Scale up and pharmacokinetic study of a novel mutated chimeric tissue plasminogen activator (mt-PA) in rats

Abstract

Because of high mortality caused by cardiovascular diseases, various fibrinolytic agents with diverse pharmacokinetic and pharmacodynamic properties have been developed. A novel mutated chimeric tissue plasminogen activator (mt-PA) was developed by the removal of first three domains of t-PA, insertion of GHRP sequence and mutation towards resistance to plasminogen activator inhibitor-1 (PAI-1). Mt-PA protein was expressed in Expi293F cells. The expression level of mt-PA was found to be 5000 IU/mL. Following purification, the pharmacokinetic properties of mt-PA were evaluated in three doses in rats. Data related to mt-PA were best fitted to two compartment model. With the increase in dose, the Area Under the plasma concentration-time Curve (AUC_0→∞) increased. The elimination half-life (t_1/2) of mt-PA was in the range of 19.1-26.1 min in three doses while that of Alteplase was 8.3 min. The plasma clearance (CLp) of mt-PA ranged from 3.8 to 5.9 mL/min in three doses, which was several times lower than that of Alteplase (142.6 mL/min). The mean residence time (MRT) of mt-PA ranged from 23.3-31.8 min in three doses, which was 4-5 times greater than that of Alteplase (6 min). Mt-PA showed extended half-life and mean residence time and is a good candidate for further clinical studies.

Figure 1. Gel-electrophoresis, SDS-PAGE and Western blotting analysis of mt-PA.

(a) Gel-electrophoresis analysis of the mt-PA- pTracer-SV40 Plasmid by SmaI restriction enzyme digestion on 1% agarose gel. (Lane 1) DNA Ladder 1 Kb. (Lane 2) pTracer-SV40 Plasmid without the mt-PA gene. (Lane 3) pTracer-SV40 Plasmid without the mt-PA gene digested by SmaI restriction enzyme. (Lane 4) pTracer-SV40 Plasmid with the mt-PA gene. (Lane 5) pTracer-SV40 Plasmid with the mt-PA gene digested by SmaI restriction enzyme. (b) The mt-PA expression analysis in transfected Expi293F cells supernatant on a 12% SDS-PAGE gel. (Lane 1) Supernatant from non-transfected Expi293F cell culture medium as negative control. (Lane 2) The mt-PA Expi293F cell culture medium. (Lane 3) Rainbow Molecular Weight Marker (code RPN756). (Lane 4) Alteplase as positive control. (c) Western blotting detection of the mt-PA expression in the supernatant of transfected Expi293F cells. (Lane 1) Supernatant from non-transfected Expi293F cell culture medium (negative control). (Lane 2) The mt-PA Expi293F cell culture medium. (Lane 3) Rainbow Molecular Weight Marker (code RPN756). (Lane 4) Alteplase (full length form). The samples in a (gel) and b (blot) derived from the same experiment and the gel and blot were processed in parallel.

Figure 2. The analysis of the affinity chromatography fractions of the transfected Expi293F cells supernatant on SDS-PAGE gel.

(Lane 1) Prior to the purification of supernatant. (Lane 2) Flow- through. (Lane 3) Unstained Protein MW Marker (Thermo Scientific). (Lane 4–10) The fractions of second elution buffer. The fractions of first elution buffer was cropped. The full-length gel is presented in Supplementary Figure S1.

Figure 3. SDS-PAGE and Western blotting analysis of mt-PA after purification.

(a) Detection of mt-PA after the 2nd step of purification using SDS-PAGE procedure. (Lane 1) Before size exclusion chromatography. (Lane 2) First peak. (Lane 4) Mt-PA. (Lane 5) Unstained Protein MW Marker (Thermo Scientific). (b) Western blotting analysis of formulated purified mt-PA. (Lane 1) Alteplase as positive control. (Lane 2) Prestained Protein Marker (Fermentas). (Lane 3) Supernatant from non-transfected Expi293F cell culture medium (negative control). (Lane 4) Formulated purified mt-PA with 43 kDa size.

Figure 4. Plasma activity of mt-PA and Alteplase after their intravenous administration to rats.

(a) Plasma activity of a 100 IU mt-PA after i.v. bolus injection to three rats. (b) Plasma activity of a 200 IU mt-PA after i.v. bolus injection to four rats. (c) Plasma activity of a 400 IU mt-PA after i.v. bolus injection to six rats. (d) Plasma activity of a 4000 IU Alteplase after i.v. bolus injection to six rats. (e) The comparison of mean (Mean ± SD) plasma activity of 400 IU mt-PA and 4000 IU Alteplase after intravenous administration to rats (n = 6).

Figure 5. Box plot of parameter model estimates of individual values over different treatment and dosing amount were determined from a pharmacokinetics model in rat.

Abbreviations: A, the y-intercept of the distribution phase; B, the y-intercept of the elimination phase; α, the distribution slope; β, the elimination slope.