Methods for treating vascular disorders
A vascular and disease technology, applied in the field of medical science, can solve problems beyond the physiological toxicity range of r-aPC
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preparation example 1
[0032] human protein C
[0033] Recombinant human protein C (rHPC) is produced in human kidney 293 cells by techniques well known to those skilled in the art, see, eg, US Patent 4,981,952 to Yan, the entire teaching of which is incorporated herein by reference. The gene encoding human protein C is disclosed and claimed in US Patent 4,775,624 to Bang et al., the entire teachings of which are incorporated herein by reference. The plasmid used to express human protein C in 293 cells is the plasmid pLPC disclosed in US Patent 4,992,373 to Bang et al., the entire teaching of which is incorporated herein by reference. The construction of plasmid pLPC is also described in European Patent 0445939 and Grinnell et al., 1987, Bio / Technology 5: 1189-1192, the entire teachings of which are incorporated herein by reference. Briefly, this plasmid was transfected into 293 cells, stable transformants were identified, subcultured and grown in serum-free medium. After fer...
preparation example 2
[0038] Activation of recombinant human protein C
[0039] Thrombin was coupled to activated CH-Sepharose (Sepharose) 4B (Pharmacia) at pH 7.5 in the presence of 50 mM HEPES at 4°C. The coupling reaction was carried out on the resin already packed into the column, using about 5000 units of thrombin / ml resin. The thrombin solution was circulated through the column for about 3 hours before adding MEA at a concentration of 0.6 ml / l of circulating solution. The solution containing MEA was recirculated for 10-12 hours to ensure complete blocking of unreacted amine on the resin. After blocking, the thrombin-coupled resin was washed with 10 times volume of 1M NaCl, 20mM Tris, pH 6.5 to remove non-specifically bound proteins, and used for activation reaction after equilibrating in activation buffer.
[0040] Purified rHPCs were formulated in 5 mM EDTA (to chelate any residual calcium ions) and diluted to a concentration of 2 mg / ml with 20 mM Tris, pH 7.4 or 20 mM T...
Embodiment 1
[0046] Human plasma concentration of aPC
[0047] Within 24 hours, 6 patients received 1 mg / m 2 / hour or about 0.024mg / kg / hour received aPC intravenous infusion. The aPC used was a lyophilized preparation containing 10 mg aPC, 5 mM Tris acetate buffer and 100 mM NaCl, redissolved with 2 ml of water and adjusted to pH 6.5.
[0048] Plasma concentrations of aPC were measured using an immunocapture-amidolytic assay. Blood was collected in the presence of citrate anticoagulant and benzamidine, a reversible inhibitor of aPC. The enzyme was captured from plasma by using an aPC-specific mouse monoclonal antibody, C3, immobilized on a microplate. This inhibitor was removed by washing and the amidolytic activity of aPC was detected with an oligopeptide chromogenic substrate. After incubation at 37°C for 16-20 hours, the absorbance was measured at 405 nm, and the data were analyzed using a weighted linear curve fitting algorithm. The concentration of aPC estima...
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