The sulfatase enzymes, N-acetylgalactosamine-4-sulfatase (arylsulfatase B (ASB)) and galactose-6-sulfatase (GALNS) hydrolyze sulfate groups of CS. Deficiencies of ASB and GALNS are associated with the mucopolysaccharidoses. To determine if expression of ASB and GALNS impacts on glycosaminoglycans (GAGs) and proteoglycans beyond their association with the mucopolysaccharidoses, we modified the expression of ASB and GALNS by overexpression and by silencing with small interference RNA in MCF-7 cells. Content of total sulfated GAG (sGAG), chondroitin 4-sulfate (C4S), and total chondroitin sulfates (CSs) was measured following immunoprecipitation with C4S and CS antibodies and treatment with chondroitinase ABC. Following silencing of ASB or GALNS, total sGAG, C4S, and CS increased significantly. Following overexpression of ASB or GALNS, total sGAG, C4S, and CS declined significantly. Measurements following chondroitinase ABC treatment of the cell lysates demonstrated no change in the content of the other sGAG, including heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Following overexpression of ASB and immunoprecipitation with C4S antibody, virtually no sGAG was detectable. Total sGAG content increased to 23.39 (؎1.06) g/mg of protein from baseline of 12.47 (؎0.68) g/mg of protein following ASB silencing. mRNA expression of core proteins of the CS-containing proteoglycans, syndecan-1 and decorin, was significantly up-regulated following overexpression of ASB and GALNS. Soluble syndecan-1 protein increased following increases in ASB and GALNS and reduced following silencing, inversely to changes in CS. These findings demonstrate that modification of expression of the lysosomal sulfatases ASB and GALNS regulates the content of CSs. N-Acetylgalactosamine-4-sulfatase (arylsulfatase B, ASB)2 and galactose-6-sulfatase (GALNS) are two important lysosomal enzymes that hydrolyze sulfate groups from the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and dermatan sulfate. Deficiencies of ASB and GALNS activity are associated with the congenital mucopolysaccharidoses (MPS) MaroteauxLamy syndrome (MPS VI) and Morquio syndrome (MPS IVA), respectively. In these lysosomal storage diseases, there is accumulation of highly sulfated, unmetabolizable glycosaminoglycans and proteoglycans. The impact of sulfatase activity on metabolism of GAGs and the proteoglycans (PGs) with which they are associated has not been widely explored, beyond the congenital storage diseases.Recently, we reported that ASB activity is reduced in uncorrected cystic fibrosis cells, consistent with the accumulation of highly sulfated GAG in pulmonary secretions in cystic fibrosis patients. Following correction of cystic fibrosis transmembrane conductance regulator in pulmonary epithelial cell lines, ASB activity increased significantly (1). Also, we found marked reductions in arylsulfatases A and B, galactose-6-sulfatase, and steroid sulfatase in malignant mammary cell lines (MCF-7, T47D, and HCC 1937), compared with normal primary mammary epi...

The study objectives were to refine the population pharmacokinetics (PK) model, determine microbial clearance, and assess short-term pulmonary outcomes of multiple-dose azithromycin treatment in preterm infants at risk for Ureaplasma respiratory colonization. Fifteen subjects (7 of whom were Ureaplasma positive) received intravenous azithromycin at 20 mg/kg of body weight every 24 h for 3 doses. Azithromycin concentrations were determined in plasma samples obtained up to 168 h post-first dose by using a validated liquid chromatography-tandem mass spectrometry method. Respiratory samples were obtained predose and at three time points post-last dose for Ureaplasma culture, PCR, antibiotic susceptibility testing, and cytokine concentration determinations. Pharmacokinetic data from these 15 subjects as well as 25 additional subjects (who received either a single 10-mg/kg dose [n ‫؍‬ 12] or a single 20-mg/kg dose [n ‫؍‬ 13]) were analyzed by using a nonlinear mixed-effect population modeling (NONMEM) approach. Pulmonary outcomes were assessed at 36 weeks post-menstrual age and 6 months adjusted age. Bronchopulmonary dysplasia (BPD) is the major pulmonary morbidity in infants born preterm and is characterized by arrested alveolar development and chronic inflammation. Studies of human infants and experimental animal models indicate that the central event in BPD pathogenesis is the interruption of normal developmental signaling during early stages of lung development by lung injury that may be initiated in utero by intrauterine infection with a subsequent dysregulated inflammatory response (1-3). A recent meta-analysis of 39 studies confirmed that respiratory tract colonization with the genital mycoplasma species Ureaplasma parvum and Ureaplasma urealyticum increased the risk for development of BPD in extremely low gestation infants (4). It has not been established whether eradicating Ureaplasma spp. from the respiratory tract of preterm infants prevents or attenuates Ureaplasma infection-mediated lung injury.Azithromycin, an azalide antibiotic, has anti-inflammatory properties and antimicrobial activity against Ureaplasma spp. in in vitro (5, 6) and in in vivo experimental models (7-9). Although the efficacies of azithromycin and a related macrolide, clarithromycin, to prevent BPD have been assessed in single-center studies of preterm infants (10-13), the optimal dosing regimens for these antibiotics have not been determined in pharmacokinetics (PK) and pharmacodynamic studies, and the impacts on long-term pulmonary and neurologic outcomes are unknown. Our first steps to address these questions have been to conduct studies in the at-risk population to determine the optimal dose, safety, and in vivo anti-infective efficacy of azithromycin in preparation for future phase III randomized, placebo-controlled trials (14, 15). We previously characterized the PK of a single dose of intravenous (i.v.) azithromycin (10 or 20 mg/kg of body weight) in preterm infants (14). We demonstrated both doses were safe in infants who

Aminopeptidase N/CD13 (Anpep) is a membrane-bound protein that catalyzes the formation of natriuretic hexapeptide angiotensin IV (ANG IV) from ANG III. We previously reported that Anpep is more highly expressed in the kidneys of Dahl salt-resistant (SR/Jr) than salt-sensitive (SS/Jr) rats, Anpep maps to a quantitative trait locus for hypertension, and that the Dahl SR/Jr rat contains a functional polymorphism of the gene. This suggests that renal Anpep may be linked to salt sensitivity; however, its effect on renal Na handling has not been determined. Here, we examined regulation of basolateral Na(+)-K(+)-ATPase, a preeminent basolateral Na(+) transporter in proximal tubule cells, by Anpep in LLC-PK1 cells. Treatment of the cells with Anpep siRNA increased total cellular Na(+)-K(+)-ATPase activity and basolateral Na(+)-K(+)-ATPase abundance by approximately twofold. Conversely, Anpep overexpression reduced Na(+)-K(+)-ATPase activity and basolateral abundance by approximately 50%. Similar effects were observed after treatment with ANG IV (10 nM, x30 min and 12 h). ANG IV receptor (AGTRIV) knockdown via specific siRNA relieved the decreases in basolateral Na(+)-K(+)-ATPase levels and activity induced by Anpep overexpression. In sum, these results demonstrate that Anpep reduces basolateral Na(+)-K(+)-ATPase levels via ANG IV/AGTRIV signaling. This novel pathway may be important in renal adaptation to high salt.

Cocaine use disorder (CUD) remains a significant public health challenge. Levo-tetrahydropalmatine (L-THP), a well-tolerated and non-addictive compound, shows promise for the management of CUD. Its pharmacologic profile includes blockade at dopamine and other monoamine receptors and attenuation of cocaine self-administration, reinstatement, and rewarding properties in rats. This study evaluated the safety of L-THP in human cocaine users and its influence on the safety and pharmacokinetics (PK) of cocaine. Twenty-four cocaine-using adult men were randomized to receive L-THP (30 mg BID orally) or placebo double-blind for 4 days, with an intranasal cocaine (40 mg) challenge on the fourth day. Safety and tolerability were evaluated using vital signs, EKG, clinical laboratory tests, and standardized self-report instruments. Peripheral venous blood was collected periodically and later assayed for L-THP and cocaine using highly sensitive and specific ultra-performance liquid chromatography-fluorescence detection (UPLC-FLD) methods. Twenty subjects completed the study, of whom 19 provided complete PK data. The short 3.5-day course of L-THP was safe and well tolerated and did not affect cocaine’s PK or its acute cardiovascular effects. The cocaine AUC0→∞ was 211.5 and 261.4 h*ng/ml, and the Cmax was 83.3 and 104.5 ng/ml for the L-THP and placebo groups respectively. In addition there were no significant difference in the number of side effects reported in each group (L-THP group: 22 [48%], Placebo group: 24 [52%]), or vital signs including, heart rate, blood pressure, complete blood count or EKG. These findings suggest that oral THP has promise for further development as a treatment for CUD.

Towards the development of potent and selective inhibitors of melanoma cells containing active ERK signaling, we herein report on the pharmacophore determination and optimization of the ERK docking domain inhibitor (Z)-3-(2-aminoethyl)-5-(4-ethoxybenzylidene)thiazolidine-2,4-dione.