Title : An alternative to krystexxa: Polysarcosine bioconjucation to uricase, biodistribution, toxicology and pharmacokinetics
PEGylation was one of the first successful technologies able to improve the pharmacokinetic of therapeutic agents and it has been applied in the clinic for more than 25 years.
In early studies, PEG has been considered a biologically inert material. However, it can become immunogenic, resulting in the formation of specific Ab that cause rapid elimination upon repeated administration. This “accelerated blood clearance” (ABC) phenomenon was observed in recent clinical trials as the case of Krystexxa, a recombinant mammalian urate oxidase (Uc) covalently conjugated to PEG (10 kDa), which was approved by the FDA in 2010 to treat refractive gout.
This increasing awareness of anti-PEG antibodies generation is reflected in FDA calling for measurement of anti-PEG antibody responses in new drugs that incorporate PEG, as well as in researchers focusing on the use of alternative polymers.
Here we use Polysarcosine (PSar), a non-ionic hydrophilic polypeptoid, as an alternative to PEG for protein conjugation, using Uc as a model protein. PSar has a highly hydrophilic character which results in a large hydrodynamic volume and a consequent resistance, as PEG, to protein absorption. Moreover, PSar is presented to be an excellent candidate to overcome PEG limitations, offering increased water solubility, immune evasion ability and low immunogenicity. Thus, in order to mimic Krystexxa conjugate, we synthesized a PSar 11 kDa, with a similar hydrodynamic volume as PEG 10 kDa, and then both polymers were conjugated to Uc undergoing the same bioconjugation process.
As a first approximation to validate our approach, polymer fate and toxicity of PSar(11kDa) and PEG(10kDa) were evaluated in a preclinically relevant model by intravenous injection. The quantification of both polymers in key tissues and fluids was achieved, after extraction optimization, using GPC-RI-MALS and UPLC- MALDI-TOF systems. PSar did not produce, unlike PEG, any significant change in pro-inflammatory cytokines concentration. Moreover, its concentration in blood, after an initial decrease, remained constant until 4 hours from administration, unlike PEG that became undetectable during the same time, probably due to the ABC phenomenon. Additionally, both PEG and PSar were not detected in organs tissues with the exception of kidneys and livers for PEG.
Then, the two bioconjugates were characterized in terms of conjugation efficiency by TNBS assay, and residual Uc activity, by measuring the oxidation of uric acid to allantoin (UV-VIS). Afterwards, PEG-Uc and PSar-Uc half-lives were studied in rats' model after a single IV dose. The residual Uc activity was measured in plasma samples as previously described. Both PEG and PSar had a similar conjugation efficiency to Uc, 39 and 40 % respectively, which did not result in a significant decrease of protein activity for both conjugates. PSar-Uc showed a half-life increase (from 3 hours to 80 hours) compared to the native Uc, similar to that observed for PEG-Uc.
PSar not only had a similar behavior compared to PEG in terms of conjugation efficiency and ability to increase Uc half-life but also showed to be less immunogenic, confirming to be an excellent alternative to PEG.
Audience Take Away:
- Audience will become aware of the need to find PEG alternatives in clinic. It is important that academy gets aligned with market necessity in order to focus research on solving actual modern problems.
- In this work, methods to extract both PEG and PSar and quantify them were developed. These results can totally be applied by other researchers for their studies in this field.
- Yes, this job provides a solution to replace PEG in an approved product such as Krystexxa. The positive results obtained in terms of activity, biodistribution and toxicity show how this product is an alternative that, if further characterized, could be approved for clinical studies.