Improvement of pharmacokinetic, immunological and stability properties of asparaginase by conjugation to linear and branched monomethoxy poly( ethylene glycol)

Summary Pretargeting techniques using the avidin-biotin system have shown encouraging results in both diagnostic and therapeutic clinical trials. It has been shown that in cancer therapy the ideal agent to be used for pretargeting should have a plasma half-life longer than avidin and lower immunogenicity than streptavidin in order for these procedures to be applied safely and repeatedly in patients. We prepared a recombinant form of avidin with no carbohydrates and avidins, biochemically modified either by decreasing the positive charges with succinic anhydride or by linking polyethylene glycol (PEG) at three different molar ratios and evaluated their in vivo behaviour after i.p. administration in mice. The succinylation and PEGylation of avidin increased the plasma half-life proportionally to the degree of protein modification. The procedures, however, affected the biotin binding to some extent. The biodistribution studies showed that, for all six time points (ranging from 20 min to 18 h post-injection), the liver and kidney to blood ratios were lower for PEGylated avidins than native, recombinant and succinyl avidin. Recombinant and low PEGylated avidin evoked an immune response in all mice after at least three injections. Native, recombinant and succinyl avidins showed higher serum titres than PEGylated avidins. In conclusion, the conjugation of avidin to PEG chains (n = 7) originates a compound with a suitable blood clearance, low immunogenicity and concurrent low cross-reactivity with avidin.

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“…In fact, previous studies had demonstrated that mPEG chains did not affect the coating of avidin to the wells (Veronese et al, 1996).…”

Section: Biochemical and Functional Characteristicsmentioning

confidence: 94%

Biochemical modifications of avidin improve pharmacokinetics and biodistribution, and reduce immunogenicity

Chinol

Casalini²,

Maggiolo³

et al. 1998

Br J Cancer

110

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“…The DBA/2 mouse was specifically chosen to be the host for the implanted tumor cells because L5178Y cells had been demonstrated to be tumorigenic in this mouse strain [31]. In addition, previous investigations had used this cell line and mouse combination to examine the success of various asparaginase therapy, therefore, a history of success using this combination has already been demonstrated and documented [32]. Moreover, another advantage of using DBA/ 2 mouse strain was that it did not require any special food or housing conditions associated with immune deficient mice such as NOD/SCID.…”

Section: Survival Of Dba/2 Mice After L5178y Treated With Tat-asnasementioning

confidence: 99%

PTD-modified ATTEMPTS system for enhanced asparaginase therapy: A proof-of-concept investigation

Kwon

Liang

et al. 2008

Journal of Controlled Release

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Macromolecular drugs such as proteins and gene products are presumably the most desirable therapeutic agents due to their unmatched substrate specificity and reaction efficiency. Yet, clinical use of these drugs has met with limited success, primarily due to the impermeable nature of the cell membrane that restricts cellular drug uptake to only small (<600 Da) and hydrophobic molecules. The recent discovery of the protein transduction domain (PTD) membrane-penetrating peptides, such as HIV-TAT, has finally offered the possibility of resolving this cell-membrane barrier for macromolecular drug delivery. Via covalent linkages, these PTD peptides have been shown to ferry the attached macromolecular species across membranes of all cell types, both in vitro and in vivo. Nevertheless, the lack of selectivity for PTD-mediated internalization restricts the application of this cell uptake method in clinical practice, due to concerns of inducing systemic toxicity caused by the carried drugs.Presented herein is a modified version of our previously established "ATTEMPTS" approach in delivery of macromolecular drugs, which integrates the cell-penetrating PTDs into a heparin/ protamine-regulated delivery system. In vitro findings using asparaginase (ASNase) as a model macromolecular anti-tumor agent were able to validate the feasibility of this delivery system. The chemically constructed TAT-ASNase conjugates not only were able to translocate into the MOLT-4 cells and elicit the cytotoxic effects, but also this PTD-mediated intracellular drug uptake could be regulated (with on/off control) by the addition of heparin and protamine. This modified ATTEMPTS system therefore presents a new avenue of treatment of various types of cancers and other diseases with macromolecular drugs. In vitro characterization and a preliminary proof-of-concept animal investigation that demonstrates the feasibility of this PTD-mediated ASNase therapeutic system is subsequently described.

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“…However, studies that directly compare linear polymers to (related) branched ones are rare. In two important studies, Veronese and co-workers compared the activity, pH and temperature stability, and proteolytic digestion of ASNase (from Erwinia Carotivora) and three other enzymes modified with either linear mPEG or double-branched mPEG (mPEG 2 ) 33,34 . Although, for the most part, catalytic activity was similar for the mPEG and mPEG 2 bioconjugates, all of the enzymes modified with mPEG 2 were more resistant to proteolysis.…”

Section: Circulation Time and Bioactivity Of Asnase In Vivomentioning

confidence: 99%

Semi-permeable coatings fabricated from comb-polymers efficiently protect proteins in vivo

et al. 2014

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In comparison to neutral linear polymers, functional and architecturally complex (that is, non-linear) polymers offer distinct opportunities for enhancing the properties and performance of therapeutic proteins. However, understanding how to harness these parameters is challenging, and studies that capitalize on them in vivo are scarce. Here we present an in vivo demonstration that modification of a protein with a polymer of appropriate architecture can impart low immunogenicity, with a commensurably low loss of therapeutic activity. These combined properties are inaccessible by conventional strategies using linear polymers. For the model protein L-asparaginase, a comb-polymer bio-conjugate significantly outperformed the linear polymer control in terms of lower immune response and more sustained bioactivity. The semi-permeability characteristics of the coatings are consistent with the phase diagram of the polymer, which will facilitate the application of this strategy to other proteins and with other therapeutic models.

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Improvement of pharmacokinetic, immunological and stability properties of asparaginase by conjugation to linear and branched monomethoxy poly( ethylene glycol)

Cited by 88 publications

References 23 publications

Biochemical modifications of avidin improve pharmacokinetics and biodistribution, and reduce immunogenicity

Biochemical modifications of avidin improve pharmacokinetics and biodistribution, and reduce immunogenicity

PTD-modified ATTEMPTS system for enhanced asparaginase therapy: A proof-of-concept investigation

Semi-permeable coatings fabricated from comb-polymers efficiently protect proteins in vivo

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