6 research outputs found
Pharmacokinetics and pharmacodynamics of thiopurines in an in\ua0vitro model of human hepatocytes: Insights from an innovative mass spectrometry assay
AIM: To apply an innovative LC-MS/MS method to quantify thiopurine metabolites in human hepatocytes and to associate them to cytotoxicity.
METHODS:
Immortalized human hepatocytes (IHH cells) were treated for 48 and 96 h, with 1.4
7 10-4 M azathioprine and 1.1
7 10-3 M mercaptopurine, concentrations corresponding to the IC50 values calculated after 96 h exposure in previous cytotoxicity analysis. After treatments, cells were collected for LC-MS/MS analysis to quantify 11 thiopurine metabolites with different level of phosphorylation and viable cells were counted by trypan blue exclusion assay to determine thiopurines in vitro effect on cell growth and survival. Statistical significance was determined by analysis of variance (ANOVA).
RESULTS:
Azathioprine and mercaptopurine had a significant time-dependent cytotoxic effect (p-value ANOVA = 0.012), with a viable cell count compared to controls of 55.5% and 67.5% respectively after 48 h and 23.7% and 36.1% after 96 h; no significant difference could be observed between the two drugs. Quantification of thiopurine metabolites evidenced that the most abundant metabolite was TIMP, representing 57.1% and 40.3% of total metabolites after 48 and 96 h. Total thiopurine metabolites absolute concentrations decreased over time: total mean content decreased from 469.9 pmol/million cells to 83.6 pmol/million cells (p-value ANOVA = 0.0070). However, considering the relative amount of thiopurine metabolites, TGMP content significantly increased from 11.4% cells to 26.4% (p-value ANOVA = 0.017). A significant association between thiopurine effects and viable cell counts could be detected only for MeTIMP: lower MeTIMP concentrations were associated with lower cell survival (p-value ANOVA = 0.011). Moreover, the ratio between MeTIMP and TGMP metabolites directly correlated with cell survival (p-value ANOVA = 0.037).
CONCLUSION:
Detailed quantification of thiopurine metabolites in a human hepatocytes model provided useful insights on the association between thioguanine and methyl-thioinosine nucleotides with cell viability
Identification and Characterization of a Novel Family of Cysteine-Rich Peptides (MgCRP-I) from Mytilus galloprovincialis
We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis. These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling
Expression Pattern of Long Non-Coding RNA Growth Arrest-Specific 5 in the Remission Induction Therapy in Childhood Acute Lymphoblastic Leukemia
Background: Long non-coding RNA growth arrest-specific 5 (GAS5) is deregulated in many cancers because of its role in cell growth arrest and apoptosis. Additionally, GAS5 interacts with glucocorticoid receptor, making it a potential pharmacotranscription marker of glucocorticoid (GC) therapy. In this study, we aimed at analysing GAS5 expression in the remission induction therapy phase of childhood acute lymphoblastic leukemia (ALL), in which GCs are mandatorily used, and to correlate it with therapy response.
Methods: GAS5 expression was measured in peripheral blood mononuclear cells taken from 29 childhood ALL patients at diagnosis, on day 15 and day 33 ofremission induction therapy using RT-qPCR methodology.
Results: Our results have shown interindividual differences in GAS5 expression at all time points. For each ALL patient, GAS5 expression was higher on day 15 in comparison to its level at diagnosis (p<0.0005). On day 33, the level of GAS5 expression decreased in comparison with day 15 (p<0.0005), but it was still significantly higher than at diagnosis for the majority of patients (p=0.001). Patients whose number of blasts on day 8 was below 100 per mL of peripheral blood had a higherGAS5 expression at diagnosis (p=0.016), and lower ratio day 15/diagnosis (p=0.009). Conclusions: Our results suggest that the expression level of GAS5 could be a potential marker of therapy response in remission induction therapy of childhood ALL
Emerging molecular mechanisms underlying cancer metastasis: the rising role of the long non-coding RNA GAS5
Metastasis is a multistep process, wherein cells in a primary tumor acquire invasive properties and disseminate throughout the body to establish secondary tumors at distant organs. During the metastatic cascade a complex interaction of signals are involved and identification of novel players in this process is crucial for cancer prognosis and for the development of agents effective on late stage malignancies. Although most of the studies in the literature focus on protein modulators of metastasis, the relevance of non-coding RNAs is now being appreciated. Long non-coding RNAs (lncRNAs) are commonly referred to as non-protein-coding RNA transcripts longer than 200 nucleotides. Emerging evidence has shown that lncRNAs are dysregulated in multiple cancer types and have a critical role in the process of metastasis (1). Of particular interest in this regard is the lncRNA GAS5 (growth arrest- speci c transcript 5) which is down-regulated and inversely correlates with clinicopathological characteristics such as tumor size and metastasis progression in various human cancers, such as renal clear cells and bladder cancer (2,3). However, studies on melanoma are still scarce
Glucocorticoid Receptor Interacting Co-regulators: Putative Candidates for Future Drug Targeting Therapy
Glucocorticoids (GCs) are largely used in different inflammatory, autoimmune and proliferative diseases. To date their mechanism of action is not completely clear and more studies are necessary, in particular to explain the great interindividual variability in clinical response. In this panorama the glucocorticoid receptor (GR) has an important role: in fact it regulates the pharmacological response thanks to the capability to interact with different molecules (DNA, RNA, ncRNA and proteins) that are known to influence its activity. In this review our aim is to highlight the knowledge about the role of protein-protein, RNA-protein interactions and epigenetic modifications on the GR and the consequent response to GCs. The characteristics of these interactions with the GR and their effects on the pharmacological activity of GCs will be examined. This information could contribute to the prediction of individual sensitivity to steroids through the identification of new markers of GC resistance. In addition this knowledge may be used in developing new strategies for targeted therapy
Causes of Treatment Failure in Children With Inflammatory Bowel Disease Treated With Infliximab: A Pharmacokinetic Study
Objectives:
Anti-tumor necrosis factor antibodies have led to a revolution in the treatment of inflammatory bowel diseases (IBD); however, a sizable proportion of patients does not respond to therapy. There is increasing evidence suggesting that treatment failure may be classified as mechanistic (pharmacodynamic), pharmacokinetic, or immune-mediated. Data regarding the contribution of these factors in children with IBD treated with infliximab (IFX) are still incomplete. The aim was to assess the causes of treatment failure in a prospective cohort of pediatric patients treated with IFX.
Methods:
This observational study considered 49 pediatric (median age 14.4) IBD patients (34 Crohn disease, 15 ulcerative colitis) treated with IFX. Serum samples were collected at 6, 14, 22 and 54 weeks, before IFX infusions. IFX and anti-infliximab antibodies (AIA) were measured using enzyme linked immunosorbent assays. Disease activity was determined by Pediatric Crohn's Disease Activity Index or Pediatric Ulcerative Colitis Activity Index.
Results:
Clinical remission, defined as a clinical score 3.11\u200a\u3bcg/mL emerged as the strongest predictor of sustained clinical remission. AIA concentrations were correlated inversely with IFX concentrations and directly with adverse reactions.
Conclusions:
Most cases of therapeutic failure were associated with low serum drug levels. IFX trough levels at the end of induction are associated with sustained long-term response