Category: Formulation and Quality
Purpose: Renal ischemia/reperfusion injury may result in acute kidney failure, delayed renal function, and early mortality after kidney transplantation and hemorrhagic shock. During reperfusion after renal ischemia, reactive oxygen species (ROS) are readily generated in the kidney and cause oxidative damage. ROS induce intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) which recruit leukocytes to the region with ischemia/reperfusion injury and cause a secondary disorder.
On the other hand, we recently developed the l-serine (Ser)-modified polyamidoamine (PAMAM) dendrimer. It is a highly potent renal-targeting drug carrier (Proc Natl Acad Sci U S A. 115(41):10511-10516 (2018)). In mice, ~82% of the intravenously administered Ser-PAMAM dose accumulated in the kidney. Therefore, the conjugation of reduced thiols to Ser-PAMAM may target the delivery of reduced thiols to the kidney and prevent renal ischemia/reperfusion injury (Figure 1).
The aim of the present study was to develop a kidney-targeting reduced thiol conjugated to Ser-PAMAM to prevent renal ischemia/reperfusion injury. To this end, we conjugated Cys and Ser to PAMAM (Ser-PAMAM-Cys). The Cys and Ser were covalently bound to the PAMAM amine moiety as a reduced thiol and a renal targeting ligand, respectively. Next, the ROS- and free radical-scavenging ability and renal targeting efficacy of Ser-PAMAM-Cys were investigated. Finally, we examined the efficacy of Ser-PAMAM-Cys at preventing ROS-mediated kidney failure in a mouse renal ischemia/reperfusion injury model.
Methods: Ser-PAMAM-Cys was synthesized by reacting Ser and Cys with third-generation (G3) PAMAM according to the HBTU-HOBt method. The mean diameter and zeta-potential saline (PBS, pH 7.4) were measured at 1 mg/mL in PBS with a Zetasizer Nano at 25 °C. Ser-PAMAM-Ser was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The number of thiol groups on PAMAM was determined by Ellman’s method and used as an index of Cys content. The hydrogen peroxide, hydroxyl radical, free radical-scavenging ability of Ser-PAMAM-Cys was evaluated by the BES-H2O2 probe, luminol probe, 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, respectively. For the tissue distribution experiments, 111In-labeled Ser-PAMAM-Cys was intravenously administered into mice at 1 mg/kg. To visualize its distribution, near-infrared fluorescence (NIR)-labeled Ser-PAMAM-Cys was intravenously administered to mice. Preventive effect of Ser-PAMAM-Cys in a mouse model of renal ischemia reperfusion injury was evaluated by measuring the plasma creatinine (Cre) and blood urea nitrogen (BUN) levels. Effect of Ser-PAMAM-Cys on inflammatory factors induced by renal ischemia/reperfusion was also evaluated using quantitative real-time PCR.
Results: The mean diameter of Ser-PAMAM-Cys was 4.44 ± 0.23 nm. The zeta-potential of Ser-PAMAM-Cys was 13.40 ± 0.89 mV. The mass of Ser-PAMAM-Cys was 10,207 kDa which corresponds to ~32 molecules of conjugated Ser and Cys. The average number of Cys on Ser-PAMAM-Cys was ~6.4 (Cys content: 20%). Ser-PAMAM-Cys effectively scavenged DPPH radical and ROS (hydrogen peroxide and hydroxyl radical) in PBS. In addition, ~64% of 111In-labeled Ser-PAMAM-Cys accumulated in mouse kidney 3 h after intravenous administration. An IVIS (in vivo imaging system) study indicated that near-infrared fluorescence dye (NIR)-labeled Ser-PAMAM-Cys specifically accumulated in the kidney (Figure 2). In a mouse renal ischemia/reperfusion injury model, increases in the kidney damage markers Cre and BUN were significantly inhibited by intravenous administration of Ser-PAMAM-Cys. In contrast, Cys injection had no statistically significant effect of preventing Cre or BUN elevation relative to the control. Furthermore, in the histological examination, Ser-PAMAM-Cys administration decreased ischemia/reperfusion-induced damage and maintained the renal structure in the treated mice comparable to that of the naïve mice (Figure 3). Ser-PAMAM-Cys also effectively downregulated the inflammatory factors gelatinase-associated lipocalin (NGAL), IL-18, ICAM-1, and VCAM-1 in the renal ischemia/reperfusion injury model.
Conclusion: These results indicate that Ser-PAMAM-Cys is a promising kidney-targeting ROS scavenger which could prevent ischemia/reperfusion-induced renal failure.
Satoru Matsuura– Kyoto Pharmaceutical University, Kyoto, Kyoto, Japan
Masaki Morishita– Assistant Professor, Kyoto Pharmaceutical University, Kyoto City, Kyoto, Japan
Akira Yamamoto– Professor, Kyoto Pharmaceutical University, Kyoto City, Kyoto, Japan