Renal Drug Delivery With Low-Molecular-Weight Proteins: The Effect of Charge Modifications on the Body Distribution of Drug-Lysozyme Conjugates

Abstract

Low-molecular-weight proteins (LMWPs) can be used as drug carriers for renal drug delivery. The covalent attachment of drug molecules is commonly achieved via reaction to the primary amino groups of the protein. However, these positively charged groups are also important for the renal reabsorption of proteins. In this study, we investigated the effect of the modification of primary amino groups on the pharmacokinetics of drug-LMWP conjugates, using the organic anion fluorescein isothiocyanate (FITC) as a model drug and the LMWP lysozyme (LZM) as a drug carrier. We prepared a series of conjugates with different amounts of primary amino groups: a more negatively charged conjugate was prepared by derivatizing the primary amino groups of LZM with succinic anhydride, while a more positively charged conjugate was prepared by derivatizing the carboxylic acid groups with ethylendiamine. The final conjugates had an average amount of primary amino groups of 0.2 (FITC-Suc-LZM), 6.5 (FITC-LZM), or 8.5 (FITC-cat-LZM). We studied the renal handling of the conjugates in freely moving Wistar rats. After i.v. injection of 33 mg/kg conjugate, the FITCLZM and the FITC-Suc-LZM conjugates were incompletely reabsorbed in the kidney, resulting in the excretion of the intact conjugate into the urine (29 4% versus 45 4% of the injected dose, p < 0.05). In contrast, the FITC-cat-LZM conjugate was not excreted into the urine (0.3 0.1% of the injected dose, p < 0.001 distributed selectively to the kidney, and significant amounts of this versus FITC-LZM). An organ distribution study with 125I radiolabeled cat-LZM showed that the cationized LMWP accumulated in the kidney (12 0 . 3% of the dose). However, cat-LZM no longer LMWP were found in the liver and spleen (53 1% and 5 2%, respectively). We conclude that charge modifications influence the biodistribution of drug-LZM conjugate. First, reducing the amount of primary amino groups results in decreased tubular reabsorption and increased excretion of the conjugate into the urine. Finally, increasing the amount of primary amino groups results not only in improved renal reabsorption, but also in an increased extrarenal distribution of the conjugate and therefore in a partial loss of renal selectivity.