L-Serine–modified polyamidoamine dendrimer as a highly potent renal targeting drug carrier Satoru Matsuuraa,1, Hidemasa Katsumia,1,2, Hiroe Suzukia, Natsuko Hiraia, Hidetaka Hayashia, Kazuhiro Koshinob, Takahiro Higuchib,c, Yusuke Yagid, Hiroyuki Kimurad, Toshiyasu Sakanea, and Akira Yamamotoa aDepartment of Biopharmaceutics, Kyoto Pharmaceutical University, 607-8414 Kyoto, Japan; bDepartment of Bio-Medical Imaging, National Cerebral and Cardiovascular Center Research Institute, 565-8565 Osaka, Japan; cDepartment of Nuclear Medicine, Wuerzburg University, 97080 Wuerzburg, Germany; and dDepartment of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, 607-8414 Kyoto, Japan Edited by Robert M. Stroud, University of California, San Francisco, CA, and approved August 28, 2018 (received for review May 11, 2018) Effective delivery of drug carriers selectively to the kidney is conjugate of superoxide dismutase and PVD, synthesized from challenging because of their uptake by the reticuloendothelial 4to4′-azobis(4-cyanovaleric acid), N-vinyl-2-pyrrolidone, and system in the liver and spleen, which limits effective treatment of dimethylmaleic anhydride via radical copolymerization, accu- kidney diseases and results in side effects. To address this issue, mulated predominantly in the kidney after i.v. injection in mice. we synthesized L-serine (Ser)–modified polyamidoamine den- Although these ligands were effective for renal targeting, the drimer (PAMAM) as a potent renal targeting drug carrier. Approx- immunogenicity of lysozyme as an exogenous protein is a con- imately 82% of the dose was accumulated in the kidney at 3 h cern, and PVD exhibits size polydispersity because it is synthe- 111 after i.v. injection of In-labeled Ser-PAMAM in mice, while i.v. sized via a classical radical reaction; moreover, the number of 111 injection of In-labeled unmodified PAMAM, L-threonine modi- functional groups available for chemical modifications is limited. fied PAMAM, and L-tyrosine modified PAMAM resulted in kidney Furthermore, PVD is hardly metabolized after administration. accumulations of 28%, 35%, and 31%, respectively. Single-photon The present study is a tissue distribution study of various types emission computed tomography/computed tomography (SPECT/ 111 of amino acid-modified dendrimers for kidney-targeted drug CT) images also indicated that In-labeled Ser-PAMAM specifi- delivery. Our aim was to develop a renal targeting system using cally accumulated in the kidneys. An intrakidney distribution study L-serine (Ser) modification, and to characterize the relationship showed that fluorescein isothiocyanate-labeled Ser-PAMAM accu- PHARMACOLOGY between the physicochemical properties and the tissue distribution mulated predominantly in renal proximal tubules. Results of a cel- of Ser-modified macromolecules, with the goal of establishing a lular uptake study of Ser-PAMAM in LLC-PK1 cells in the presence strategy for the rational design of Ser-modified macromolecules as of inhibitors [genistein, 5-(N-ethyl-N-isopropyl)amiloride, and lyso- zyme] revealed that caveolae-mediated endocytosis, micropinocy- drug carriers and their use as therapeutics for kidney diseases. To this end, we selected polyamidoamine dendrimer (PAMAM) as a tosis, and megalin were associated with the renal accumulation of – Ser-PAMAM. The efficient renal distribution and angiotensin- macromolecule (12 14) and examined the tissue distribution of converting enzyme (ACE) inhibition effect of captopril (CAP), an Ser-modified PAMAM (Ser-PAMAM) after i.v. injection in mice ACE inhibitor, was observed after i.v. injection of the Ser-PAMAM- in terms of PAMAM generation, physicochemical properties, CAP conjugate. These findings indicate that Ser-PAMAM is a prom- and dose. Furthermore, the intrakidney distribution, delivery ising renal targeting drug carrier for the treatment of kidney dis- eases. Thus, the results of this study demonstrate efficient renal Significance targeting of a drug carrier via Ser modification. Delivery of most drug carriers to the kidney is limited because drug delivery | renal targeting | L-serine | dendrimer of their uptake by the reticuloendothelial system in the liver and spleen. We have developed L-serine (Ser)–modified poly- he kidney plays an important role in maintaining the ho- amidoamine dendrimer (PAMAM) as a potent renal targeting Tmeostasis of body fluids, and filters waste products and extra drug carrier for the treatment of kidney diseases. Pharmaco- water from the blood to produce urine (1–3). Various drugs, such kinetic and single-photon emission computed tomography/ as angiotensin-converting enzyme (ACE) inhibitors, steroids, computed tomography studies indicated that Ser modification and immunosuppressive agents, have been developed for the results in efficient kidney targeting of PAMAM. Ser-PAMAM treatment of kidney diseases, including renal cancer, glomerular accumulated predominantly in proximal tubules, a pattern as- disease, and acute and chronic renal failure. However, delivering sociated with the pathogenesis of renal cell carcinoma and these drugs selectively to the kidney is difficult, which limits ef- chronic renal failure. Efficient renal distribution and pharma- fective treatment of kidney diseases and results in side effects. cologic effect of captopril was observed after i.v. injection of Thus, there is an urgent need for an effective renal targeting the Ser-PAMAM-captopril conjugate. Thus, our results demon- system that can improve the therapeutic efficacy of drugs for strate successful kidney targeting of a drug carrier via Ser kidney diseases. modification. Of the various strategies available, conjugation of drugs with Author contributions: H. Katsumi designed research; S.M., H. Katsumi, H.S., N.H., H.H., targeting ligands via chemical modification appears to be a K.K., T.H., Y.Y., and H. Kimura performed research; K.K., T.H., Y.Y., and H. Kimura con- promising approach for renal drug targeting (4–6). However, tributed new reagents/analytic tools; S.M., H. Katsumi, T.S., and A.Y. analyzed data; and chemically modified conjugates are generally distributed in the S.M., H. Katsumi, and A.Y. wrote the paper. liver and spleen because of uptake by the reticuloendothelial The authors declare no conflict of interest. system (7, 8). Several studies have demonstrated the successful This article is a PNAS Direct Submission. use of lysozyme, a low molecular weight protein that is filtered in Published under the PNAS license. the glomerulus and reabsorbed in the proximal tubules, and poly 1S.M. and H. Katsumi contributed equally to this work. (vinylpyrrolidone-codimethyl maleic acid) (PVD) as renal tar- 2To whom correspondence should be addressed. Email: [email protected]. geting ligands (9–11). Haas et al. (9) reported that a conjugate of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. naproxen and lysozyme was taken up in proximal convoluted 1073/pnas.1808168115/-/DCSupplemental. tubules via endocytosis. Kamada et al. (11) reported that a www.pnas.org/cgi/doi/10.1073/pnas.1808168115 PNAS Latest Articles | 1of6 Downloaded by guest on October 2, 2021 Table 1. Physiochemical properties of PAMAM derivatives Ser-PAMAMs rapidly disappeared from the blood circulation, Compound Mean diameter, nm Mean ζ potential, mV and the plasma retention of Ser-PAMAMs was inversely pro- portional to the generation of PAMAM. Approximately 47.9%, PAMAM (G4) 4.20 ± 0.09 4.56 ± 0.81 81.7%, and 47.2% of the dose was accumulated in the kidney at Ser-PAMAM (G2) 2.50 ± 0.12 6.04 ± 0.31 180 min after i.v. injection of Ser-PAMAM (G2), Ser-PAMAM Ser-PAMAM (G3) 4.03 ± 0.29 4.76 ± 0.70 (G3), and Ser-PAMAM (G4), respectively. Although Ser- Ser-PAMAM (G4) 4.39 ± 0.26 24.77 ± 0.67 PAMAMs accumulated slightly in the liver (∼4.15%), no sig- Thr-PAMAM (G3) 4.15 ± 0.35 2.58 ± 1.36 nificant radioactivity was detected in the spleen, heart, or lungs Tyr-PAMAM (G3) 3.17 ± 0.35 5.26 ± 3.00 (Fig. 1 D–F). Ser-PAMAM (G3)-CAP 4.75 ± 0.27 3.43 ± 0.61 Table 2 shows the pharmacokinetics parameters of Ser-PAMAM (G3), Thr-PAMAM (G3), Tyr-PAMAM (G3), and PAMAM (G4). The hepatic uptake clearance (CLliver) of Ser-PAMAM (G3) was route to the kidney, and mechanism of renal uptake of Ser- almost equivalent to that of Thr-PAMAM and much lower than PAMAM were investigated after i.v. injection in mice. Finally, that of PAMAM (G4) and Tyr-PAMAM (G3). The renal uptake ∼ the tissue distribution and pharmacologic effects of captopril (CAP), clearance (CLkidney) of Ser-PAMAM (G3) was 4.87 mL/h, which an ACE inhibitor, was examined in mice after i.v. injection of a was almost 79.1% of the total body clearance. A B Ser-PAMAM-CAP conjugate, in which multi-CAP molecules were Fig. 2 and show the in vivo and ex vivo biodistribution images covalently bound to Ser-PAMAM through disulfide linkages. of near- infrared (NIR) fluorescence dye-labeled Ser-PAMAM [NIR-labeled Ser-PAMAM (G3) and NIR-labeled PAMAM (G4)], Results obtained using the IVIS imaging system (PerkinElmer) after i.v. Table 1 shows the physiochemical properties of PAMAM, L-tyrosine– injection in HR-1 mice. Fluorescence intensity derived from NIR- PAMAM (G4) was almost absent in vivo, with weak signals de- modified PAMAM (Tyr-PAMAM), L-threonine–modified PAMAM tected in the liver and kidney ex vivo. In contrast, high fluores- (Thr-PAMAM), Ser-modified PAMAM (Ser-PAMAM), and CAP- cence intensity derived from Ser-PAMAM (G3) was specifically conjugated Ser-PAMAM (Ser-PAMAM-CAP). For this study, we observed in the kidney at 60 min after i.v. injection. selected the second, third, and fourth generations of PAMAM Fig. 2C shows the biodistribution image of 111In-labeled Ser- – (G2, G3, and G4) as bioinert dendrimer backbones (12 14). The PAMAM (G3), obtained using single-photon emission computed ∼ – mean diameters of PAMAM derivatives were 2 5 nm. PAMAM tomography/computed tomography (SPECT/CT) after i.v. in- derivatives had a positive charge ranging from 2.58 to 24.77 mV, jection in ddY mice.