Selective and predicable amine conjugation sites by kinetic characterization under excess reagents.

The site selectivity for lysine conjugation on a native protein is difficult to control and characterize. Here, we applied mass spectrometry to examine the conjugation kinetics of Trastuzumab-IgG (Her-IgG) and α-lactalbumin under excess linker concentration ([L]0) based on the modified Michaelis-Menten equation, in which the initial rate constant per amine (kNH2 = Vmax/NH2/KM) was determined by the maximum reaction rate (Vmax/NH2) under saturated accessible sites and initial amine-linker affinity (1/KM). Reductive amination (RA) displayed 3-4 times greater Vmax/NH2 and a different panel of conjugation sites than that observed for N-hydroxysuccinimide ester (NHS) chemistry using the same length of polyethylene glycol (PEG) linkers. Moreover, faster conversion power rendered RA site selectivity among accessible amine groups and a greater tunable range of linker/protein ratio for aldehyde-linkers compared to those of the same length of NHS-linkers. Single conjugation with high yield or poly-conjugations with site homogeneity was demonstrated by controlling [L]0 or gradual addition to minimize the [L]0/KM ratio. Formaldehyde, the shortest aldehyde-linker with the greatest 1/KM, exhibited the highest selectivity and was shown to be a suitable probe to predict conjugation profile of aldehyde-linkers. Four linkers on the few probe-predicted hot spots were elucidated by kinetically controlled RA with conserved drug efficacy when conjugated with the payload. This study provides insights into controlling factors for homogenous and predictable amine bioconjugation.

Electrospun nanofibers in oral drug delivery.

In order to enhance the delivery of drugs with limited absorption due to poor solubility/dissolution, approaches are being developed to improve the dissolution rates and solubility of drug molecules. These approaches include identification of water-soluble salts of parent drugs, preparation of stable amorphous drug formulations, inclusion of solubility-enhancing agents in the dosage form, and particle size reduction. Technologies to reduce drug particle size to sub-micrometer range are being applied to product development more frequently. Electrospinning is being considered as one of the technologies which can produce nanosized drugs incorporated in polymeric nanofibers. In vitro and in vivo studies have demonstrated that the release rates of drugs from these nanofiber formulations are enhanced compared to those from original drug substance. This technology has the potential to be used for enhancing the oral delivery of poorly soluble drugs.

Synthesis of analogs of L-valacyclovir and determination of their substrate activity for the oligopeptide transporter in Caco-2 cells.

L-Valacyclovir, a prodrug of acyclovir, is a substrate for the peptide transporter (PepT1) in the intestinal mucosa, which accounts for its higher than expected oral bioavailability. The substrate activity of L-valacyclovir for PepT1 is surprising, particularly when one considers that the molecule has the structural features of a nucleoside rather than a peptide. In an attempt to better understand the structure-transport relationships (STR) for the interactions of L-valacyclovir with PepT1, analogs of this molecule with structural changes in the guanine moiety were synthesized and their substrate activity for PepT1 in Caco-2 cell monolayers was determined. The analogs synthesized include those that had the guanine moiety of L-valacyclovir substituted with purine, benzimidazole, and 7-azaindole. All three analogs (purine, benzimidazole, and 7-azaindole) exhibited affinity for PepT1 in binding studies, but only the purine analog (as the L-valine ester) showed PepT1-associated transcellular transport across Caco-2 cell monolayers. The benzimidazole and 7-azaindole analogs (as their L-valine esters) were rapidly metabolized by esterase when applied to the apical surface of Caco-2 cells, which probably explains their low penetration as the intact prodrugs via PepT1.