Anti-inflammatory effects of intravenous methotrexate associated with lipid nanoemulsions on antigen-induced arthritis

Abstract

Objective: To test the hypothesis that intravenous use of methotrexate associated with lipid nanoemulsions can achieve superior anti-inflammatory effects in the joints of rabbits with antigen-induced arthritis compared with commercial methotrexate.

Methods: Arthritis was induced in New Zealand rabbits sensitized with methylated bovine serum albumin and subsequently intra-articularly injected with the antigen. A nanoemulsion of methotrexate labeled with 3H-cholesteryl ether (4 mg/kg methotrexate) was then intravenously injected into four rabbits to determine the plasma decaying curves and the biodistribution of the methotrexate nanoemulsion by radioactive counting. Additionally, the pharmacokinetics of the methotrexate nanoemulsion were determined by high-pressure liquid chromatography. Twenty-four hours after arthritis induction, the animals were allocated into three groups, with intravenous injection with saline solution (n=9), methotrexate nanoemulsion (0.5 µmol/kg methotrexate, n=7), or commercial methotrexate (0.5 µmol/kg, n=4). The rabbits were sacrificed 24 h afterward. Synovial fluid was then collected for protein leakage and cell content analyses and synovial membranes were collected for histopathological analysis.

Results: The methotrexate nanoemulsion was taken up mainly by the liver and the uptake by arthritic joints was two-fold greater than that by control joints. The methotrexate nanoemulsion treatment reduced leukocyte influx into the synovial fluid by nearly 65%; in particular, mononuclear and polymorphonuclear cells were reduced by 47 and 72%, respectively. In contrast, cell influx was unaffected following treatment with commercial methotrexate. Protein leakage into the arthritic knees of the rabbits was also more limited following methotrexate nanoemulsion treatment than following commercial methotrexate treatment.

Conclusions: The intravenous methotrexate nanoemulsion showed anti-inflammatory effects on the synovia of arthritic joints that were clearly superior to the effects of a commercial methotrexate preparation. This result is conceivably due to greater methotrexate uptake by the joints when the drug is associated with a nanoemulsion.

Conflict of interest statement

No potential conflict of interest was reported.

Figures

Figure 1-

Decay curve of didodecyl MTX associated with an LDE after a bolus injection of 3.6 mg/kg drug and pharmacokinetic parameters obtained from the curve. The concentration of MTX in the plasma was determined by HPLC. The analysis was performed using a two-compartment open model. t1/2β: elimination half-life; AUC: area under the plasma concentration-time curve; Vss: volume of distribution at steady state; CL: total body clearance. The results are expressed as the mean ± SE of data obtained from 4 rabbits.

Figure 2-

Tissue uptake of LDE labeled with 3H-cholesteryl oleyl ether. The tissue samples were excised for radioactivity counting 24 h after intravenous injection of the labeled LDE into the rabbits with AIA. Contralateral synovia without arthritis induction were used as controls.

Figure 3-

Leukocyte count (monocytes: white column; polymorphonuclear cells: gray column) in the synovial fluid of rabbits 48 h after AIA induction. The animals received commercial MTX (0.5 µmol/kg), LDE-MTX (0.5 µmol/kg) or saline solution intravenously 24 h after AIA induction. * p<0.05 compared with the control saline-treated rabbits with AIA. The results are expressed as the mean ± SEM. The data have been subjected to repeated analysis of variance. Post-analysis was conducted using Newman-Keuls multiple comparison tests. In all analyses, p<0.05 was considered statistically significant.

Figure 4-

Vascular permeability, as assessed by the Evans Blue method, in joint fluid collected from the joints of rabbits 48 h after AIA induction. The animals received intravenous commercial MTX (0.5 µmol/kg), LDE-MTX (0.5 µmol/kg) or saline solution 24 h after the induction of arthritis. *p<0.05 compared with the control saline-treated rabbits with AIA. The results are expressed as the mean ± SEM. The data have been subjected to repeated analysis of variance. Post-analysis was conducted using Newman-Keuls multiple comparison tests. In all analyses, p<0.05 was considered statistically significant.