Background: Glucosamine sulfate is used for the treatment of arthrosis, especially osteoarthritis of the knee joint. The available evidence suggests differences in its pharmacokmetics in Chinese subjects compared with non-Chinese subjects.Objective: The aim of this study was to compare the pharmacokinetics and relative bioavailability of a test and reference formulation of glucosamine sulfate 500 mg after single oral administration in healthy Chinese volunteers.Methods: This open-label, randomized-sequence, single-dose, 2-way crossover study was performed at the First Hospital of Nanjing Medical University, Nanjing, China. Eligible subjects were healthy male volunteers who were randomly assigned at a 1:1 ratio to receive a single 500-mg dose of the test or reference capsule formulation, followed by a 1-week washout period and administration of the alternate formulation. The study drugs were administered after a 12-hour overnight fast. Glucosamine sulfate was assayed using a liquid-chromatography tandem mass spectrometry method. For analysis of pharmacokinetic properties, including C-max, AUC(0-t), and AUC(0-infinity), blood samples were obtained at intervals over a 14-hour period after study drug administration. The formulations were considered bioequivalent if the log-transformed ratios of Cmax and AUC were within the predetermined equivalence range (70%-143% for C-max and 80%-125% for AUC) as established by the State Food and Drug Administration (SFDA) of China. Tolerability was assessed by monitoring vital signs and laboratory tests (hematology; blood biochemistry, hepatic function, and urinalysis), and by questioning subjects about adverse events (AEs).Results: Twenty-two healthy male Chinese subjects were enrolled (mean [range] age, 24 [22-26] years; weight, 63.9 [58.5-69.3] kg; height, 172 [1.67-177] cm); all completed the study. No period or sequence effect was observed. The 90% CIs for the log-transformed ratios of C-max, AUC(0-t), and AUC(0-infinity) were 93.4 to 127.3, 92.4 to 114.5, and 92.7 to 114.6, respectively (all, P = NS). The AUC(0-infinity) of the test and reference formulations was 1.83 (0.66) and 1.77 (0.72) mu g/h/mL, respectively. No AEs were observed or reported during the study.Conclusions: In this small study in healthy male Chinese volunteers, a single 500-mg dose of the test formulation met the SFDA's regulatory definition for bioequivalence to the reference formulation. Both formulations were well tolerated. (Clip Ther. 2009; 31:1551-1558) (C) 2009 Excerpta Medica Inc.

BACKGROUND:The role of the carotid baroreflex in blood pressure regulation has been known for a long time but its effects were thought to be short lived. Recent data indicate that stimulation of carotid baroreceptors may lower blood pressure not only for short periods of time, but also in the long run. OBJECTIVE/METHODS:Recent advances in technology permitted the development of a new device (Rheos) that addresses problems with older devices. Several questions remain to be addressed before Rheos can be used widely, and several potential clinical applications remain to be clarified. This review examines these issues and comprehensively describes this therapeutic approach. RESULTS/CONCLUSIONS:The carotid baroreceptor reflex is probably not completely in control of blood pressure. Baroreflexes are one of many control systems acting in concert.

A sensitive and specific liquid chromatography-tandem-mass spectrometry method was developed and validated for the simultaneous determination of clopidogrel and its carboxylic acid metabolite (SR26334) in human plasma using nateglinide and pioglitazone as internal standards. Analytes were extracted from 0.50 mL of plasma using diethyl ether–n-hexane (4:1, v/v). Chromatographic separation was performed on a Teknokroma C18 column with a mobile phase of methanol–water (containing 0.1% formic acid) (80:20, v/v) at a flow rate of 0.20 mL min−1 within 5.6 min. Linearity was established over the concentration range of 0.005–5 ng mL−1 for clopidogrel and 20–2,500 ng mL−1 for SR26334. Intra- and inter-batch standard deviations were less than 9.2% and the accuracy of this assay was found to fall within an acceptable range ≤10.0%. The method was successfully applied to the therapeutic drug monitoring of clopidogrel.

The role of mitochondrial injury in the pathogenesis of complications of uremia is incompletely defined, although diminished bioenergetic capacity and the accumulation of mitochondrial DNA (mtDNA) mutations have been reported. This study was undertaken to evaluate the prevalence of mtDNA injury in 180 patients who had ESRD and were enrolled into the baseline phase of the HEMO study and to relate these markers to all-cause mortality. The mitochondrial injury markers studied in peripheral blood mononuclear cells were the mtDNA copy number per cell, measured by quantitative PCR, and the presence of the mtDNA(4977) mutation. After frequency-matching healthy control subjects for age, mtDNA copy number was lower among older dialysis patients compared with older healthy subjects (P = 0.01). A one-log increase in mtDNA copy number was independently associated with a decreased hazard for mortality (adjusted hazard ratio 0.64; 95% confidence interval 0.46 to 0.89). The mtDNA(4977) deletion was present in 48 (31%) patients and was independently associated with a decreased hazard for mortality (adjusted hazard ratio 0.33; 95% confidence interval 0.19 to 0.56). In summary, the mtDNA(4977) seems to predict survival in ESRD, but a reduced mitochondrial copy number seems to predict a poor outcome. Although further exploration of these associations is needed, evaluation of mitochondrial DNA copy number and somatic mtDNA mutations may provide simple genomic biomarkers to predict clinical outcomes among patients with ESRD.

BACKGROUNDInterleukin-6 (IL-6) is a mediator and marker of the chronic inflammatory process that is responsible for much of the morbidity and mortality seen in hemodialysis (HD) patients. This study evaluated circulating plasma IL-6 as a predictor of all-cause mortality and cardiovascular mortality and studied its relationship to prevalent comorbidity and hypoalbuminemia, in a cohort of stable HD patients enrolled in the HEMO study.METHODSClinical data included demographic, medical, and routine laboratory parameters. Comorbidities were graded using the Index of Co-Existing Diseases (ICED). Outcomes of interest were all-cause mortality and cardiovascular mortality. Blood samples were drawn at enrollment and annually, and plasma IL-6 levels measured with high-sensitivity enzyme-linked immunosorbent assay.RESULTSMedian plasma IL-6 level in 206 patients was 7.9 pg/mL (range, 0.1 to 90.3 pg/mL) and was higher in patients with vascular disease ( P = 0.03), higher ICED scores ( P = 0.01), and lower Karnofsky indices ( P < 0.01). Serum albumin was inversely related to plasma IL-6 levels ( P = 0.03, r = -0.16). Unadjusted median survival time was 1,209 days in the lowest quartile of plasma IL-6 and 806 days in the highest ( P = 0.02, log rank test). A 1-log increase in plasma IL-6 was associated with a 1.19-fold higher adjusted risk for all-cause mortality ( P = 0.04; 95% confidence interval, 1.01 to 1.40) and a 1.43-fold higher adjusted risk of cardiovascular mortality ( P = 0.02; 95% confidence interval, 1.06 to 1.92). Hazard ratio estimates were higher when IL-6 levels over time were incorporated as a time-dependent covariate.CONCLUSIONPlasma IL-6 levels are strongly associated with comorbidity in HD patients and are a powerful predictor of cardiovascular and all-cause mortality.

Background: Although cytokines play a pivotal role in the inflammatory responses that mediate the severity of acute renal failure (ARF), the importance of pro- and anti-inflammatory cytokine gene promoter polymorphisms has been unexplored.Methods: We prospectively evaluated the relationship of single nucleotide polymorphism in the promoter region of tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) to mortality in 61 patients with ARF requiring intermittent hemodialysis. Cytokine genotyping was performed on leukocytes using PCR techniques. Cox proportional-hazards regression analysis was used to explore these relationships.Results: The mean (+/-SD) APACHE II score was 24 +/- 7, MOF score 2 +/- 1, and 64% had sepsis. The TNF-alpha high producer genotype (-308 A-allele carrier) was associated with a higher risk of death after adjustment for the APACHE II score (HR = 2.5; P = 0.04), and the IL-10 intermediate/high producer genotype (-1082 G-allele carrier) was associated with a lower risk of death after adjustment for the MOF score (HR = 0.36; P = 0.03). Considering combinations of genotypes, the TNF-alpha high and IL-10 low producer genotype combination was associated with a similar to6-fold increased risk of death compared to the TNF-alpha-low and IL-10 intermediate/high producer genotype combination, after adjustment for either APACHE II (P = 0.004), MOF score (P = 0.004) or sepsis (P = 0.006).Conclusions: TNF-alpha and IL-10 gene polymorphisms are related to the risk of death among patients with ARF who require dialysis. Larger studies are needed to confirm these relationships. (C) 2004 Elsevier Ltd. All rights reserved.

BACKGROUNDAtherosclerotic vascular disease is a leading cause of morbidity and mortality in patients with end-stage renal disease (ESRD) on maintenance hemodialysis (HD). Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that inhibits the atheromatous process. We studied coding region polymorphisms of the TGF-beta1 gene (+869 T --> C at codon 10 and +915 G --> C at codon 25) as genetic susceptibility factors for prevalent vascular disease and cardiac outcomes in a cohort of HD patients enrolled in the HEMO Study.METHODSGenotyping was carried out using polymerase chain reaction-sequence specific primer (PCR-SSP) methods with a cytokine genotyping tray. Prevalent vascular disease was coded from the Index of Disease Severity (IDS) scores for ischemic heart disease (IHD), peripheral vascular disease (PVD), cerebrovascular disease (CVD), and congestive heart failure (CHF), 0 indicating absence, and 1 to 3 increasing grades of severity. The presence of any vascular disease (VD) (i.e., any degree of IHD/PVD/CVD), and the number of coexistent vascular system diseases per patient were derived. Cardiac outcomes, one of the secondary outcomes of the HEMO Study, were expressed as a composite of the first hospitalization for, or death from, cardiac causes.RESULTSThe cohort consisted of 183 patients at enrollment, 56% male, 44% African American (AA), and 40% diabetic. The mean age was 62.4 +/- 12.2 years, and median dialysis vintage 2.02 years. The most frequent genotype at codon 10 was T/C (67%), and at codon 25 was G/G (72%). IHD was present in 52% of patients; 65% had at least one vascular system involvement, and 31% had 2 or more. On both univariate and multivariate analysis, the G/C genotype at codon 25 was significantly associated with the presence and extent of vascular disease at enrollment. The median time to cardiac outcome, defined as a composite of the first hospitalization for, or death from, cardiac causes, was 411 days in patients with the G/C genotype compared with 851 days in those with the G/G genotype (P= 0.03). Patients with the G/C genotype had a 1.6-fold increased hazard for cardiac outcomes after adjustment for baseline covariates (P= 0.04).CONCLUSIONThe G/C substitution at codon 25 was associated with an increased risk for prevalent vascular disease, new onset cardiac morbidity, and cardiac mortality in HD patients, and may be a genetic susceptibility factor for the development of atherosclerosis. Further studies are required to evaluate the role of TGF-beta1 as a candidate gene.

Background. Polymorphonuclear leukocyte (PMNL) production of reactive oxygen species (ROS) has been linked to hemodialysis (HD) associated morbidity. The effect of dialyzer membrane type and reuse on PMNL function has not been clearly defined.Methods. The present report is a cross-sectional study undertaken in a cohort of patients undergoing regular HD, at enrollment into the Hemodialysis (HEMO) Study, to study the association between patient and dialysis-related factors and PMNL function. PMNL function was assessed by measuring PMA- and N-formyl methionyl-leucyl-phenylalanine (fMLP) -induced respiratory burst, and phagocytic activity toward Staphylococcus aureus.Results. PMNL from patients dialyzed with polysulphone (PS) or cuprophane (CU) membranes showed higher PMA-induced respiratory burst activity compared with those exposed to substituted cellulose (cellulose acetate, cellulose triacetate, CA/CT) membranes, regardless of dialyzer reuse. The use of bleach as a cleansing agent during reuse was associated with higher PMA- induced PMNL superoxide production, as was the use of renalin when compared to aldehydes. In a subgroup of patients using PS dialyzers, reuse itself was associated with higher fMLP-induced superoxide production. The type of bleach-germicide combination during reuse showed that use of renalin as a germicide was also associated with higher PMNL phagocytosis index. The number of years on HD correlated inversely with PMA- induced PMNL superoxide response. Weaker PMNL response to fMLP was associated with greater comorbidity and poor functional status as quantified by Index of Coexisting Diseases (ICED) and Karnofsky scores, respectively.Conclusion. Our results indicate that dialyzer membrane type and the reuse process influence the oxidative response of PMNL among HD patients. The implications of these observations on clinical morbidity need to be further evaluated in prospective studies.

BACKGROUND Middle molecules such as beta2-microglobulin (beta2M) and advanced glycation end products (AGE)-modified proteins contribute to inflammation in uremia. The BetaSorb column is a new adsorptive device, which contains copolymeric beads, suitable for removal of beta2M and other middle molecules. We assessed the effect of this column on the bioreactivity of uremic plasma, as measured by cytokine responsiveness. METHODS Uremic plasma was perfused in vitro through the column (10 mL/min) and samples were collected after 10 to 30 passes. Endotoxin-stimulated tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) production by THP-1-derived monocytes was measured following brief exposure to uremic plasma. beta2M levels were measured. The contribution of AGE-modified proteins to the bioreactivity of uremic plasma was explored. RESULTS TNF-alpha and IL-10 production markedly decreased after 30 passes (629 +/- 78 vs. 144 +/- 62 pg/mL; 207 +/- 25 vs. 117 +/- 23 pg/mL; P=0.04). The column removed beta2M efficiently with a marked decline in plasma levels by 99% after 30 passes. Neutralization of AGE receptor (RAGE) resulted in a further reduction in the bioreactivity of uremic plasma. This was observed with nonperfused, as well as perfused, uremic plasma, suggesting that AGE-modified proteins were biologically active and still present after perfusion. CONCLUSION The sorbent beads removed uremic solute(s) that prime monocytes to enhanced cytokine production. Removal of beta2M was efficient, and of native and AGE-modified middle molecules likely.

Background: Interleukin-10 (IL-10), a potent regulatory monokine produced by activated mononuclear cells, provides an efficient autocrine mechanism for controlling proinflammatory cytokine synthesis. We hypothesized that defective synthesis of IL-10 could contribute to the inflammatory state in hemodialysis (HD) patients due to impaired feedback inhibition of proinflammatory cytokine production. Methods: We compared peripheral blood mononuclear cell (PBMC) synthesis and transcription of IL-10 and TNF-α in 12 patients with end-stage renal disease on long-term maintenance HD and a control group of 10 healthy subjects. Results: The synthesis of IL-10 by unstimulated PBMC was detectable in 5 of 12 (42%) HD patients as compared to 7 of 10 (70%) controls (p = 0.02). IL-10 synthesis in response to endotoxin (ET) by PBMC from HD patients was significantly lower when compared to the robust response in the control group (p = 0.008). Among the HD patients, there was a positive correlation between ET-stimulated IL-10 synthesis and the duration of time on dialysis. Unstimulated and ET-stimulated synthesis of TNF-α by PBMC did not differ between the 2 groups. In the HD patients, there was an inverse correlation between TNF-α and IL-10 synthesis by ET-stimulated PBMC, suggesting a regulatory effect of IL-10 on PBMC TNF-α synthesis. There was also an inverse correlation between plasma albumin and ET-stimulated TNF-α synthesis by PBMC among HD patients. TNF-α mRNA expression did not differ in HD patients relative to healthy controls. In contrast, when IL-10 mRNA from ET-stimulated PBMC was quantified, there was marked difference between the 2 groups indicating a transcriptional defect in IL-10 synthesis in PBMC from HD patients. Conclusion: Our observations indicate a marked abnormality in IL-10 synthesis by PBMC from HD patients probably related to a transcriptional defect. Low PBMC IL-10 synthesis may contribute to a chronic inflammatory state in these patients by defective feedback inhibition of proinflammatory cytokine production.

Recent technological advances have led to the development of a new adsorption column that allows removal of β-2 microglobulin (β2M) from the blood (BetciSorb™, RenalTech Int., New York, NY). The aim of this study was to assess the effect ot BetaSorb™ on the bioreactivity of uremic plasma mianly cytokine and hydrogen peroxide (H2O2) production by THP-1-moiiocytes. Plasma obtained pre dialysis from an ESRD patient at 5 distinct time points was used for in vitro perfusion experiments. Plasma was perfused over the column at a flow rate of 10 ml/min and samples were collected after 10, 20 and 30 passes. LPS-stimulated TNF-α and IL-10 production and H2O2 generation by THP-1-monocytes were measured following brief exposure to uremic plasma. β2M removal by the column was also examined. The column removed β2M efficiently as demonstrated by a marked decrease in β2M level by 91%, 98% and 99% after 10, 20 and 30 passes, respectively. Moreover, TNF-α and IL-10 production by THP-1-monocytes markedly decreased after 30 passes over the column while H2O2 production did not significantly change after 30 passes.TableThese results indicated that the BetaSorb™ perfusion column removes uremic solute(s) that prime monocytes to enhanced production of pro-inflammatory cytokines. Although this column is capable of removing β2M efficiently, studies are under way to elucidate mechanisms behind this drastic decline in cytokine production by monocytes after in vitro perfusion.

Apoptosis, or programmed cell death, is an active form of cell death that is initiated by a number of stimuli and is intricately regulated. Apoptosis in both excessive and reduced amounts has pathophysiologic implications. Accelerated programmed cell death has been observed in leukocytes among patients with chronic renal failure (CRF). This has been ascribed in part to the retention of uremic toxins. The Fas/Fas ligand (FasL) system is a key regulatory apoptotic pathway. Membrane-bound Fas is a cell-surface receptor that transduces apoptosis after interaction with membrane-bound or soluble FasL (sFasL). By contrast, soluble Fas (sFas) binds sFasL and inhibits its activity. In an attempt to examine the balance between these soluble factors in uremia, we measured soluble sFas and sFasL levels in the serum of healthy control subjects and patients with various degrees of CRF and examined the distribution of the various molecular mass fractions of these proteins in uremic serum. In brief, serum was obtained from 15 healthy volunteers, 17 patients with CRF, 11 patients undergoing maintenance hemodialysis (HD), and 7 patients undergoing peritoneal dialysis (PD). Serum sFas and sFasL were measured by enzyme-linked immunosorbent assay, and their molecular distribution was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot. Compared with results in healthy control subjects, sFas levels were significantly higher in patients with CRF and in patients undergoing dialysis. There was a significant inverse correlation between sFas levels and creatinine clearance. Serum sFasL levels were not different among the four groups. However, the sFas-to-sFasL ratio was significantly lower in healthy control subjects as compared with patients with CRF and patients undergoing dialysis. Immunoblots and densitometric analyses of sFas and sFasL depicted a known 48-kd sFas, a known 27-kd sFasL, and a 60-kd sFas-sFasL protein aggregate signal. In conclusion, serum sFas levels are increased in patients with various degrees of CRF and may bind circulating sFasL, thereby minimizing mediation of cellular apoptosis.