Engineered tissue plasminogen activator

Modified forms of tPA have also been generated in an effort to develop a product with an improved therapeutic profile (e.g. faster acting or exhibiting a prolonged plasma half-life). Reteplase is the international non-proprietary name given to one such modified human tPA produced in recombinant E. coli cells and is sold under the tradenames Ecokinase, Retavase and Rapilysin (Table 12.5). This product's development was based upon the generation of a synthetic nucleotide sequence encoding a shortened (355 amino acid) tPA molecule. This analogue contained only the tPA domains responsible for fibrin selectivity and catalytic activity. The nucleotide sequence was integrated into an expression vector subsequently introduced into E. coli (strain K12) by treatment with calcium chloride. The protein is expressed intracellularly, where it accumulates in the form of an inclusion body. Owing to the prokaryotic production system, the product is non-glycosylated. The final sterile freeze-dried product exhibits a 2-year shelf life when stored at temperatures below 25 °C. An overview of the production process is presented in Figure 12.12.

The lack of glycosylation, as well as the absence of the EGF and K1 domains (Table 12.6), confers an extended serum half-life upon the engineered molecule. Reteplase-based products display a serum half-life of up to 20 min, facilitating its administration as a single bolus injection as opposed to continuous infusion. Absence of the molecule's Fx domain also reduces the product's fibrin-binding affinity. It is theorized that this may further enhance clot degradation, as it facilitates more extensive diffusion of the thrombolytic agent into the interior of the clot. Tenecteplase (also marketed under the tradename Metalyse) is yet an additional engineered tPA now on the market. Produced in a CHO cell line, this glycosylated variant differs in sequence to native tPA by six amino acids (Thr 103 converted to Asn; Asn 117 converted to Gln and the Lys-His-Arg-Arg sequence at position 296-299 converted to Ala-Ala-Ala-Ala). Collectively, these modifications result in a prolonged plasma half-life (to between 15 and 19 min), as well as an increased resistance to PAI-1 (plasminogen activator inhibitor 1, a natural tPA inhibitor).

Figure 12.12 Production of Ecokinase, a modified tPA molecule that gained regulatory approval in Europe in 1996. The production cell line is recombinant E. coli K12, which harbours a nucleotide sequence coding for the shortened tPA molecule. The product accumulates intracellularly in the form of inclusion bodies

Figure 12.12 Production of Ecokinase, a modified tPA molecule that gained regulatory approval in Europe in 1996. The production cell line is recombinant E. coli K12, which harbours a nucleotide sequence coding for the shortened tPA molecule. The product accumulates intracellularly in the form of inclusion bodies

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

Get My Free Ebook


Post a comment