Weekly split-dose regimen for oral methotrexate reduced polyglutamation in red blood cells in patients with rheumatoid arthritis compared with single-dose regimen: Results from a multicentered randomized control trial.

AIMS: We compared the incidence of adverse events between single and divided-dose regimens of methotrexate (MTX) by using a multicenter randomized controlled trial. METHODS: Eighty-nine patients with insufficient control on MTX 8 mg/wk were randomly assigned into single-dose (39 patients) or triple dose (39 patients) groups. The MTX dose for all patients was gradually increased to 16 mg/wk. The primary endpoint was the occurrence of liver dysfunction during the observation period (20 weeks). RESULTS: There were no differences in baseline data and MTX dose at Week 20 between groups. There was no significant difference in the incidence of liver dysfunction between groups (single dose, 3 [7.7%] patients vs. triple dose, 5 [13.2%] patients; P = .455). The incidence of adverse event increased in triple dose (single dose, 12 [30.8%] patients vs. triple dose, 20 [51.3%]), but the difference was not significant (P = .066). There was no significant difference in disease activity between groups, although MTX-triglutamate (PG3), MTX-PG4, and MTX-PG5 were significantly higher in the single dose group. CONCLUSIONS: Weekly split dosing reduced the polyglutamation of MTX. There was no significant difference in efficacy and safety between the 2 groups.

Red blood cell methotrexate polyglutamates emerge as a function of dosage intensity and route of administration during pulse methotrexate therapy in rheumatoid arthritis.

OBJECTIVE: MTX is a prodrug producing anti-arthritic effects through a folylpolyglutamate synthase-mediated activation to MTX polyglutamates (MTXPGs). Our objective was to characterize the pharmacokinetics of intracellular MTXPGs and the factors associated with their accumulation in adult RA patients treated with MTX weekly. METHODS: MTX pharmacokinetics were evaluated in 47 MTX-naïve patients enrolled in an MTX dose-escalation study for an average of 20 weeks and 223 patients enrolled in a cross-sectional study under long-term MTX therapy. Short-chain (MTXPG1-2), long-chain (MTXPG3) and very long-chain (MTXPG4-5) concentrations were measured in circulating red blood cells using liquid chromatography. Statistical analyses consisted of non-linear mixed models, multivariate regression analyses and Wilcoxon signed-rank test. RESULTS: The accumulation of MTXPG1-5 was sigmoidal and steady-state concentrations were achieved after 7 weeks of therapy. However, additional exposure and MTX dosage escalation produced a selective redistribution towards longer chain MTXPGs at the expense of shorter chain MTXPGs. Age, glomerular filtration rate and route of MTX administration were the most important predictors of MTXPG accumulation. In 10 patients, a switch from oral to parenteral MTX was associated with a 37% increase in long-chain MTXPGs, a 132% increase in very long-chain MTXPGs and a concomitant 31% reduction in disease activity (P<0.02). CONCLUSION: The selective emergence of long-chain MTXPGs is function of dose, time of exposure and hence dosage intensity. Switching from oral to parenteral MTX produces a selective accumulation of longer chain MTXPGs that are known to be more potent inhibitors of de novo purine biosynthesis than shorter chain MTXPGs.

Regulation of polyglutamic acid synthesis by glutamate in Bacillus licheniformis and Bacillus subtilis.

The synthesis of polyglutamic acid (PGA) was repressed by exogenous glutamate in strains of Bacillus licheniformis but not in strains of Bacillus subtilis, indicating a clear difference in the regulation of synthesis of capsular slime in these two species. Although extracellular gamma-glutamyltranspeptidase (GGT) activity was always present in PGA-producing cultures of B. licheniformis under various growth conditions, there was no correlation between the quantity of PGA and enzyme activity. Moreover, the synthesis of PGA in the absence of detectable GGT activity in B. subtilis S317 indicated that this enzyme was not involved in PGA biosynthesis in this bacterium. Glutamate repression of PGA biosynthesis may offer a simple means of preventing unwanted slime production in industrial fermentations using B. licheniformis.

Effects of manganese (II) on Bacillus licheniformis ATCC 9945A physiology and gamma-poly(glutamic acid) formation.

Bacillus licheniformis ATCC 9945A was cultivated in shake flasks using citrate (12 gl-1), glutamate (20 gl-1) and glycerol (80 gl-1) as carbon sources for cell growth and gamma-poly(glutamic acid) (gamma-PGA) production. The effect of the MnSO4 concentration in the medium over a range from 0.0 to 615 microns was studied. The number of viable cells increased for all concentrations of MnSO4 from approximately 10(5) to 10(9) colony-forming units (cfu) ml-1 by the early stationary phase (24 h). However, after 50 h, the cell viability decreased rapidly for relatively lower MnSO4 concentrations (0.615 and 0 microns). The utilization of carbon sources by B. licheniformis was greater for cultures containing 33.8 and 615 microns MnSO4 relative to cultures with no added MnSO4. For example, cultures with 615 microns MnSO4 utilized 37, 54 and 93% and cultures with no added MnSO4 utilized 19, 10 and 17% of glutamate, glycerol and citrate, respectively. The gamma-PGA volumetric yield increased from approximately 5 to 17 gl-1 for corresponding increases in MnSO4 concentration from 0 to 33.8 microns and then decreased at higher MnSO4 concentrations. The stereochemical content of gamma-PGA was found to vary inversely with MnSO4 concentration, and ranged from 59 to 10% L-glutamate units for MnSO4 concentrations of 0 and 615 microns, respectively. For all of the MnSO4 concentrations investigated, the gamma-PGA molecular weights decreased rapidly as the gamma-PGA volumetric yield simultaneously increased for cultivation times from 24 to approximately 50 h. Mw and Mn values after approximately 50 h cultivation times, determined by gel permeation chromatography (GPC), were 1.3 to 1.6 and 0.5 to 0.8 million g mol-1, respectively. A complex gamma-PGA molecular weight distribution that appeared bimodal by GPC analysis due to the presence of a low-molecular-weight product fraction was observed in cultures containing 33.8 and 61.5 microns MnSO4 at extended cultivation times. A high-molecular-weight fraction and the unfractionated gamma-PGA sample from the 33.8 microns MnSO4 culture contained 13 +/- 4 and 30 +/- 1% L-repeat units, respectively. A relationship between the product molecular weight and its stereochemical composition was thus established.