Novel poly(ethylene glycol) derivatives for preparation of ribosome-inactivating protein conjugates.
This study describes the synthesis, characterization, and reactivity of new methoxypoly(ethylene glycol) (mPEG) derivatives containing a thioimidoester reactive group. These activated polymers are able to react with the lysyl epsilon-amino groups of suitable proteins, generating an amidinated linkage and thereby preserving the protein's positive charge. mPEG derivatives of molecular weight 2000 and 5000 Da were used, and two spacer arms were prepared, introducing chains of different lengths between the hydroxyl group of the polymer and the thioimidate group. These mPEG derivatives were used to modify gelonin, a cytotoxic single-chain glycoprotein widely used in preparation of antitumoral conjugates, whose biological activity is strongly influenced by charge modification. The reactivity of mPEG thioimidates toward lysil epsilon-amino groups of gelonin was evaluated, and the results showed an increased degree of derivatization in proportion to the molar excesses of the polymer used and to the length of the alkyl spacer. Further studies showed that the thioimidate reactive is able to maintain gelonin's significant biological activity and immunogenicity. On the contrary, modification of the protein with N-hydroxysuccinimide derivative of mPEG strongly reduces the protein's cytotoxic activity. Evaluation of the pharmacokinetic behavior of native and PEG-grafted gelonin showed a marked increase in plasma half-life after protein PEGylation; in particular, the circulating life of the conjugates increased with increased molecular weight of the polymer used. The biodistribution test showed lower organ uptake after PEGylation, in particular by the liver and spleen.
Dipartimento di Scienza e Tecnologia del Farmaco, Via Pietro Giuria 9, 10125 Torino, Italy.
Silvia
S
Arpicco
Franco
F
Dosio
Andrea
A
Bolognesi
Chiara
C
Lubelli
Paola
P
Brusa
Barbara
B
Stella
Maurizio
M
Ceruti
Luigi
L
Cattel
D000818:Animals; D002474:Cell-Free System / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000737:chemistry; D005260:Female; D006207:Half-Life; D020128:Inhibitory Concentration 50; D051379:Mice; D008807:Mice, Inbred BALB C; D009928:Organ Specificity; D010940:Plant Proteins / Q000008:administration & dosage / Q000737:chemistry* / Q000276:immunology / Q000493:pharmacokinetics; D011092:Polyethylene Glycols / Q000008:administration & dosage / Q000737:chemistry*; D011500:Protein Synthesis Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011817:Rabbits; D054789:Ribosome Inactivating Proteins, Type 1; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D054789:Ribosome Inactivating Proteins, Type 1; D011092:Polyethylene Glycols; C025674:GEL protein, Gelonium multiflorum; C028210:monomethoxypolyethylene glycol