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The application of atom transfer radical polymerization (ATRP) for preparation of a novel class of protein-polymer bioconjugates is described, exemplified by the synthesis of a recombinant human growth hormone (rh-GH) poly(ethylene glycol) methyl ether methacrylate (PEGMA) hybrid. The rh-GH protein was activated via a bromo-ester functionalized linker and used as a macroinitiator to polymerize the hydrophilic monomer PEGMA under solely aqueous conditions at 4 degrees C. ATRP conditions resulted in controlled polymer growth from rh-GH with low-polydispersity polyPEGMA chains. The rh-GH PEGMA product exhibited properties consistent with the presence of attached hydrophilic polymer chains, namely, high stability to denaturation and proteolysis. The polymerization conditions and conjugation proceeded with retention of the biological activity of the hormone. The rh-GH PEGMA was administered subcutaneously to rats and the activity compared to native rh-GH. The rh-GH PEGMA exhibited similar activity as the native rh-GH in vivo when a daily dose of 40 microg was administered. However, when a higher dose of 120 microg was administered with 3 days between injections the bioavailability of the rh-GH PEGMA was significantly better than that of the native. The results therefore demonstrate that ATRP can be successfully used as a general alternative approach to direct polymer conjugation, namely, PEGylation, to produce PEG-like protein conjugates. This technique can be exploited to design and synthesize protein-polymer derivatives with tailored therapeutic properties.

Flurbiprofen (FLU) lipophilic prodrugs with lipoamino acids (LAA) 6a- e were synthesized for brain delivery. Chemical and plasmatic stability of prodrugs 6a- e as well as pharmacokinetic distribution studies for the prodrugs 6b and 6d were carried out. FLU prodrugs 6a- e were compared to the parent drug for their ability to inhibit binding of [F-18]FDDNP to in vitro formed beta-amyloid protein (Abeta fibrils). FLU-LAA conjugates showed a typical prodrug stability profile, being stable in PBS at pH 7.4 and releasing the active drug in plasma. Compound 6d yielded a slow accumulation of FLU in the brain. In the in vitro inhibition assay, all prodrugs except for the prodrug with the longest alkyl side chain ( 6e) were effective as inhibitors of [F-18]FDDNP binding to Abeta fibrils with EC50 values in the 10-300 nM range. The different brain accumulation kinetics shown by FLU and its LAA conjugate 6d suggested a possible slow-releasing activity of FLU by these prodrugs in the brain or a differential pharmacological effect deserving further, detailed studies on their biodistribution and pharmacological profile.

The tumor targeting properties of a new drug carrier synthesized by bioconjugation of folic acid (FA) to beta-cyclodextrins through a poly(ethylene glycol) (PEG) spacer (CD-PEG-FA) were investigated. Surface plasmon resonance demonstrated that CD-PEG-FA specifically interacts with immobilized folate binding protein (FBP) while the naked beta-cyclodextrins do not display any specific interaction. In vitro studies demonstrated that CD-PEG-FA was devoid of cell toxicity. [(3)H]-folic acid/CD-PEG-FA competition binding investigations performed with folate receptor overexpressing human epidermal carcinoma KB cells showed that CD-PEG-FA had about 14 times lower tumor cell binding capacity than free folic acid. The carrier cell trafficking properties were investigated using rhodamine-B as fluorescent probe, which possesses 3000 and 4580 M(-)(1) inclusion constants for CD-PEG-FA and beta-cyclodextrins, respectively. Cell-associated fluorescence measurements showed that CD-PEG-FA does not promote the rhodamine-B uptake into non-folate receptor expressing human lung carcinoma MCF7 cells while 19% higher accumulation in KB cells was found with respect to rhodamine-B loaded beta-cyclodextrins. Confocal laser scanning microscopy indicated the presence of cytosolic red fluorescent spots after 2 h of incubation of KB cells with rhodamine-B included CD-PEG-FA. The fluorescent dye resided primarily in small spots, namely, endosomes and multivesicular bodies. At 1 h after pulsed incubation, wider red fluorescent cellular structures appeared as a fusion of previous structures.

beta-Cyclodextrin-poly(ethylene glycol)-folic acid conjugate (CD-PEG-FA) was synthesized according to a two-step procedure: (1). synthesis of CD-PEG-NH(2) by reaction of monotosyl-activated beta-cyclodextrin with excess of 700 Da diamino-PEG; (2). synthesis of CD-PEG-FA by reaction of CD-PEG-NH(2) with succinimidyl ester-activated folic acid. The CD-PEG-NH(2) intermediate was purified by precipitation in acetone, and the CD-PEG-FA by gel permeation and C-18 reversed-phase chromatography. Both CD-PEG-NH(2) and CD-PEG-FA were analyzed by mass spectrometry, (1)H NMR, and UV-vis spectroscopy. All analytical methods confirmed the theoretical composition of the conjugates: the CD-PEG-NH(2) intermediate was composed of CD and PEG in the molar ratio of 1:1, and the CD-PEG-FA was composed of beta-cyclodextrin, PEG, and folic acid in the molar ratio of 1:1:1. The CD-PEG-FA conjugate was highly soluble in buffer (>42 mM) as compared to the unmodified beta-cyclodextrin (16.3 mM). Phase solubility diagrams of beta-estradiol revealed that drug solubility increases from 11 microM in buffer to 600 microM in the presence of beta-cyclodextrins and 5900 microM with CD-PEG-FA. However, the affinity of beta-estradiol for beta-cyclodextrins decreased about 4 times with PEG and folic acid conjugation. Stability studies carried out using chlorambucil confirmed that the conjugate partially prevents drug degradation in buffer, although this effect was considerably lower than that obtained with beta-cyclodextrin. Computer modeling studies showed that the folic acid linked to the beta-cyclodextrins through a PEG spacer could partially interact with the cyclodextrin cavity. Finally, CD-PEG-FA displayed reduced hemolytic effect as compared to unmodified beta-cyclodextrin.