Pharmacokinetics, immunogenicity and anticancer efficiency of Aspergillus flavipesl-methioninase

Methionine starvation can powerfully modulate DNA methylation, cell cycle transition, polyamines and antioxidant synthesis of tumor cells, in contrary to normal ones. Aspergillus flavipesl-methioninase was previously characterized by our studies, displaying affordable biochemical properties comparing to Pseudomonas putida enzyme (ONCASE). Thus, the objective of current study was to evaluate the catalytic properties of Af-METase in New Zealand rabbits, exploring its antitumor efficacy. In vivo, Af-METase (40.8U/ml) have T₁/₂ 19.8h, elimination constant 0.088U/h and apparent volume distribution 85U/ml. Also, Af-METase has two maxima one at A₂₈₀ₙₘ (apo-enzyme) and at A₄₂₀ₙₘ (internal Schiff base of PLP), unlike control plasma (without enzyme). The two peaks of absorption spectra were detected maximally at 15min then the absorbance at 420nm was subsequently decreased with circulation time, due to dissociation of the co-enzyme. The A₂₈₀/₄₂₀ ratio was increased from 1.69 to 5.81 with circulation time from 15 to 30h. Rabbits plasma methionine was depleted from 18.7μM (control) to 8.8μM after 1h of enzyme injection and completely omitted after 2h till 19h, assuming the sustainability of negligible levels of methionine (<2μM) in plasma of rabbits, for about 17h. Upon infusion of PLP, the T₁/₂ of Af-METase was significantly prolonged by 3.2 fold, assuming the fully reconstitution of the enzyme. The holo-AfMETase still retained its co-enzyme, completely, till 33h of PLP infusion. From spectral studies, the internal aldimine linkage of apo-Af-METase was constructed upon PLP infusion, with fully catalytic structure after less than 4h of its infusion, the A₂₈₀/₄₂₀ ratio being not relatively changed till 45h. After 25 days of last enzyme dose, the titer of IgG was increase by about 1.66 fold comparing to control (without enzyme). However, IgM was not detected along the tested challenge points. In vitro, plasma anti-Af-METase neutralizing antibodies (NAb) were assessed, with no significant reduction on activity of Af-METase by Nab. All the hematological parameters were in normal range, otherwise, the RBCs titer and platelet level was slightly increased, after 25 days of Af-METase injection, comparing to control. There is no obvious negative effect on chemistry of liver, kidney, glucose, lipids, and other electrolytes. Additionally, the anticancer activity of Af-METase was evaluated against five types of human cancer cell lines, in vitro. The enzyme showed a powerful activity against prostate (PC3), liver (HEPG2) and breast (MCF7) cancers, with IC₅₀ 0.001U/ml, 0.26U/ml and 0.37U/ml, respectively.