This study investigated the impact of allelic variation in SLCO1B1, a gene encoding for the liver‐specific solute carrier organic anion transporter family member 1B1 protein (SLCO1B1), on simvastatin ...and simvastatin acid (SVA) systemic exposure in children and adolescents. Participants (8–20 years old) with at least 1 variant SLCO1B1 c.521T>C allele (521TC, n = 15; 521CC, n = 2) and 2 wild‐type alleles (521TT, n = 15) completed a single oral dose pharmacokinetic study. At equivalent doses, SVA exposure was 6.3‐ and 2.5‐fold greater in 521CC and TC genotypes relative to 521TT (Cmax, 2.1 ± 0.2 vs 1.0 ± 0.5 vs 0.4 ± 0.3 ng/mL; P < .0001; and AUC, 12.1 ± 0.3 vs 4.5 ± 2.5 vs 1.9 ± 1.8 ng·h/mL; P < .0001). The impact of the SLCO1B1 c.521 genotype was more pronounced in children, although considerable interindividual variability in SVA exposure was observed within genotype groups. In addition, SVA systemic exposure was negligible in 25% of pediatric participants. Further investigation of the ontogeny and genetic variation of SVA formation and SLCO1B1‐mediated hepatic uptake is necessary to better understand the variability in SVA exposure in children and its clinical consequences.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
BACKGROUND
Methotrexate (MTX) is the cornerstone of treatment in the management of autoimmune arthritis, including juvenile idiopathic arthritis (JIA). Despite being in use for over 35 years the ...mechanism through which MTX inhibits inflammation remains unclear. In this study we investigate activation of JAK/STAT signaling in fibroblast‐like synoviocytes from patients with JIA and the effect of MTX.
METHODS
Synoviocytes were isolated from the synovial fluid of juvenile idiopathic arthritis patients following routine arthrocentesis and maintained in culture. Synoviocytes were treated with MTX and assessed for cellular levels of MTX and its polyglutamated metabolites, as well as intermediates of nucleotide biosynthesis, including dUMP and ZMP. Analytes were detected by UPLC‐MS/MS. JAK/STAT signaling was assessed in synoviocytes and A549 human lung carcinoma cells by Western blotting and immunofluorescence. Experiments were performed in triplicate and statistical testing was done by unpaired t test analysis.
RESULTS
MTX was actively taken up and metabolized in synoviocytes to form polyglutamated metabolites of MTX, however, MTX failed to cause measurable inhibition of de novo nucleotide synthesis by dUMP and ZMP analysis. Synoviocytes displayed a high level of STAT phosphorylation with 58.8±4.8% of total measureable STAT3 signal and 69.9±7.3% of total measureable STAT5 signal. In contrast, phosphorylation of STAT3 and STAT5 was undetectable in A549 cells and treatment with 100 ng/mL of recombinant IL‐6 resulted in a marked increase in STAT phosphorylation with 64.4±6.0% of measureable STAT3 signal and 76.6±8.5% of measureable STAT5 signal. Treatment with MTX resulted in inhibition of STAT3 and STAT5 phosphorylation by 38.1±3.4% (p<0.05) and 61.2±7.8% (p<0.05) in primary synoviocytes and 65.3±2.8 (p<0.05) and 92.8±8.1% (p<0.01) in IL‐6 stimulated A549 cells, respectively. In comparison, phosphorylation of STAT3 and STAT5 in response to IL‐6 stimulation in A549 cells was decreased by 91.6±11.3% (p<0.001) and 94.5±12.3% (p<0.001) by 50 μM of JAK Inhibitor I (CAS 457081‐03‐7). In A549 cells inhibition of STAT phosphorylation by MTX was completely reversed by co‐treatment with 100 μM folinic acid.
CONCLUSION
These findings support constitutive activation of JAK/STAT signaling in fibroblast‐like synoviocytes isolated from patients with JIA that is inhibited by treatment with MTX. Although the underlying biochemical mechanism through which MTX inhibits JAK/STAT signaling is not known, it appears to be dependent on the anti‐folate activity of MTX. Together, this data supports inhibition of JAK/STAT signaling as a possible mechanism of anti‐inflammatory activity of MTX.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Nicotinamide phosphoribosyltransferase (NAMPT) is a pleiotropic protein implicated in the pathogenesis of acute respiratory distress syndrome, aging, cancer, coronary heart diseases, diabetes, ...nonalcoholic fatty liver disease, obesity, rheumatoid arthritis, and sepsis. However, the underlying molecular mechanisms of NAMPT in these physiological and pathological processes are not fully understood. Here, we provide experimental evidence that a Nampt gene homozygous knockout (Nampt
) resulted in lethality at an early stage of mouse embryonic development and death within 5-10 days in adult mice accompanied by a 25.24±2.22% body weight loss, after the tamoxifen induction of Nampt
× Cre mice. These results substantiate that Nampt is an essential gene for life. In Nampt
mice versus Nampt
mice, biochemical assays indicated that liver and intestinal tissue NAD levels were decreased significantly; histological examination showed that mouse intestinal villi were atrophic and disrupted, and visceral fat was depleted; mass spectrometry detected unusual higher serum polyunsaturated fatty acid containing triglycerides. RNA-seq analyses of both mouse and human pediatric liver transcriptomes have convergently revealed that NAMPT is involved in key basic cellular functions such as transcription, translation, cell signaling, and fundamental metabolism. Notably, the expression of all eight enzymes in the tricarboxylic acid cycle were decreased significantly in the Nampt
mice. These findings prompt us to posit that adult Nampt
mouse lethality is a result of a short supply of ATP from compromised intestinal absorption of nutrients from digested food, which leads to the exhaustion of body fat stores.
Objective
Folates exist as a fluctuating pool of polyglutamated metabolites that may serve as a clinical marker of methotrexate (MTX) activity. This study was undertaken to evaluate circulating ...folate content and folate polyglutamate distribution in juvenile idiopathic arthritis (JIA) patients and in a cell culture model based on MTX exposure and folate supply.
Methods
Blood, plasma, and red blood cell (RBC) measurements of MTX and folates were obtained from previously published data sets and an additional analysis of JIA patients receiving MTX (n = 98) and those not receiving MTX (n = 78). Erythroblastoid cells maintained in culture were exposed to MTX and grown under varying levels of folic acid supplementation. Samples were analyzed for cellular folate and MTX content.
Results
Circulating folate levels were lower in JIA patients receiving MTX, with reduced levels of blood, plasma, and RBC 5‐methyl‐tetrahydrofolate (5mTHF) (P < 0.0001). Average polyglutamate chain length (Gluavg) of RBC 5mTHF was elevated in JIA patients receiving MTX (median ± interquartile range 5.63 ± 0.15 versus 5.54 ± 0.11 in those not receiving MTX; P < 0.001) and correlated with both RBC MTX accumulation (P = 0.02) and reduced plasma 5mTHF levels (P = 0.008). MTX exposure and folate deprivation in erythroblastoid cells resulted in a depletion of bioactive folate species that was associated with a shift to higher Gluavg values for several species, most notably tetrahydrofolate (THF) and 5,10‐methylene‐tetrahydrofolate (CH2THF). Increased Gluavg resulted from the depletion of short‐chain and the accumulation of long‐chain glutamate species.
Conclusion
Our findings indicate that folate content and polyglutamate distribution are responsive markers of MTX activity and folate supply in vivo and in vitro, and may provide novel clinical markers of pharmacologic activity of MTX.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
•A UHPLC-MS/MS assay for the detection of pravastatin and metabolites in human plasma was developed.•The method was validated and proved to be fast, sensitive, specific.•Pharmacokinetic parameters of ...pravastatin in dyslipidemic children were determined.•Pravastatin pharmacokinetics are highly variable in this patient population.
An ultra high pressure liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) method for the simultaneous quantitation of pravastatin and major metabolites, 3′α-hydroxy-pravastatin, pravalactone and 3′α-hydroxy-pravalactone, in human plasma has been developed and validated. Aliquots of (100μL) plasma in EDTA were diluted in pH 4.5 (0.1M buffer) to stabilize the analytes and subjected to hydrophilic lipophilic balance (HLB) solid phase extraction on 96 well μelution plates. Extracted samples were evaporated to dryness and reconstituted in pH 4.5 buffer. Chromatographic separation was performed on a Cortecs™ C18 column (2.1×100mm, 1.8μm), using gradient elution with a blend of acetonitrile and 10mM methylammonium acetate buffer (pH 4.5) at a flow rate of 0.4mL/min. Mass spectrometric detection was performed using multiple reaction monitoring (MRM) switching between positive/negative electrospay ionization (ESI). Pravastatin, 3′α-hydroxy-pravastatin, and internal standards 2H3-pravastatin, and 2H3-3′α-hydroxy-pravastatin were monitored in negative ESI mode at ion transitions m/z 423.2→321.1 and 426.2→321.1, respectively. Positive ESI mode was used for the detection of pravalactone, 3′α-hydroxy-pravalactone, and internal standards 2H3-pravalactone, and 2H3-3′α-hydroxy-pravalactone at ion transitions m/z 438.2→183.1 and 441.2→269.1 respectively. The method was linear for all analytes in the concentration range 0.5–200nM with intra- and inter-day precisions (as relative standard deviation) of ≤5.2% and accuracy (as relative error) of ≤8.0% at all quality control levels. The method was successfully applied to the investigation of pharmacokinetic properties of pravastatin and its metabolites in children after an oral dose of 20–40mg.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Methotrexate (MTX) efficacy in autoimmune arthritis is variable and unpredictable resulting in the need for the identification of biomarkers to guide drug therapy. This study utilizes the ...collagen-induced arthritis mouse model to investigate erythrocyte MTX disposition and anti-folate activity as biochemical markers of efficacy in autoimmune arthritis. Following induction of arthritis, DBA/1J mice were treated with once-weekly subcutaneous MTX at varying doses over a period of 40 days. At the completion of the study tissue samples were analyzed for MTX and folate content and assessed for their relationship with MTX efficacy. MTX treatment resulted in a reduction in disease activity that was variable and dose-dependent. Erythrocyte accumulation of MTX and its polyglutamate metabolites were dose proportionate, however, polyglutamate metabolites represented a mean ± S.E.M. of 8.9 ± 0.4% of total erythrocyte MTX, which is markedly lower than previously observed in humans and failed to display any significant association with MTX efficacy. MTX treatment resulted in reductions in erythrocyte 5-methyl-tetrahydrofolate (5mTHF) levels that were similar to those previously observed in human studies. Disease induction was associated with a decrease in liver 5mTHF and increased formyl-tetrahydrofolate (fTHF) that was normalized in MTX treated mice. MTX efficacy was associated with reductions in erythrocyte 5mTHF (P = 0.04) and increases in liver 5mTHF (P = 0.0001). Together, these findings demonstrate a relationship between alterations in tissue folate levels and MTX efficacy, and supports erythrocyte levels of 5mTHF as a marker of MTX efficacy in autoimmune arthritis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Generation 5 poly(amidoamine) dendrimer nanoparticles conjugated with folic acid and methotrexate (G5-MTX-FA) for targeted treatment of cancer are of recent interest. The increased efficacy of these ...nanodevices over the free methotrexate has been shown in vitro and in vivo. The heterogeneous nature of this nanoparticle together with possible release of active compounds complicated the method development. This work presents a bioanalytical assay for the detection of nanoparticle-conjugated methotrexate, released methotrexate, and its main plasma metabolite 7-hydroxymethotrexate in rat plasma. Determination of G5-MTX-FA-associated methotrexate occurred by a reductive cleavage of the C9-N10 bond in methotrexate, resulting in a highly fluorescent 2,4-diamino-6-methylpteridine reporter molecule that could be measured by reversed-phase chromatography and fluorescence detection. It was found that reduction should occur directly in the plasma matrix to avoid irreversible adsorption of the nanodevice during sample preparation. The method was linear over a range from 50 to 10,000 nM G5-MTX-FA utilizing 100 μL of plasma. Nanoparticle-released methotrexate and its metabolite 7-hydroxymethotrexate were determined by reversed-phase chromatography followed by online post-column photochemical derivatization and fluorescence detection. The method was specific for these analytes irrespective of nanoparticle concentration. Sample preparation consisted of perchloric acid protein precipitation followed by a strong anion exchange solid-phase extraction. Limits of quantification were about 50 nM for methotrexate and 10 nM for 7-hydroxymethotrexate. Preliminary pharmacokinetic profiles of intravenous and subcutaneous administered G5-MTX-FA in rats were obtained. These data indicated that less than 0.1% of the methotrexate mass is released from the nanoparticle in plasma.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Trimethoprim (TMP) has been widely used since the 1960s, both alone and in combination with sulfamethoxazole. Unfortunately, information regarding the role that cytochrome P450 enzymes (P450s) play ...in the formation of TMP primary metabolites is scarce. Hence, we undertook in vitro studies to identify and more fully characterize the P450s that catalyze formation of six TMP primary metabolites: TMP 1-N-oxide (1-NO-TMP) and 3-N-oxide (3-NO-TMP), 3'- and 4'-desmethyl-TMP, a benzylic alcohol (Cα-OH-TMP), and an N-acetyl cysteine (NAC) adduct of TMP (Cα-NAC-TMP). Formation kinetics for each TMP metabolite in human liver microsomes (HLMs) were consistent with single-enzyme Michaelis-Menten kinetics, and Km values were markedly above (≥10-fold) the therapeutic concentrations of TMP (50 µM). The combined results from correlation studies between rates of metabolite formation and marker P450 activities in a panel of HLMs along with inhibition studies utilizing selective P450 inhibitors incubated with pooled HLMs suggested that 1-NO-TMP, Cα-NAC-TMP, and Cα-OH-TMP were predominantly formed by CYP3A4. In contrast, 3-NO-TMP was formed predominantly by CYP1A2 in HLMs and inhibited by α-naphthoflavone. 4'-Desmethyl-TMP, which is believed to be a reactive TMP metabolite precursor, was formed by several P450s, including CYP3A4, correlated with multiple P450 activities, but was inhibited primarily by ketoconazole (up to 50%), suggesting that CYP3A4 makes a major contribution to TMP 4'-demethylation. TMP 3'-demethylation was catalyzed by multiple P450s, including CYP2C9, correlated with CYP2C9 activity, and was inhibited by sulfaphenazole (up to 40%). Overall, CYP2C9 and CYP3A4 appear to be the most significant contributors to TMP primary metabolism.
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