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|Title:||Modification of recombinant human interleukin-11 to enhance pharmacologic properties and therapeutic potential||Authors:||Yu, Kuo-ming||Advisors:||Wong, Wing-tak (ABCT)||Keywords:||Interleukins
Thrombocytopenia -- Treatment
|Issue Date:||2017||Publisher:||The Hong Kong Polytechnic University||Abstract:||Many new medical interventions have been attempted to treat chemotherapy-induced thrombocytopenia but all have ended up with halts in preclinical phase or discontinuation in clinical development, leaving repeated administrations of recombinant human interleukin-11 (rhIL-11) the only medicine available together with platelet transfusion as the gold standard treatment regimen. This unmet medical need could be fulfilled by the novel conjugated rhIL-11, modified with a single 40-KD branched polyethylene glycol (PEG) chain linked to the N-terminal amine, thus providing a long-lasting half-life in vivo, enhanced efficacy and reduced side-effects with regard to dilutional anemia in animal studies. To achieve this, a novel, cost-effective and amiable manufacturing process for rhIL11 isolated from secretory yeast medium has been attempted and successfully developed. This scalable process employed the liquid two-phase extraction of the protein, followed by a refolding process and a two-column step purification process comprising a cation-exchanger and a hydrophobic interaction column in tandem at high sample loads. The overall recovery was about 20~25% with a product purity greater than 95% and impurities mainly comprised of oxidative rhIL-11. Such a novel process resolved problems such as unamiable recovery process, low yield, and N-terminal heterogeneity inherent in the E. coli expression system.
In order to provide sufficient investigational agent for preclinical development exploring efficacy and potential toxicity in animal models, a robust process for production of the PEGylated IL-11 was successfully optimized and scaled-up, yielding a sufficiently large quantity of the medicinal product with consistent quality. The PEGylated drug product presented a specific activity at 1.0~1.4x106 U/mg, which carried approximately 12~14% residual bioactivity of the unmodified rhIL-11. The drug product was found stable at 2~8°C. Deamindation was found to be the primary degradation pathway upon long-term storage. The addition of PEG moiety onto rhIL-11 in 1:1 molar ratio did not alter its secondary structure of rhIL-11, and the conjugation occurred at the N-terminal amine at a high occupancy of 81~86% with the remaining conjugation sites at Lys-174. Such a facile and economic approach of qualitative and quantitative determination of conjugation occupancy employing stable isotope-dimethylation has not been reported. Preclinical assessment of the pharmacology and toxicology properties of the drug was performed employing Sprague Dawley rats and cynomolgus monkeys. Pharmacodynamics studies confirmed that a single subcutaneous injection of the drug in myelosuppressed rats successfully prevented the occurrence of severe thrombocytopenia while exhibited ameliorated dilutional anemia. Non-clinical toxicology studies revealed improved toxicity profiles when compared with those treated with un-modified rhIL-11. The elimination half-life particularly in monkey was extended to 67 hours, thus making monthly clinical regimen feasible. A single exaggerated dose at 15 mg/kg in rats and 6.25 mg/kg in monkeys elicited reversible adverse events without leading to mortality or morbidity. Multiple administration on a weekly basis for continuous 4 weeks at 0.3 mg/kg in monkeys was found tolerable and non-immunogenic. Bones were directly affected upon receiving the drug, whereas transient toxicities in liver, kidney, heart and lung were resulted from fluid retention. The addition of PEG moiety did not elicit additional off-target toxicities. Preclinical evaluations of pharmacology and toxicology support a safe while effective starting dose at 10 µg/kg in human subjects for the planned clinical phase I trial, deploying treatment cohorts of the conventional 3+3 design.
|Description:||xxii, 318 pages : color illustrations
PolyU Library Call No.: [THS] LG51 .H577P ABCT 2017 Yu
|URI:||http://hdl.handle.net/10397/70342||Rights:||All rights reserved.|
|Appears in Collections:||Thesis|
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