Quantitation of vancomycin and its crystalline degradation product (CDP-1) in human serum by high performance liquid chromatography

The delayed clearance of vancomycin results in accumulation of vancomycin crystalline degradation product, CDP-1, in the bodies of renally impaired patients. The 2 isomers, CDP-1-M (major) and CDP-1-m (minor), of CDP-1 are antibiotically inactive but cross-react with some immunoassays that use polyclonal antibodies resulting in falsely elevated results. A high performance liquid chromatographic (HPLC) method was developed to quantitate vancomycin and CDP-1 in the serum of renal patients. After solid phase extraction of 200 μl serum, the separation of vancomycin, the 2 isomers of CDP-1 and the internal standard (cefazolin) was accomplished by gradient HPLC on a reversed phase C18 column with detection at 210 nm. Linearity was established from 1 to 25 and 25 to 100 μg ml⁻¹ vancomycin and 1 to 25 μg ml⁻¹ CDP-1. Coefficients of variation for vancomycin and CDP-1 were 3.3–8.6% (n=10) and 2.8–5.2% (n=8).     

Bioequivalence evaluation of two brands of meloxicam tablets (Promotion and Mobicox): pharmacokinetics in a healthy female Mexican population

We conducted a randomized, crossover study in 23 healthy young female volunteers to compare the bioavailability of two brands of meloxicam (7.5 mg) tablets and to obtain pharmacokinetic parameters of this molecule in Mexican population not reported previously. Two tablets (15 mg) were administered as a single dose on 2 treatment days separated by a 1-week washout period. After dosing, serial blood samples were collected for a period of 72 h. Plasma harvested was analyzed for meloxicam by a modified and validated high-performance liquid chromatography (HPLC) method previously reported. Pharmacokinetic parameters AUC(0-t), AUC(0-alpha), C(max), T(max), k(e), MRT and t(1/2) were determined from plasma concentrations of both formulations, resulting in a C(max) 120% larger than and a T(max) 65% faster than those reported in other populations. AUC(0-t), AUC(0-alpha), and C(max) were statistically tested for bioequivalence after log transformation data in a non-balanced design, and no significant differences were found either in 90% classical confidence interval (90% CI) or in Schuirmann test (p < 0.05); thus, we concluded that bioequivalence existed between both formulations.     

阿托伐他汀对高血压脑出血患者血清神经元特异性烯醇化酶和S100B蛋白水平的影响及疗效观察

目的：探讨阿托伐他汀对高血压脑出血患者血清神经元特异性烯醇化酶（NSE）和S100B蛋白水平的影响及疗效观察.方法：选取高血压脑出血患者84例,随机分为观察组42例和对照组42例.两组患者均予以监控生命体征、控制颅压和血压、止血等常规治疗.观察组患者加用口服阿托伐他汀钙20 mg,qd,连用14d.评定两组患者治疗后的临床疗效及不良反应,并比较两组治疗前后血清NSE和S100B蛋白水平的变化.结果：治疗14 d后,两组患者血清NSE和S100B蛋白水平均有明显下降（P＜0.05或0.01）,且观察组下降幅度较对照组更明显（P＜0.05）;同时观察组患者的临床总有效率为92.86％,明显高于对照组的76.19％（P＜0.05）,两组患者治疗中均未发生明显药品不良反应.结论：阿托伐他汀治疗高血压脑出血的具有肯定的疗效,安全性较优,作用可能与其降低血清NSE和S100B水平,保护大脑神经元细胞密切相关.     

Comparison of gentamicin C1 and (C1a,C2)-levels in patients

Summary A high performance liquid chromatographic (HPLC) assay method has been used to measure gentamicin serum concentrations in patients receiving gentamicin complex. The HPLC method resolves gentamicin C₁ from the other two components, C_1a and C₂; gentamicin C_1a and C₂ cochromatograph. In the analysis of 46 serum samples collected from 16 patients it was found that the mean ratio (PHR) of the peak height of gentamicin C₁ to the height of the peak due to components C_1a and C₂ was 0.53±0.05; this value agreed well with the PHR's usually found from the HPLC analysis of aqueous solutions of gentamicin complex or of gentamicin dosage forms. In an additional two patients, the HPLC analysis of a sample of the gentamicin dosage form administered, a urine sample, and serum samples, resulted in almost identical PHR's for the respective patients. Finally, similar results were obtained from an experiment in a rabbit. It was concluded that the disposition of all three components of gentamicin complex are the same or very similar.     

The biliary excretion and enterohepatic circulation of benzo(a)pyrene and its metabolites in the rat

The enterohepatic circulation of benzo(a)pyrene (BP) has been investigated in the rat with a view to determining the availability of potentially toxic metabolities to tissues within this cycle. Some 60% of the dose of [¹⁴C]-BP (3 μmoles kg^?1, i.v.) is excreted in bile in 6 hr, with less than 3% in urine. The biliary metabolities are mainly polar conjugates; only 8% of the ¹⁴C in 2 hr bile samples can be directly extracted into ethyl acetate. However, following hydrolysis by β-glucuronidase some 40% of the ¹⁴C is extractable at pH 7. The extract consisted of polar metabolites (polyhydroxylated and/or conjugated; 37.5%), BP 4,5-diol (16.8%), BP 3,6-quinone (5.9%). 9-hydroxy BP (5.4%) and 3-hydroxy BP (5.3%) as indicated by co-chromatography with authentic standards on reversed phase HPLC, together with several unidentified metabolites. The proximate carcinogen BP 7, 8-diol was not detected. Biliary metabolites of [¹⁴C]-BP undergo enterohepatic recirculation in the rat; following the intraduodenal infusion of bile containing metabolites of [¹⁴C]-BP into bile duct cannulated rats, approximately 20% of the dose is absorbed and excreted in bile in 30 hr, with only 1% in urine. The pattern of metabolites in this bile is very similar to that in bile from rats administered [¹⁴C]-BP i.v. Following a single i.v. dose of [¹⁴C]-BP (3 μmoles kg^?1) to rats with re-entrant bile duct cannulae, which allowed intermittent collection of bile over a period of several days with minimal interference to the enterohepatic circulation, the proximate carcinogen BP 7, 8-diol was detected in recirculating bile. Biliary metabolites of BP, which have recently been shown to be mutagenic, can thus traverse the intestine to undergo enterohepatic circulation in the rat.     

Pharmacokinetics of inhibition of adenosine deaminase by erythro-9-(2-hydroxy-3-nonyl)adenine in CBA mice

The pharrnacokinetics oferythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) inhibition of adenosine deaminase (ADA) was measured in vivo in CBA mice. The in vivo assay utilized injection of 10–100 nmoles [2-³H]adenosine and measurement of blood ³H₂O 20 min later. A single oral dose of EHNA (50 mg/kg) totally inhibited ADA for 4 hr and caused a large increase in conversion of [2-³H]adenosine to [2-³H]ATP. EHNA (3 mg/kg) decreased deamination by 50% for 2–6 hr, depending on the dose of adenosine used. Mice dosed with EHNA (100 mg/kg) once daily for 7 days showed the same ADA recovery rate as mice dosed only once. High single oral doses of EHNA had no effect on blood ATP and GTP pools.     

Understanding the Increased Aggregation Propensity of a Light-Exposed IgG1 Monoclonal Antibody Using Hydrogen Exchange Mass Spectrometry,Biophysical Characterization,and Structural Analysis

This work compares the conformational stability, backbone flexibility, and aggregation propensity of monomer and dimer fractions of an IgG1 monoclonal antibody (mAb) generated on UVA light exposure for up to 72 h collected by preparative size-exclusion chromatography, compared with unstressed control. UVA light exposure induced covalent aggregation, and fragmentation as measured by size-exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and extensive oxidation of specific methionine residues (Met 257, Met 433, and Met 109) in both size fractions identified by reverse phase chromatography coupled to mass spectrometry. Compared with unstressed mAb, both the monomer and dimer fractionated from 72 h UVA light–exposed mAb had decreased thermal melting temperatures (T_m1) by 1.4°C as measured by differential scanning calorimetry, minor changes in tertiary structure as measured by near-UV CD, increased monomer loss, and aggregation on accelerated storage at 35°C. Hydrogen/deuterium exchange mass spectrometry identified local segments with increased flexibility in C_H2 and C_H3 domains of both size fractions, and decreased flexibility in few segments of F_ab and C_H1 domains in the dimer fraction. Segment 247-256 in heavy chain, an established aggregation hotspot in IgG1 mAbs had large increase in flexibility in both size fractions compared with unstressed mAb.     

Degradation of nociceptin (orphanin FQ) by mouse spinal cord synaptic membranes is triggered by endopeptidase-24.11: an in vitro and in vivo study

We analyzed spinal metabolic pathway of nociceptin/orphanin FQ related to pain-transmission or modulation in the both in vitro and in vivo experiments. Nociceptin was degraded by spinal synaptic membranes. Major metabolites of nociceptin were free phenylalanine, nociceptin (1-13) and nociceptin (14-17). Both the degradation of nociceptin and the accumulation of the major cleavage metabolites, nociceptin (1-13) and nociceptin (14-17), were strongly inhibited by a metal chelator and also by specific inhibitors of endopeptidase-24.11, thiorphan and phosphoramidon. Furthermore, purified endopeptidase-24.11 hydrolyzed nociceptin at the cleavage site (Lys(13)-Leu(14) bond) identical to that by spinal synaptic membranes. Recently, we have found that nociceptin, injected intrathecally at small doses (fmol order) elicits a behavioral response consisting of scratching, biting and licking in mice. In the present study, we have examined the effect of peptidase inhibitors on the behavioral response elicited by intrathecal injection of nociceptin in mice. Phosphoramidon simultaneously injected with nociceptin additively enhanced nociceptin-induced behavioral response, whereas the nociceptin-induced behavioral response was unaffected by either bestatin, an aminopeptidase inhibitor or captopril, an angiotensin-converting enzyme inhibitor. However, the nociceptin effect was potentiated by combined injection of phosphoramidon and bestatin, indicating that inhibition of aminopeptidase may also contribute to inducing the behavioral response to nociceptin. These data suggest that endopeptidase-24.11 plays a major role in initial stage of nociceptin metabolism at the spinal cord level in mice.     

On the biomarkers and mechanisms of konzo, a distinct upper motor neuron disease associated with food (cassava) cyanogenic exposure

Konzo is a self-limiting central motor-system disease associated with food dependency on cassava and low dietary intake of sulfur amino acids (SAA). Under conditions of SAA-deficiency, ingested cassava cyanogens yield metabolites that include thiocyanate and cyanate, a protein-carbamoylating agent. We studied the physical and biochemical modifications of rat serum and spinal cord proteins arising from intoxication of young adult rats with 50-200 mg/kg linamarin, or 200 mg/kg sodium cyanate (NaOCN), or vehicle (saline) and fed either a normal amino acid- or SAA-deficient diet for up to 2 weeks. Animals under SAA-deficient diet and treatment with linamarin or NaOCN developed hind limb tremors or motor weakness, respectively. LC/MS-MS analysis revealed differential albumin carbamoylation in animals treated with NaOCN, vs. linamarin/SAA-deficient diet, or vehicle. 2D-DIGE and MALDI-TOF/MS-MS analysis of the spinal cord proteome showed differential expression of proteins involved in oxidative mechanisms (e.g. peroxiredoxin 6), endocytic vesicular trafficking (e.g. dynamin 1), protein folding (e.g. protein disulfide isomerase), and maintenance of the cytoskeleton integrity (e.g. α-spectrin). Studies are needed to elucidate the role of the aformentioned modifications in the pathogenesis of cassava-associated motor-system disease.     

Transport of N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2)

N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine (Ac-DCVC) and S-(1,2-dichlorovinyl)-l-cysteine (DCVC) are the glutathione conjugation pathway metabolites of a common industrial contaminant and potent nephrotoxicant trichloroethylene (TCE). Ac-DCVC and DCVC are accumulated in the renal proximal tubule where they may be secreted into the urine by an unknown apical transporter(s). In this study, we explored the hypothesis that the apical transport of Ac-DCVC and/or DCVC may be mediated by the multidrug resistance associated protein 2 (Mrp2, ABCC2), which is known to mediate proximal tubular apical ATP-dependent transport of glutathione and numerous xenobiotics and endogenous substances conjugated with glutathione. Transport experiments using membrane vesicles prepared from mouse proximal tubule derived cells expressing mouse Mrp2 utilizing ATPase assay and direct measurements of Ac-DCVC/DCVC using liquid chromatography/tandem mass-spectrometry (LC/MS/MS) demonstrated that mouse Mrp2 mediates ATP-dependent transport of Ac-DCVC. Expression of mouse Mrp2 antisense mRNA significantly inhibited the vectorial basolateral to apical transport of Ac-DCVC but not DCVC in mouse proximal tubule derived cells endogenously expressing mouse Mrp2. The results suggest that Mrp2 may be involved in the renal secretion of Ac-DCVC.