Determination of cyclophosphamide in human urine by HPLC coupled to tandem mass spectrometry

Biological samples urine are much more complex due to presence of proteins, salts, acids, bases and various organic compounds with similar chemistry to analytes of interest. As a result, the extraction methods for biological samples have been difficult. The main task was to remove maximum interfering substance from urine matrix and conversion of analytes into a more suitable form for injection. Therefore, optimization of SPE method was done by changing different parameter such as different type and amount of sorbent, composition and volume of elution, washing solvent. The use of 24 well SPE plate for matrix purification significantly allows the use of small urine and solvent volumes, reduces the sample preparation time and ensures a high throughput, thus allowing the routine biological monitoring of CP as indices of human exposures. In order to investigate the effectiveness of the SPE method tests were repeated on Strata-X cartridge and method showed good recovery (above 75 %) of CP. After optimizing SPE method, CP separation conditions of the HPLC-MS/MS analysis was achieve by changing different parameter such as suitable mobile phase and its composition, flow rate, scan type, polarity. The LC separation was performed on Lichrocart® 100 RP-18 column (125 4 mm, particle size of 5 μm) with 1 mM formic acid with ammonia buffer pH 3 and methanol using gradient program at a flow rate 1 mL/min with total run time 15 min. A reversed phase HPLC system was interfaced with an ESI source coupled to tandem mass spectrometry. The triple quadrupole mass spectrometer was operated in positive ion mode and MRM was used for analysis of CP. LOD and LOQ were 0.27 ng/mL and 0.54 ng/mL. A sensitive, specific and accurate HPLC/MS/MS has been developed for monitoring CP in urine samples. The use of reversed phase HPLC coupled to tandem mass spectrometry has facilitated the analysis of CP in short retention time. Fifteen urine samples were collected from medical personnel of Medical University of Gdansk, who were exposed with CP during their work in pharmacy. SPE-HPLC-MS/MS method was applied for the analysis of health workers and all urine samples from university workers has CP below analytical limit of detection

Pharmacokinetics of hydroxyurea therapy in African children with sickle cell anemia: a NOHARM ancillary PK study

Background: Hydroxyurea is an important disease-modifying therapy for sickle cell anemia (SCA). Through induction of fetal hemoglobin and other mechanisms, hydroxyurea reduces the number and severity of vaso-occlusive painful crises and episodes of acute chest syndrome, while also lowering the frequency of transfusions and hospitalizations, and even reducing mortality. In the US, hydroxyurea is recommended widely for both children and adults with SCA, although variation in treatment response is well-recognized. In sub-Saharan Africa, which has the greatest global burden of SCA, hydroxyurea is not used widely, due in part to a lack of data regarding its safety and efficacy. Pharmacokinetics (PK) analysis of hydroxyurea has also not been performed to date in Africa, but would provide important data regarding the variable responses to hydroxyurea treatment in low-resource settings. Objectives. There were three main goals of the NOHARM Ancillary PK Study: (1) to evaluate the feasibility of hydroxyurea sparse sampling PK collection and analysis in a low-resource clinical setting; (2) to determine the PK parameters for a cohort of young African children with SCA receiving hydroxyurea at an average oral daily dose of 20 mg/kg; and (3) to compare the PK parameters of African children collected and analyzed in the NOHARM trial with those of US children with SCA from the earlier HUSTLE study that included African-American children with SCA. Methods: NOHARM (NCT01976416) is a prospective, randomized, placebo-controlled, double-blinded phase 3 trial that is investigating the effects of hydroxyurea on the incidence and severity of malaria in very young children living in Uganda with SCA. After a year of blinded treatment, all participants were offered open-label hydroxyurea at an average oral daily dose of 20 ± 2.5 mg/kg, using either a 5-day or 7-day per week treatment regimen. After written informed consent for the ancillary PK study, hydroxyurea analysis was performed using a published sparse-sampling technique based on Bayesian estimation with collections pre-dose and then at T=15 minutes, 60 minutes, and 180 minutes. These four timed finger-prick sample collections over 3 hours used a volumetric absorptive microsampling device that collects exact 10 uL aliquots. All samples were stored at -80°C until analysis of hydroxyurea by LC-MS/MS (ug/mL) using a stable isotope internal standard, performed at Cincinnati Children\u27s Hospital. Population PK analysis was performed using nonlinear mixed effect modeling (NONMEM 7.2). Allometric scaling was used to correct for differences in size. Results: A total of 100 children were enrolled in the NOHARM ancillary PK study (average age 3.8 ± 0.9 years; 55 males), and 98 of these children had three evaluable post-dose samples. Individual PK profiles were best described by a one-compartment model with first order absorption. The dose-normalized hydroxyurea concentrations in NOHARM were comparable between the 5-day and 7-day dosing regimens and similar to published HUSTLE data for 96 children, except for higher T=180 hydroxyurea concentrations and overall higher hydroxyurea exposure in the NOHARM cohort. The higher blood hydroxyurea concentrations were the result of reduced drug clearance in NOHARM, possibly influenced by younger age. Population PK parameter estimates of oral clearance in the NOHARM and HUSTLE cohorts were 9.8 L/h/70kg versus 12.9 L/h/70kg, respectively (p\u3c0.01). Conclusions: The NOHARM PK study demonstrates the feasibility of collecting timed samples for analysis of hydroxyurea exposure, even in the low-resource setting of Uganda. Using a sparse sampling technique, young children with SCA had individual PK parameters calculated and a population PK model established. The oral drug clearance was lower in NOHARM than HUSTLE, with resulting higher hydroxyurea exposure, perhaps reflecting differences in age, nutrition, glomerular filtration, and genetic modifiers that warrant further investigation

Novel genetic loci that influence fetal hemoglobin expression in children with sickle cell anemia

Introduction: Elevated levels of fetal hemoglobin (HbF) are known to ameliorate both the morbidity and mortality of sickle cell anemia (SCA). Sustained post-natal HbF expression is heritable and regulated by multiple quantitative trait loci. Previous genomic studies have identified three major gene loci (BCL11A, HBS1L-MYB, and HBG2) that account for ~40% of HbF variation in SCA, but additional genetic modifiers remain to be discovered. We performed a genome wide association study (GWAS) using DNA collected from multiple cohorts of children with SCA, to identify novel genes and variants involved in HbF expression. Methods: We analyzed genomic DNA from 1009 children with SCA and pre-treatment steady-state HbF levels who enrolled in prospective research trials from the United States (HUSTLE, SWiTCH, TWiTCH), the Caribbean (SACRED) or sub-Saharan Africa (REACH, NOHARM). Whole blood DNA was first genotyped using the H3Africa SNP array (Illumina) that identifies over 2.2 million single nucleotide variants (SNVs) across the genome. Most samples also underwent whole exome sequencing (WES) using NimbleGen VCRome 2.1 capture reagents and the Illumina HiSeq2500 platform analysis, which identifies coding variants in all known exons. Results: From the combined SNP and WES dataset, 8 BCL11A variants passed genome wide significance (p\u3c10-8) in the discovery analysis, and 1,048 additional variants were identified with nominal HbF association (p\u3c0.001). We found that 173 of these novel variants had sustained association in at least one of the replication cohorts (p\u3c0.05). We selected 20 variants with the strongest and most consistent associations with HbF from the discovery and replication analyses for further verification (Table 1). Conclusions: Our large GWAS of HbF with diverse global cohorts of children with SCA from Africa, the United States, and the Caribbean validated the strong associations of HbF with common genetic variants near the BCL11A and HBS1L-MYB gene loci. We also identified two novel gene loci, ITGA1 and RUNX1T1, that have statistical associations with HbF expression. The RUNX1T1 gene is a broad transcriptional corepressor known to impact myeloid differentiation in hematopoiesis, while ITGA1 encodes the integrin alpha subunit of a cell-surface receptor involved in cell-cell adhesion and inflammation

Genetic variants that influence fetal hemoglobin expression from hydroxyurea treatment

Introduction: Hydroxyurea is a potent therapeutic agent for sickle cell anemia (SCA), and treatment at maximum tolerated dose (MTD) is becoming the standard of care. Hydroxyurea exerts its disease-modifying effects primarily through induction of fetal hemoglobin (HbF), although the cellular and molecular mechanisms by which hydroxyurea increases HbF expression remain unclear. Children with SCA treated with hydroxyurea at MTD have substantial phenotypic variation, however, as some have higher HbF responses than others. We hypothesized that unknown quantitative trait loci modulate the pharmacological induction of HbF, so we performed a large genome wide association study (GWAS) of hydroxyurea-associated HbF responses for children with SCA treated prospectively with dose escalation to MTD. Methods: We analyzed genomic DNA from 831 children with SCA enrolled in pediatric research trials from the US (HUSTLE, SWiTCH, TWiTCH), the Caribbean (EXTEND, SACRED) and sub-Saharan Africa (REACH, NOHARM); all of these trials reported robust treatment responses with average HbF \u3e20%. Study participants received hydroxyurea with dose escalation to MTD based on mild myelosuppression. Whole blood DNA was genotyped using the H3Africa SNP array (Illumina) with whole exome sequencing (WES) using NimbleGen VCRome 2.1 capture reagents and the Illumina HiSeq2500 platform. A transformed z-score for each study cohort gave a standardized measure of HbF induction relative to their steady-state level and their treatment HbF level at MTD. Results: In the discovery GWAS step, no variant passed genome wide significance (p\u3c10-8) for the MTD HbF phenotype, including no significant associations with known genetic modifiers of endogenous HbF (BCL11A, HBS1L-MYB, HBG2). A total of 2057 low frequency and common SNVs had at least nominal association (p\u3c0.001) with the hydroxyurea treatment responses, of which 44 were also significant (p\u3c0.05) and with the same direction of association with HbF induction in the replication cohort. In the final verification step, these 44 significant variants were then tested in additional independent SCA cohorts with at least three demonstrating a strong effect. Conclusions: This large GWAS using global cohorts of children with SCA and robust prospective HbF phenotype data has identified genetic predictors of HbF hydroxyurea treatment responses. Three novel genetic loci, PTPRD, RPH3AL, and ELL2 have SNVs associated with lower HbF responses. PTPRD is a protein tyrosine phosphatase receptor involved in cellular processes such as cell growth and differentiation, while RPH3AL, a rabphilin 3A like protein, is known to be involved in calcium-ion-dependent exocytosis. ELL2 is an elongation factor for RNA polymerase II and could modify RNA processing under the cytostatic effects of hydroxyurea

PI3K delta contributes to ER stress-associated asthma through ER-redox disturbances: the involvement of the RIDD-RIG-I-NF-kappa B axis

Hyperactivation of phosphoinositol 3-kinase (PI3K) has been suggested to be a potential mechanism for endoplasmic reticulum (ER) stress-enhanced airway hyperresponsiveness, and PI3K inhibitors have been examined as asthma therapeutics. However, the regulatory mechanism linking PI3K to ER stress and related pathological signals in asthma have not been defined. To elucidate these pathogenic pathways, we investigated the influence of a selective PI3K delta inhibitor, IC87114, on airway inflammation in an ovalbumin/lipopolysaccharide (OVA/LPS)-induced asthma model. In OVA/LPS-induced asthmatic mice, the activity of PI3K, downstream phosphorylation of AKT and activation of nuclear factor-kappa B (NF-kappa B) were all significantly elevated; these effects were reversed by IC87114. IC87114 treatment also reduced the OVA/LPS-induced ER stress response by enhancing the intra-ER oxidative folding status through suppression of protein disulfide isomerase activity, ER-associated reactive oxygen species (ROS) accumulation and NOX4 activity. Furthermore, inositol-requiring enzyme-1 alpha (IRE1 alpha)-dependent degradation (RIDD) of IRE1 alpha was reduced by IC87114, resulting in a decreased release of proinflammatory cytokines from bronchial epithelial cells. These results suggest that PI3K delta may induce severe airway inflammation and hyperresponsiveness by activating NF-kappa B signaling through ER-associated ROS and RIDD-RIG-I activation. The PI3K delta inhibitor IC87114 is a potential therapeutic agent against neutrophil-dominant asthma.1

Characterization of <it>Salvia Miltiorrhiza </it>ethanol extract as an anti-osteoporotic agent

<p>Abstract</p> <p>Background</p> <p><it>Salvia miltiorrhiza </it>(SM) has long been used as a traditional oriental medicine for cardiovascular disease. Accumulating evidence also indicates that SM has anti-osteoporotic effects. This study was conducted to examine the SM-induced anti-osteoporotic effect and its possible mechanisms with various doses of SM.</p> <p>Methods</p> <p>We studied Sprague-Dawley female rats aged 12 weeks, divided into six groups: sham-operated control (SHAM), OVX rats supplemented with SM (1, 3, 10 and 30 mg/kg) orally for 8 weeks. At the end of the experiment, blood samples were collected and biochemistry analysis was performed. Specimens from both tibia and liver were processed for light microscopic examination. DEXA and μ-CT analyses of the tibia were also performed.</p> <p>Results</p> <p>SM treatment significantly ameliorated the decrease in BMD and trabecular bone mass according to DEXA and trabecular bone architecture analysis of trabecular bone structural parameters by μ-CT scanning. In serum biochemical analysis, SM decreased the released TRAP-5b, an osteoclast activation marker and oxidative stress parameters including MDA and NO induced by OVX.</p> <p>Conclusions</p> <p>The preventive effect of SM was presumably due to its anti-oxidative stress partly via modulation of osteoclast maturation and number. In current study, SM appears to be a promising osteoporosis therapeutic natural product.</p

An Involvement of Oxidative Stress in Endoplasmic Reticulum Stress and Its Associated Diseases

The endoplasmic reticulum (ER) is the major site of calcium storage and protein folding. It has a unique oxidizing-folding environment due to the predominant disulfide bond formation during the process of protein folding. Alterations in the oxidative environment of the ER and also intra-ER Ca2+ cause the production of ER stress-induced reactive oxygen species (ROS). Protein disulfide isomerases, endoplasmic reticulum oxidoreductin-1, reduced glutathione and mitochondrial electron transport chain proteins also play crucial roles in ER stress-induced production of ROS. In this article, we discuss ER stress-associated ROS and related diseases, and the current understanding of the signaling transduction involved in ER stress