Towards a novel haemoglobin-based oxygen carrier: Euro-PEG-Hb, physico-chemical properties, vasoactivity and renal filtration

Portörõ, István; Kocsis, László; Hermán, Péter; Caccia, Dario; Perrella, Michele; Ronda, Luca; Bruno, Stefano; Bettati, Stefano; Micalella, C.; Mozzarelli, Andrea; Varga, Attila; Vas, Mária; Lowe, K. C.; Eke, András

doi:10.1016/j.bbapap.2008.03.005

Cited by 44 publications

(35 citation statements)

References 25 publications

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“…In order to change the target aminoacid for the PEGylation reaction, while using the same PEG derivative (MAL-PEG), we took advantage of the extension arm-facilitated PEGylation, a two-step procedure which has already been used in PEGylation of human hemoglobin to increase the number of reaction sites (33,44) while avoiding the derivatization of cysteines with crucial role in protein activity (42,45). Dr Urad was pretreated with 2-iminothiolane (IMT), a cyclic molecule that reacts with the primary amino groups of lysine and N-terminal residues to form sulfhydryl groups.…”

Section: Resultsmentioning

confidence: 99%

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease

et al. 2017

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PurposeBecause of the evolutionary loss of the uricolytic pathway, humans accumulate poorly soluble urate as the final product of purine catabolism. Restoration of uricolysis through enzyme therapy is a promising treatment for severe hyperuricemia caused by deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). To this end, we studied the effect of PEG conjugation on the activity and stability of the enzymatic complement required for conversion of urate into the more soluble (S)-allantoin.MethodsWe produced in recombinant form three zebrafish enzymes required in the uricolytic pathway. We carried out a systematic study of the effect of PEGylation on the function and stability of the three enzymes by varying PEG length, chemistry and degree of conjugation. We assayed in vitro the uricolytic activity of the PEGylated enzymatic triad.ResultsWe defined conditions that allow PEGylated enzymes to retain native-like enzymatic activity even after lyophilization or prolonged storage. A combination of the three enzymes in an appropriate ratio allowed efficient conversion of urate to (S)-allantoin with no accumulation of intermediate metabolites.ConclusionsPharmaceutical restoration of the uricolytic pathway is a viable approach for the treatment of severe hyperuricemia.Electronic supplementary materialThe online version of this article (doi:10.1007/s11095-017-2167-6) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease

et al. 2017

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“…1,4,9,10 However, clinical trials with diPEG/Hb conjugates have presented side effects, especially because these complexes do not deliver oxygen efficiently due to both their lack of cooperativity and their high oxygen affinity. 11 Conformation of diPEG/Hb conjugates, depending on PEG molecular weight (M w ), have been suggested to play a key role in such physiological changes. Nevertheless, they still remain misunderstood.…”

Section: ■ Introductionmentioning

confidence: 99%

Conformation of the Poly(ethylene Glycol) Chains in DiPEGylated Hemoglobin Specifically Probed by SANS: Correlation with PEG Length and in Vivo Efficiency

et al. 2015

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Cell-free hemoglobin (Hb)-based oxygen carriers have long been proposed as blood substitutes but their clinical use remains tricky due to problems of inefficiency and/or toxicity. Conjugation of Hb with the biocompatible polymer poly(ethylene glycol) (PEG) greatly improved their performance. However, physiological data suggested a polymer molecular weight (Mw) threshold of about 10 kDa, beyond which the grafting of two PEG chains no longer improves efficiency and nontoxicity of diPEG/Hb conjugates. We used small-angle neutron scattering and contrast variation, which are the only techniques able to probe separately the conformation of PEG chains and Hb protein within the complex, to investigate the role of PEG chain conformation in diPEGylated Hb conjugates as a function of the polymer Mw. We found out that the structure of Hb tetramer is not modified by the polymer grafting. Similarly, with a constant grafting of two chains per protein, there is no significant change of the Gaussian conformation between free and grafted PEG below ∼10 kDa, the complex being well described by the "dumbbell" model. However, beyond that threshold, the radius of gyration of grafted PEG is significantly smaller than that of the free polymer, showing a compaction of the PEG chains, either in the "dumbbell" model or in the "shroud" one. In the latter model, the polymer may be wrapped on the surface of the protein spreading a protective "shielding" effect over a larger fraction of the protein. Both proposed models are in good agreement with the physiological data reported in the literature.

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“…The protection of the thiol of Cys-93(b) is also accomplished, when the EAF PEGylation is carried out under deoxy conditions (Portöro et al 2008). When the Cys-93(b) is reversibly protected under the oxy conditions, stability of the tetrameric structure of resulting EAF P5K6 Hb, wherein all EAF PEGylation reactions are targeted to the e-amino groups of Hb, is indistinguishable from the unmodified Hb .…”

Section: Extension Arm Chemistry Facilitates the Attenuation Of Pegylmentioning

confidence: 99%

“…The presence of extension arm between the amino group and the domain of the PEG chains minimizes any potential interactions between the molecular surface of Hb and the PEGchains. This apparently, reduces the impact of the PEG chains on the weakening of the interdimeric interactions of the Hb tetramer as compared to the conjugation of PEG chains directly to the amino groups of the protein without the intervention of extension arms (Portöro et al 2008) has been reported to be higher than MP4 of Sangart, even though both have been generated using EAF PEGylation protocols. The primary difference between the two products is the site selectivity of PEGylation at Cys-93(b).…”

Section: Extension Arm Chemistry Facilitates the Attenuation Of Pegylmentioning

confidence: 99%

“…Euro PEG Hb (Portöro et al 2008) is another prototype EAF PEGylated Hb and is generated under the deoxy conditions. Just as in Einstein approach, 2-iminothiolane mediated one-step extension arm chemistry is used for the generation of Euro PEG Hb as well, but maleimido propionalimido PEG 5K is used instead of maleimido phenyl urethane of PEG.…”

Section: Hemospan and Euro-peg Hbmentioning

confidence: 99%

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Design of Nonhypertensive Conjugated Hemoglobins as Novel Resuscitation Fluids

Acharya

Intaglietta

Tsai

et al. 2013

Hemoglobin-Based Oxygen Carriers as Red Cell Substitutes and Oxygen Therapeutics

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Towards a novel haemoglobin-based oxygen carrier: Euro-PEG-Hb, physico-chemical properties, vasoactivity and renal filtration

Cited by 44 publications

References 25 publications

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease

Conformation of the Poly(ethylene Glycol) Chains in DiPEGylated Hemoglobin Specifically Probed by SANS: Correlation with PEG Length and in Vivo Efficiency

Design of Nonhypertensive Conjugated Hemoglobins as Novel Resuscitation Fluids

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