Pinocytosis (cell sipping) has been thought to be a nonspecific, nonsaturable, non-carrier-mediated form of membrane transport via vesicular uptake of bulk fluid into cells from the surrounding medium (22, 23). This mechanism is most relevant to large particles and polymer conjugates. The term "pinocytosis" has fallen from favor and one suspects that many events previously ascribed to nonspecific pinocytosis are now recognized as being due to specific receptor-mediated endocytosis. Endocytosis is specific and intrinsic to the mechanism of action of many macromolecular drugs. This process is also used to deliver small molecules as prodrugs, and mediates the distribution and clearance of many contemporary pharmacological agents, including many biotechnology products, most peptide hormones, and cytokines (e.g., insulin, growth hormone, erythropoetin, granulocyte colony-stimulating factor, and interleukins) (24).
Receptor-mediated endocytosis plays an important role in the pharmacokinetics and nephro- and ototoxicity of aminoglycoside antibiotics. As was shown in Chapter 3 (Figure 3.6), gentamicin exhibits flip-flop kinetics, wherein elimination appears as the initial phase, followed by a very slow distribution phase (25). The first phase corresponds to clearance from plasma by glomerular filtration, the second phase to redistribution of drug from the tissues, particularly kidney, back into the central compartment. After glomerular filtration, aminoglycosides are taken up via endocytosis at the brush border by renal proximal tubule epithelial cells (26). The accumulation of antibiotic (as much as 10% of the dose) in these cells results in lysosomal disruption and cell necrosis, producing dose-limiting nephrotoxicity. However, the uptake is saturable, so that, for a given total intravenous dose, accumulation in the kidney is lower when multiple intermittent doses are given rather than when a continuous dose is infused over the same time period (27). This allows far greater peak therapeutic concentrations than could be tolerated otherwise, a clinically important consideration because aminogly-cosides exhibit peak concentration-dependent bactericidal effects (28). The optimum dose and interval for various aminoglycosides remain areas of ongoing research (29, 30).
The endocytosis of aminoglycosides via clathrin-coated pits is thought to involve initial binding of the polybasic cationic drugs to anionic lipids. Recently, megalin (also known as gp330 and as low-density lipoprotein receptor-related protein-2), a receptor protein on the brush border, has been implicated (31). Megalin knockout mice accumulate only about 5% as much of an intraperitoneal gentamicin dose in their kidneys as do wild-type mice. This protein is involved in the uptake of many low molecular weight proteins containing positively charged regions, including vitamin-binding proteins, lipoproteins, hormones, and also calcium. Competition for megalin binding between calcium and aminoglycosides may be the basis for the ability of oral calcium loading to attenuate aminoglycoside nephrotoxicity. The megalin receptor is most highly expressed in proximal renal tubule cells. It is also expressed in an eclectic assortment of other cells, including the epithelium of the inner ear, which may explain ototoxicity associated with long-term aminoglycoside treatment (32, 33).
Transcytosis is the receptor-mediated uptake of a ligand on one side of the cell, vesicular transport across the cell, and exocytosis of the vesicle contents on the opposite side. This process is responsible for the uptake of the iron-binding protein transferrin (Tf) across the blood-brain barrier (BBB) by the transfer-rin receptor (TfR). Monoclonal antibodies that recognize the transferrin receptor (mABTfR) are also carried across the cell and have been used to deliver various cargos. An early demonstration used mABTfR conjugated to avidin to deliver vasoactive intestinal peptide (VIPa) disulfide-linked to biotin. Reduc-tases in the brain cleaved the disulfide linkage, releasing VIPa to express its pharmacological effect (Figure 14.3) (34, 35).
Applications of transcytosis have been extended to additional receptors, cargos, and delivery sites (36). The TfR has been used to deliver 111In-labeled DTPA-EGF-PEG-biotin-streptavidin-mABTfR (DTPA = the metal chelator diethylenetriaminepentaacetic acid; EGF = endothelial cell-derived growth factor; PEG = polyetheylene glycol) across the BBB, where binding to cells expressing EGF receptor (EGFR) was useful
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