like proteinase. By using a high pressure liquid chromatography assay, SARS 3C-like proteinase was shown to cut the 11 peptides covering all of the 11 cleavage sites on the viral polyprotein with different efficiency. The two peptides corresponding to the two self-cleavage sites are the two with highest cleavage efficiency, whereas peptides with non-canonical residues at P2 or P1′ positions react slower. The P2 position of the substrates seems to favor large hydrophobic residues. Secondary structure studies for the peptide substrates revealed that substrates with more β-sheetlike structure tend to react fast. This study provides a basic understanding of the enzyme catalysis and a full substrate specificity spectrum for SARS 3C-like proteinase, which are helpful for structural-based inhibitor design against SARS and other coronavirus.

Sex differences in nevirapine disposition in HIV-infected patients.

To evaluate the role of sex in nevirapine pharmacokinetics, we analysed the pharmacokinetic profile of nevirapine in 11 male and 11 female HIV-positive patients chronically treated with nevirapine 400 mg once a day. Statistically significant inter-group differences in the plasma concentration and fluctuations between that and the concentration just before the next dose, at steady-state, were observed. Variations in lean body mass and composition affecting nevirapine distribution might account for inter-sex discrepancies in nevirapine plasma levels.

Ensuring Supplies of Quality Diethylcarbamazine Citrate (DEC)

In 1997, the World Health Organization (WHO) made a commitment to eliminate lymphatic filariasis. The WHO Global Program to Eliminate Lymphatic Filariasis (WHO-FIL) needed a reliable supply of diethylcarbamazine citrate (DEC) of known acceptable quality at an affordable price, so in August 1999, it started the DEC Project. Today's standards required development of a modern stability-indicating assay method for DEC and for DEC tablet dissolution. ADD Advanced Drug Delivery Technologies (Switzerland) developed a high-pressure liquid chromatography assay, which was independently validated and is in the United States Pharmacopoeia 25 (2002). After a global search, the project found that almost all existing and potential DEC active pharmaceutical ingredient (API) and tablet manufacturers are in low-income countries. The project constructed an audit team to conduct on-site audits to assess good manufacturing practices according to European Union standards. National/state inspectors accompany the audit team. The team prequalified one DEC API manufacturer and three DEC tablet manufacturers. The project plans to increase the number of prequalified DEC manufacturers. Now, WHO-FIL only purchases from prequalified manufacturers. Consolidation of several national program DEC requirements into a limited international competitive bid reduced the price for DEC tablets between 30% and 45%, compared to previous small-scale WHO purchases.

An Antisense Oligonucleotide to 1-cys Peroxiredoxin Causes Lipid Peroxidation and Apoptosis in Lung Epithelial Cells

1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, reduces phospholipid hydroperoxides as well as organic peroxides and H(2)O(2). To determine the physiological function(s) of 1-cysPrx, we have used an antisense strategy to suppress endogenous 1-cysPrx in L2 cells, a rat lung epithelial cell line. A 25-base antisense morpholino oligonucleotide was designed to bind a complementary sequence overlapping the translational start site (-18 to +7) in the rat 1-cysPrx mRNA, blocking protein synthesis. Treatment with an antisense oligonucleotide for 48 h resulted in approximately 60% suppression of the 1-cysPrx protein content as measured by immunoblot analysis and an approximately 44% decrease of glutathione peroxidase activity as compared with random oligonucleotide treated and control (vehicle only) cells. Accumulation of phosphatidylcholine hydroperoxide in plasma membranes was demonstrated by high pressure liquid chromatography assay for conjugated dienes (260 pmol/10(6) cells for antisense versus 70 pmol/10(6) cells for random oligonucleotide and control cells) and by fluorescence of diphenyl-1-pyrenylphosphine, a probe for lipid peroxidation. The percentage of cells showing positive staining for annexin V and propidium iodide after antisense treatment was 40% at 28 h and 80% at 48 h. TdT-mediated dUTP nick end labeling assay at 48 h indicated DNA fragmentation in antisense-treated cells that was blocked by prior infection with adenovirus encoding 1-cysPrx or by pretreatment with a vitamin E analogue. The results indicate that 1-cysPrx can function in the intact cell as an antioxidant enzyme to reduce the accumulation of phospholipid hydroperoxides and prevent apoptotic cell death.

Pitfalls in cefepime titration from human plasma: plasma- and temperature-related drug degradation in vitro.

While developing a high-pressure liquid chromatography assay for cefepime in plasma, we observed significant drug degradation at 20 and 37 degrees C but not at 4 degrees C. This plasma-related degradation persisted after protein removal. This warrants caution regarding cefepime assays for pharmacokinetic and pharmacodynamic studies of cefepime in vitro and in vivo.

Urinary cortisol as an additional tool to assess the welfare of pregnant sows kept in two types of housing.

The use of urinary cortisol (UC) as an additional tool to evaluate sows welfare was assessed in two experiments. In a preliminary methodological experiment, the kinetics of cortisol excretion in urine was studied during an Adreno Cortico Trophic Hormone (ACTH) challenge test in 10 pregnant sows. In a second experiment, 96 primiparous sows of an experimental unit were assigned to two different housing systems: 48 animals were housed in individual pens (IP) and 48 animals in collective pens (CP) with 6 animals per pen. UC was measured at the beginning and at the end of pregnancy and compared with other welfare indicators such as behaviour or skin damage. In both experiments, UC was measured using a high pressure liquid chromatography assay. In experiment 1, UC was constant on the day before injection of ACTH, with no variations related to circadian rhythm. It began to rise 2 h after the injection, peaked between 2 to 5 h after then returned to the basal concentration on the day after the injection. In experiment 2, UC concentrations were not different between CP- and IP-housed sows but they were higher in sows exhibiting the less stereotypies in comparison with sows exhibiting the most stereotypies. The results of this study suggest that UC is a good indicator of acute stress, more convenient than plasma cortisol measurement since it is a non-invasive method avoiding restraint or catheterisation of sows. They also suggest that UC could also give additional information on the assessment of chronic stress and improve the evaluation of animal welfare if used in conjunction with other welfare indicators.

Therapeutic drug monitoring of HIV protease inhibitors using high-performance liquid chromatography with ultraviolet or photodiode array detection.

Published data suggest that therapeutic drug monitoring of human immunodeficiency virus protease inhibitors would improve the management of antiretroviral therapy. The authors have developed a high-pressure liquid chromatographic assay allowing simultaneous determination of six protease inhibitors (ritonavir, saquinavir, indinavir, nelfinavir, amprenavir, and lopinavir), using carbamazepine as internal standard. Detection was based on a dual wavelength ultraviolet spectrophotometer and can be improved by the use of a photodiode array detector. Monitoring was performed 1 month after initiation of therapy or in instances of therapeutic failure, side effects, suspicion of noncompliance, drug interactions, or malabsorption. Trough concentrations were 0.15 to 13.6 mg/L for ritonavir, 0.06 to 9.7 mg/L for indinavir, 0.03 to 5.5 mg/L for saquinavir, and 0.15 to 4.15 mg/L for nelfinavir. Concentrations below the limit of quantification were observed in 63/438 (14%) of the patients. Target concentrations are not well established, and reported in vitro inhibitory concentrations may be of limited value. The authors therefore chose to compare observed concentrations with mean plasma concentrations reported in clinical trials. Observed saquinavir and indinavir concentrations were often below or close to these target concentrations, particularly when used as a single protease inhibitor. Concentration-controlled studies should now be used to select proper target concentrations for each protease inhibitor, either prescribed alone or in combination.