Investigation of the effects of altered receptor binding activity on the clearance of erythropoiesis-stimulating proteins: Nonerythropoietin receptor-mediated pathways may play a major role

Agoram, Balaji; Aoki, Ken; Doshi, Sameer; Gegg, Colin V.; Jang, Graham; Molineux, Graham; Narhi, Linda O.; Elliott, Steve

doi:10.1002/jps.21578

Cited by 35 publications

(34 citation statements)

References 40 publications

(57 reference statements)

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“…Because of the two additional N-linked carbohydrate chains, the molecular weight increased from 30 to 37 kDa and the carbohydrate part of the new molecule increased from 40% to 51%. Importantly, the maximum number of sialic-acid residues increased from 14 to 22, resulting in an increase in serum half-life [46]. Molecules with carbohydrate contents greater than darbepoetin alfa (N53 and AMG114) and polyethylene glycol-linked darbepoetin alfa (peg-Aranesp) have also been examined, and these have a further increase in serum half-life.…”

Section: Successful Development Of Darbepoetin Alfamentioning

confidence: 99%

“…To determine whether EPOR-mediated degradation explained the reduced clearance of darbepoetin alfa in vivo, experiments with rHuEPO analogs with different receptor binding and clearance properties were performed [46]. Pharmacokinetic characteristics of an EPO analog (NM385), which had no detectable EPOR binding activity, showed only a modest difference in clearance compared with rHuEPO.…”

Section: Successful Development Of Darbepoetin Alfamentioning

confidence: 99%

See 1 more Smart Citation

Discovery and basic pharmacology of erythropoiesis-stimulating agents (ESAs), including the hyperglycosylated ESA, darbepoetin alfa: an update of the rationale and clinical impact

Kiss¹,

Elliott

Jedynasty³

et al. 2010

Eur J Clin Pharmacol

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Cloning of the human erythropoietin (EPO) gene and development of the first recombinant human erythropoietin (rHuEPO) drug were truly breakthroughs. This allowed a deeper understanding of the structure and pharmacology of rHuEpo, which in turn inspired the discovery and development of additional erythropoiesisstimulating agents (ESAs). In vivo specific activity and serum half-life of rHuEPO are influenced by the amount and structure of the attached carbohydrate. Increased numbers of sialic acids on carbohydrate attached to rHuEPO correlated with a relative increase in in-vivospecific activity and increased serum half-life. The effect of increasing the number of sialic-acid-containing carbohydrates on in-vivo-specific activity was explored. Initial research focused on solving the problem of how the protein backbone could be engineered so a cell would add more carbohydrate to it. Additional work resulted in darbepoetin alfa, a longer-acting molecule with two additional carbohydrate chains.

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Section: Successful Development Of Darbepoetin Alfamentioning

confidence: 99%

Section: Successful Development Of Darbepoetin Alfamentioning

confidence: 99%

Discovery and basic pharmacology of erythropoiesis-stimulating agents (ESAs), including the hyperglycosylated ESA, darbepoetin alfa: an update of the rationale and clinical impact

Kiss¹,

Elliott

Jedynasty³

et al. 2010

Eur J Clin Pharmacol

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“…Extended terminal half-life due to attachment of 30 kDa PEG is in agreement with findings from previous studies. 12,34 The attachment of a 30 kDa PEG to the E31C analog results in a five-fold increase in half-life, as reported by Agoram et al 12 It has been shown that PEGylation influences protein clearance and plays a crucial role in their elimination from the circulation. 6 A 10-fold decrease in clearance of PEGylated E31C analog with 30 kDa PEG is comparable with another study in which a 30 kDa PEG attached to the NM385 variant.…”

Section: Dovepressmentioning

confidence: 86%

“…The five Sprague-Dawley rats (300 g ± 30 g weight) in each group received a single intravenous dose of 30 µg protein/kg. 12 Blood samples were collected from the lateral tail vein at 15 minutes, 1 hour, 8 hours, 24 hours, 48 hours, 96 hours, 120 hours, and 144 hours after the injection. The collected samples were centrifuged at 1500 g for 12 minutes, and separated plasma was analyzed using an EPO ELISA kit (Roche Diagnostics GmbH).…”

Section: Biological Assays and Pharmacokinetic Studiesmentioning

confidence: 99%

“…11 For example, the only marketed PEGylated form of rhEPO, CERA ® , was generated by conjugation of 30 kDa methoxy-PEG via amide bonds to the N-terminal or the amino groups of lys-52 or lys-45. 12,13 These drawbacks can be successfully compensated by chemoselective cysteine PEGylation, which is a strong tool for improvement of pharmacokinetic properties of therapeutic proteins. However, the addition of free cysteine residue to protein is not simple and occurs commonly in cysteine PEGylation because of the rare existence of free cysteine residue in the proteins.…”

Section: Introductionmentioning

confidence: 99%

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Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin

Cohan

Madadkar-Sobhani

Khanahmad³

et al. 2011

IJN

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Background: Recombinant human erythropoietin (rhEPO) is considered to be one of the most pivotal pharmaceutical drugs in the market because of its clinical application in the treatment of anemia-associated disorders worldwide. However, like other therapeutic proteins, it does not have suitable pharmacokinetic properties for it to be administrated at least two to three times per week. Chemoselective cysteine PEGylation, employing molecular dynamics and graphics in in silico studies, can be considered to overcome such a problem. Methods: A special kind of EPO analog was elicited based on a literature review, homology modeling, molecular dynamic simulation, and factors affecting the PEGylation reaction. Then, cDNA of the selected analog was generated by site-directed mutagenesis and subsequently cloned into the expression vector. The construct was transfected to Chinese hamster ovary/dhfr − cells, and highly expressed clones were selected via methotrexate amplification. Ion-immobilized affinity and size exclusion (SE) chromatography techniques were used to purify the expressed analog. Thereafter, chemoselective PEGylation was performed and a nanosize PEGylated EPO was obtained through dialysis. The in vitro biologic assay and in vivo pharmacokinetic parameters were studied. Finally, E31C analog Fourier transform infrared, analytical SE-high-performance liquid chromatography, zeta potential, and size before and after PEGylation were characterized. Results: The findings indicate that a novel nanosize EPO31-PEG has a five-fold longer terminal half-life in rats with similar biologic activity compared with unmodified rhEPO in proliferation cell assay. The results also show that EPO31-PEG size and charge versus unmodified protein was increased in a nanospectrum, and this may be one criterion of EPO biologic potency enhancement. Discussion: This kind of novel engineered nanosize PEGylated EPO has remarkable advantages over rhEPO.

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Time‐dependent clearance and hematological pharmacodynamics upon erythropoietin multiple dosing in rats

Ait‐Oudhia

Scherrmann

Krzyzanski

2010

Biopharm & Drug Disp

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The pharmacokinetics (PK) and pharmacodynamics (PD) of recombinant human erythropoietin (rHuEPO) upon its repeated administrations were investigated. Two groups (A and B) of normal Wistar rats received rHuEPO intravenously at 450 or 1350 IU/kg thrice weekly for 2 and 6 weeks. PK studies were conducted following days 0 and 4 for group (A) and days 0, 17 and 28 for group (B), then, washout PK were assessed on days 11 and 36 for both groups. Reticulocytes (RET), red blood cells (RBC) and hemoglobin (Hb) were evaluated daily until day 14, then every 2 days until day 30 for group (A) and 59 for group (B). The total clearance CL(Total) increased with the dose but decreased over time. Its decay reached 20% and 55% between the first and last full PK in both treatment arms. RET peaked on day 5 and were 77.6% and 87.3% higher than baselines for the two dosing regimen. Their nadirs occurred on days 22 and 55 and were 37.9% and 47.3% below normal values. Hb peaked on days 10 and 34 and was 28.9% and 38.6% above the baseline level, its nadirs occurred on days 25 and 57 and were 13.1% and 16% below baselines. Control animals showed stable baselines over the study but with moderate variability. In conclusion, rHuEPO exhibits a nonlinear PK with a time-dependent decrease of its CL(Total). During exposure, RET, RBC and Hb showed a tolerance effect. After exposure, the rebound was characterized for RET, RBC, but not Hb.

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Investigation of the effects of altered receptor binding activity on the clearance of erythropoiesis-stimulating proteins: Nonerythropoietin receptor-mediated pathways may play a major role

Cited by 35 publications

References 40 publications

Discovery and basic pharmacology of erythropoiesis-stimulating agents (ESAs), including the hyperglycosylated ESA, darbepoetin alfa: an update of the rationale and clinical impact

Discovery and basic pharmacology of erythropoiesis-stimulating agents (ESAs), including the hyperglycosylated ESA, darbepoetin alfa: an update of the rationale and clinical impact

Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin

Time‐dependent clearance and hematological pharmacodynamics upon erythropoietin multiple dosing in rats

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