Leflunomide is a novel, isoxazol based disease-modifying anti-rheumatic drug (DMARD) for the treatment of rheumatoid arthritis (RA). Its mechanism differs from other DMARDs in that it inhibits de novo pyrimidine synthesis by inhibiting the enzyme dihydroorotate dehydrogenase (DHODH). It is a pro-drug and undergoes rapid conversion to its active form in vivo, A77-1726. A77-1726 inhibits the mitochondrial enzyme DHODH, which plays a key role in the de novo synthesis of pyrimidine ribonucleotide uridine monophosphate (rUMP). Leflunomide prevents clonal expansion of activated lymphocytes by interfering with the cell cycle progression due to inadequate production of rUMP and utilizing mechanisms involving p53. The relative lack of toxicity of A77-1726 on non-lymphoid cells may be due to the ability of these cells to fulfill their ribonucleotide requirements by use of salvage pyrimidine pathway, which makes them less dependent on de novo synthesis. Several phase II clinical trials of patients with RA revealed that leflunomide was effective and well tolerated. Large-scale phase III clinical trials have shown that leflunomide (20mg/day) provided a statistically significant clinical benefit and prevention of radiographic progression in comparison to placebo. The clinical benefits of leflunomide were similar to or greater than methotrexate and sulfasalazine. Now, many multi-national studies are in progress and planning, including combination therapy with other DMARD. In future, those studies will provide us more information about the effectiveness and potential adverse effect of leflunomide.
Antirheumatic Agents ; Arthritis, Rheumatoid ; Cell Cycle ; Humans ; Lymphocytes ; Methotrexate ; Oxidoreductases ; Sulfasalazine ; Uridine Monophosphate

Leflunomide is a novel, isoxazol based disease-modifying anti-rheumatic drug (DMARD) for the treatment of rheumatoid arthritis (RA). Its mechanism differs from other DMARDs in that it inhibits de novo pyrimidine synthesis by inhibiting the enzyme dihydroorotate dehydrogenase (DHODH). It is a pro-drug and undergoes rapid conversion to its active form in vivo, A77-1726. A77-1726 inhibits the mitochondrial enzyme DHODH, which plays a key role in the de novo synthesis of pyrimidine ribonucleotide uridine monophosphate (rUMP). Leflunomide prevents clonal expansion of activated lymphocytes by interfering with the cell cycle progression due to inadequate production of rUMP and utilizing mechanisms involving p53. The relative lack of toxicity of A77-1726 on non-lymphoid cells may be due to the ability of these cells to fulfill their ribonucleotide requirements by use of salvage pyrimidine pathway, which makes them less dependent on de novo synthesis. Several phase II clinical trials of patients with RA revealed that leflunomide was effective and well tolerated. Large-scale phase III clinical trials have shown that leflunomide (20mg/day) provided a statistically significant clinical benefit and prevention of radiographic progression in comparison to placebo. The clinical benefits of leflunomide were similar to or greater than methotrexate and sulfasalazine. Now, many multi-national studies are in progress and planning, including combination therapy with other DMARD. In future, those studies will provide us more information about the effectiveness and potential adverse effect of leflunomide.
Antirheumatic Agents ; Arthritis, Rheumatoid ; Cell Cycle ; Humans ; Lymphocytes ; Methotrexate ; Oxidoreductases ; Sulfasalazine ; Uridine Monophosphate

Uridine-cytidine kinase, an important catalyst in the compensation pathway of nucleotide metabolism, can catalyze the phosphorylation reaction of cytidine to 5'-cytidine monophosphate (CMP), but the reaction needs NTP as the phosphate donor. To increase the production efficiency of CMP, uridine-cytidine kinase gene from Thermus thermophilus HB8 and polyphosphate kinase gene from Rhodobacter sphaeroides were cloned and expressed in Escherichia coli BL21(DE3). Uridine-cytidine kinase was used for the generation of CMP from cytidine and ATP, and polyphosphate kinase was used for the regeneration of ATP. Then, the D403 metal chelate resin was used to adsorb Ni²⁺ to form an immobilized carrier, and the immobilized carrier was specifically combined with the recombinant enzymes to form the immobilized enzymes. Finally, single-factor optimization experiment was carried out to determine the reaction conditions of the immobilized enzyme. At 30 °C and pH 8.0, 60 mmol/L cytidine and 0.5 mmol/L ATP were used as substrates to achieve 5 batches of high-efficiency continuous catalytic reaction, and the average molar yield of CMP reached 91.2%. The above method has the advantages of low reaction cost, high product yield and high enzyme utilization rate, and has good applied value for industrial production.
Cytidine Monophosphate ; metabolism ; Escherichia coli ; genetics ; Industrial Microbiology ; methods ; Phosphotransferases (Phosphate Group Acceptor) ; metabolism ; Uridine Kinase

BACKGROUND: Uracil nucleotides are stored in platelets and all other cells, and are released into the extracellular space upon stimulation. They show various biological responses but their actions and mechanism are not well understood. This study was conducted to investigate the effects of uridine 5'-triphosphate(UTP) on vascular tone and to identify the characteristics of their receptors. METHODS: Aortic ring preparation were made from the rat descending thoracic aorta. Endo-thelial cells were preserved or removed by gentle rubbing, The basal tension of aortic ring was lgm and isometric contraction were recorded on polygraph using force transducer. RESULTS: In aortic ring Precontracted by 100nM norepinephrine, UTP induced dual effect with various concentrations. UTP elicited endothelium-dependent relaxation at low concentrations(100nM-10microM), and endothelium-independent contraction at high concentrations(more than 30microM). Among uracil nucleotides, UDP was as much effective as UTP in vascular tone, but UMP and uridine were not. UTP(pA50 6.15) was more potent than ATP(5.17), ITP(4.75) and other nucleotides(TTP, GTP, CTP). At basal tension, UTP induced relaxation at low concentrations and contraction at hige concentrations in endothelium-intact ring. But in endothelium-removed ring, UTP elicited only contraction. Prior treatment of aortic ring with suramin, a non-selective P2-purinoceptor blocker, inhibited UTP-Induced relaxation and contraction. Reactive blue-2, a P2gamma purinoceptor blocker, inhibited relaxation only, but alpha, beta-methylene ATP, a P2x Purinoceptor blocker, enhanced contractile response. ATP inhibited the UPT-induced relaxation, but 2-methylthio ATP did not alter the effects of UTP. It means that UTP and ATP act at the same receptor but 2-methylthio ATP does not. CONCLUSION: These results suggest that UTP-induced relaxation is mediated by nucleotide receptors on endothelium and the contraction is mediated by pyrimidinoceptors on vascular smooth muscle.
Adenosine Triphosphate ; Animals ; Aorta ; Aorta, Thoracic* ; Endothelium ; Extracellular Space ; Guanosine Triphosphate ; Isometric Contraction ; Muscle, Smooth, Vascular ; Norepinephrine ; Rats* ; Receptors, Purinergic ; Receptors, Purinergic P2X ; Relaxation ; Suramin ; Transducers ; Uracil Nucleotides ; Uridine Diphosphate ; Uridine Monophosphate ; Uridine Triphosphate ; Uridine*

No abstract available.
Antiviral Agents/*therapeutic use ; Benzimidazoles/*therapeutic use ; Female ; Fluorenes/*therapeutic use ; Hepacivirus/*classification ; Hepatitis C, Chronic/*drug therapy ; Humans ; Liver Cirrhosis/*drug therapy ; Male ; Ribavirin/*therapeutic use ; Uridine Monophosphate/*analogs & derivatives

The hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B protein, is the key viral enzyme responsible for replication of the HCV viral RNA genome. Although several full-length and truncated forms of the HCV NS5B proteins have been expressed previously in insect cells, contamination of host terminal transferase (TNTase) has hampered analysis of the RNA synthesis initiation mechanism using natural HCV RNA templates. We have expressed the HCV NS5B protein in insect cells using a recombinant baculovirus and purified it to near homogeneity without contaminated TNTase. The highly purified recombinant HCV NS5B was capable of copying 9.6-kb full-length HCV RNA template, and mini-HCV RNA carrying both 5'- and 3'-untranslated regions (UTRs) of the HCV genome. In the absence of a primer, and other cellular and viral factors, the NS5B could elongate over HCV RNA templates, but the synthesized products were primarily in the double stranded form, indicating that no cyclic replication occurred with NS5B alone. RNA synthesis using RNA templates representing the 3'-end region of HCV minus-strand RNA and the X-RNA at the 3'-end of HCV RNA genome was also initiated de novo. No formation of dimersize self-primed RNA products resulting from extension of the 3'-end hydroxyl group was observed. Despite the internal de novo initiation from the X-RNA, the NS5B could not initiate RNA synthesis from the internal region of oligouridylic acid (U)20, suggesting that HCV RNA polymerase initiates RNA synthesis from the selected region in the 3'-UTR of HCV genome.
3' Untranslated Regions/genetics ; 5' Untranslated Regions/genetics ; Animals ; Cell Line ; Gene Expression ; Genome ; Genome, Viral ; Hepacivirus/*enzymology/genetics ; RNA/biosynthesis/genetics ; RNA, Viral/genetics/metabolism ; Recombinant Proteins/genetics/isolation & purification/metabolism ; Spodoptera ; Templates, Genetic ; Uridine Monophosphate/metabolism ; Viral Nonstructural Proteins/chemistry/*genetics/isolation & purification/*metabolism

No abstract available.
Hepatitis C, Chronic ; Renal Insufficiency, Chronic* ; Sofosbuvir*

No abstract available.
Genotype* ; Hepatitis C, Chronic* ; Hepatitis, Chronic* ; Ribavirin* ; Sofosbuvir*

BACKGROUND/AIMS: Chronic hepatitis C (CHC) is a major comorbidity in patients with hemophilia. METHODS: Patients (n=30) were enrolled between September 2015 and April 2016. Twenty-six patients were genotype 1 (1b, n=21; 1a, n=5) and four patients were genotype 2a/2b. Among 21 patients with genotype 1b, Y93H resistance-associated variants (RAVs) were detected in three patients (14.3%). We evaluated sustained virologic response (SVRs) at 12 weeks, as well as relapse and safety. RESULTS: Five patients with genotype 1a and three patients with genotype 1b (RAV positive) received ledipasvir/sofosbuvir for 12 weeks. SVR12 rate was 100% (8/8). Eleven patients with genotype 1b were treatment-naïve and received daclatasvir plus asunaprevir for 24 weeks. SVR12 rate was 91% (10/11). One patient experienced viral breakthrough without RAV at 12 weeks. Seven treatment-experienced patients with genotype 1b received daclatasvir plus asunaprevir for 24 weeks. SVR12 rate was 85.7% (6/7). One patient experienced viral breakthrough with RAV (L31M, Y93H) at 12 weeks. Four patients with genotype 2a/2b received sofosbuvir plus ribavirin for 12 weeks. SVR12 rate was 100% (4/4). No serious adverse event-related discontinuations were noted. CONCLUSIONS: New direct acting antiviral treatment achieved high SVRs rates at 12 weeks in CHC patients with hemophilia without serious adverse events.
Antiviral Agents* ; Comorbidity ; Genotype ; Hemophilia A* ; Hepatitis C ; Hepatitis C, Chronic* ; Hepatitis, Chronic* ; Humans ; Recurrence ; Ribavirin ; Sofosbuvir

BACKGROUND/AIMS: The approval of sofosbuvir (SOF), a direct-acting antiviral, has revolutionized the treatment of chronic hepatitis C virus (HCV). METHODS: We assessed the sustained virological response (SVR) of SOF-based regimens in a real-world single-center setting for the treatment of chronic HCV genotype 1 (G1) patients. This was a retrospective review of chronic HCV G1 adult patients treated with a SOF-based regimen at Virginia Mason Medical Center between December 2013 and August 2015. RESULTS: The cohort comprised 343 patients. Patients received SOF+ledipasvir (LDV) (n=155), SOF+simeprevir (SIM) (n=154), or SOF+peginterferon (PEG)+ribavirin (RBV) (n=34). Of the patients, 50.1% (n=172) had cirrhosis. The SVR rate was 92.2% for SOF/LDV, 87.0% for SOF/SIM, and 82.4% for SOF/PEG/RBV. Compared with the cirrhotic patients, the patients without cirrhosis had a higher SVR (96.8% vs 85.5%, p=0.01, SOF/LDV; 98.2% vs 80.6%, p=0.002, SOF/SIM; 86.4% vs 75.0%, p=0.41, SOF/PEG/RBV). In this study, prior treatment experience adversely affected the response rate in subjects treated with SOF/PEG/RBV. CONCLUSIONS: In this single-center, real-world setting, the treatment of chronic HCV G1 resulted in a high rate of SVR, especially in patients without cirrhosis.
Adult ; Cohort Studies ; Fibrosis ; Genotype ; Hepacivirus ; Hepatitis C, Chronic* ; Hepatitis, Chronic* ; Humans ; Retrospective Studies ; Sofosbuvir ; Virginia