Site-specific N-terminus conjugation of poly(mPEG1100) methacrylates to salmon calcitonin: synthesis and preliminary biological evaluation

Sayers, Claire T.; Mantovani, Giuseppe; Ryan, Sinéad M.; Randev, Rajan K.; Keiper, Odin; Leszczyszyn, Oksana I.; Blindauer, Claudia A.; Brayden, David J.; Haddleton, David M.

doi:10.1039/b905335b

Cited by 27 publications

(33 citation statements)

References 62 publications

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“…Chemical structure, preparation, purification and characterisation of the sCTconjugated polymers has been previously published, including ion-exchange (IE), reverse-phase HPLC and MALDI-TOF [27,28]. SEC-HPLC analysis confirmed high purity and predicted the molecular weights of all sCT-P comb-shaped conjugates (Table S1).…”

Section: Characterization Of Sct-p Conjugatesmentioning

confidence: 92%

PK/PD modelling of comb-shaped PEGylated salmon calcitonin conjugates of differing molecular weights

Ryan

Frías²,

Wang

et al. 2011

Journal of Controlled Release

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Salmon calcitonin (sCT) was conjugated via cysteine-1 to novel combed-shaped endfunctionalised poly(PEG) methyl ether methacrylate) (sCT-P) comb-shaped polymers, to yield conjugates of total molecular weights (MW) inclusive of sCT: 6.5, 9.5, 23 and 40 kDa. The conjugates were characterised by HPLC and their in vitro and in vivo bioactivity was measured by cAMP assay on human T47D cells and following intravenous (i.v.) injection to rats, respectively. Stability against endopeptidases, rat serum and liver homogenates was assessed. There were linear and exponential relationships between conjugate MW with potency and efficacy respectively, however the largest MW conjugate still retained 70% of E max and an EC 50 of 3.7 nM. In vivo, while free sCT and the conjugates reduced serum [calcium] to a maximum of 15-30 % over 240 min, the half life (T½) was increased and the area under the curve (AUC) was extended in proportion to conjugate MW. Likewise, the polymer conferred protection on sCT against attack by trypsin, chymotrypsin, elastase, rat serum and liver homogenates, with the best protection afforded by sCT-P (40 kDa). Mathematical modelling accurately predicted the MW relationships to in vitro efficacy, potency, in vivo PK and enzymatic stability. With a significant increase in T½ for sCT, the 40 kDa MW comb-shaped PEG conjugate of sCT may have potential as a long-acting injectable formulation.

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Section: Characterization Of Sct-p Conjugatesmentioning

confidence: 92%

PK/PD modelling of comb-shaped PEGylated salmon calcitonin conjugates of differing molecular weights

Ryan

Frías²,

Wang

et al. 2011

Journal of Controlled Release

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“…Under these conditions unmodified sCT yields four fragments, Cys1-Lys11, Leu12-Lys18, Leu19-Arg24, and Thr25-Pro(NH2)32. This approach to determine the attachment site in sCT-polymer conjugates was conducted by analogy to that previously described by us 49 and Lee et al 54 His17 and Tyr22 residues reside on separate fragments, Leu12-Lys18 and Leu19-Arg24 respectively, and therefore the selectivity of the conjugation process could be determined by MALDI-ToF-MS analysis of the digest fragments. For both 15 N (3b)-sCT and (6)-sCT analysis of the region corresponding to the Leu19-Arg24 tyrosine-containing fragment revealed a distribution corresponding to a 15 NH2 labeled fragment (addition of 16 Da) as well as the analogous residue from the digestion of the traces of unconjugated sCT present as an impurity in the isolated (6)-sCT biohybrid material, and some secondary distributions ( Figure 6).…”

Section: Characterization Of Sct-polymer Conjugates: Attachment Sitementioning

confidence: 99%

Direct Peptide Bioconjugation/PEGylation at Tyrosine with Linear and Branched Polymeric Diazonium Salts

et al. 2012

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Direct polymer conjugation at peptide tyrosine residues is described. In this study Tyr residues of both leucine enkephalin and salmon calcitonin (sCT) were targeted using appropriate diazonium salt-terminated linear monomethoxy poly(ethylene glycol)s (mPEGs) and poly(mPEG) methacrylate prepared by atom transfer radical polymerization. Judicious choice of the reaction conditionspH, stoichiometry, and chemical structure of diazonium saltled to a high degree of site-specificity in the conjugation reaction, even in the presence of competitive peptide amino acid targets such as histidine, lysines, and N-terminal amine. In vitro studies showed that conjugation of mPEG2000 to sCT did not affect the peptide’s ability to increase intracellular cAMP induced in T47D human breast cancer cells bearing sCT receptors. Preliminary in vivo investigation showed preserved ability to reduce [Ca2+] plasma levels by mPEG2000-sCT conjugate in rat animal models.

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“…3,17,24,25,26 This method provides the ability to completely characterize preformed polymers prior to reaction with a protein. The well-defined polymers derived from RDRP techniques have allowed end group control, either by postpolymerization modifications or through the use of a functionalized initiator, leading to well-defined bioconjugates with control over the site-specificity and multiplicity of the polymers.…”

Section: ■ Introductionmentioning

confidence: 99%

Role of Polymer Architecture on the Activity of Polymer–Protein Conjugates for the Treatment of Accelerated Bone Loss Disorders

et al. 2015

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Polymers of similar molecular weights and chemical constitution but varying in their macromolecular architectures were conjugated to osteoprotegerin (OPG) to determine the effect of polymer topology on protein activity in vitro and in vivo. OPG is a protein that inhibits bone resorption by preventing the formation of mature osteoclasts from the osteoclast precursor cell. Accelerated bone loss disorders, such as osteoporosis, rheumatoid arthritis, and metastatic bone disease, occur as a result of increased osteoclastogenesis, leading to the severe weakening of the bone. OPG has shown promise as a treatment in bone disorders; however, it is rapidly cleared from circulation through rapid liver uptake, and frequent, high doses of the protein are necessary to achieve a therapeutic benefit. We aimed to improve the effectiveness of OPG by creating OPG-polymer bioconjugates, employing reversible addition-fragmentation chain transfer polymerization to create well-defined polymers with branching densities varying from linear, loosely branched to densely branched. Polymers with each of these architectures were conjugated to OPG using a "grafting-to" approach, and the bioconjugates were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The OPG-polymer bioconjugates showed retention of activity in vitro against osteoclasts, and each bioconjugate was shown to be nontoxic. Preliminary in vivo studies further supported the nontoxic characteristics of the bioconjugates, and measurement of the bone mineral density in rats 7 days post-treatment via peripheral quantitative computed tomography suggested a slight increase in bone mineral density after administration of the loosely branched OPG-polymer bioconjugate.

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Site-specific N-terminus conjugation of poly(mPEG1100) methacrylates to salmon calcitonin: synthesis and preliminary biological evaluation

Cited by 27 publications

References 62 publications

PK/PD modelling of comb-shaped PEGylated salmon calcitonin conjugates of differing molecular weights

PK/PD modelling of comb-shaped PEGylated salmon calcitonin conjugates of differing molecular weights

Direct Peptide Bioconjugation/PEGylation at Tyrosine with Linear and Branched Polymeric Diazonium Salts

Role of Polymer Architecture on the Activity of Polymer–Protein Conjugates for the Treatment of Accelerated Bone Loss Disorders

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