L-Serine–Modified Polyamidoamine Dendrimer As a Highly Potent Renal Targeting Drug Carrier

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 lysozyme] 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 diseases. 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 proportional 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. Specific renal accumulation of 111In-labeled and the positive charge gradually increased in association with their Ser-PAMAM was clearly observed, although slight bladder ac- generation and amino groups. The number of CAP modifications cumulation was also observed. on PAMAM was 5.75 in Ser-PAMAM (G3)-CAP. Fig. 3 shows the microscopic images of mouse renal sections at Fig. 1 shows the plasma concentration profiles and tissue 60 min after i.v. injection of fluorescein isothiocyanate (FITC)- distribution of 111In-labeled PAMAM derivatives after i.v. in- labeled Ser-PAMAM (G3). As shown in Fig. 3 A and B, the jection in ddY mice. PAMAM (G4) and Tyr-PAMAM (G3) fluorescence intensity of FITC-labeled Ser-PAMAM (G3) was accumulated mainly in the liver and kidney tissues. Thr-PAMAM almost absent in the renal medulla, whereas high fluorescence (G3) slowly disappeared from blood circulation, and ∼34.9% of intensity was observed in the renal cortex. Furthermore, fluo- the dose accumulated in the kidney within 180 min after in- rescence from FITC-labeled Ser-PAMAM (G3) was clearly observed in the proximal tubules (Fig. 3C). jection (Fig. 1 A–C). To elucidate the effect of glomerular filtration on the renal distribution of Ser-PAMAM (G3), we performed a pharmacokinetic study of 111In-labeled Ser-PAMAM (G3) after i.v. in- A B C jection in mice with HgCl2-induced acute renal failure (ARF). 100 100 100 The glomerular filtration rate was significantly decreased after HgCl treatment, indicating that ARF was established by this 80 80 80 2 method (Fig. 4D). Although HgCl2-induced ARF had no sig- 60 60 60 nificant effect on the plasma concentration profile of Ser- 40 40 40 PAMAM (G3) (Fig. 4A), the renal accumulation and uptake 111 20 20 20 clearance of In-labeled Ser-PAMAM (G3) were decreased in B C 0 0 0 mice with HgCl2-induced ARF (Fig. 4 and ). 060120180 0 60 120 180 060120180 Fig. 5 A and B show the time course of apical-to-basolateral Time (min) Time (min) Time (min) D E F (absorptive direction) and basolateral-to-apical (secretory di- 100 100 100 rection) transport of FITC-labeled Ser-PAMAM (G3) and PAMAM (G4) across the monolayers of LLC-PK1 cells (an 80 80 80 epithelial cell line derived from proximal tubular cells of porcine 60 60 60 Tissue accumulation (% of dose) (% accumulation Tissue

Plasma concentration (% (% Plasma concentration of dose/ml) 40 40 40 Table 2. Pharmacokinetic parameters of PAMAM derivatives 20 20 20 0 0 0 Clearance, mL/h 060120180 0 60 120 180 060120180 Dose, AUC, % of Time (min) Time (min) Time (min) Compound mg/kg dose h/mL Total Liver Kidney

Fig. 1. Time courses of plasma concentration and tissue accumulation of Ser-PAMAM (G3) 1 16.25 6.16 0.10 4.87 various amino acid-modified dendrimers in mice after i.v. injection at a dose Thr-PAMAM (G3) 1 80.98 1.23 0.05 0.55 111 of 1 mg/kg. (A) In-labeled PAMAM (G4). (B) Thr-PAMAM (G3). (C) Tyr- Tyr-PAMAM (G3) 1 1.17 85.25 33.44 25.19 PAMAM (G3). (D) Ser-PAMAM (G2). (E) Ser- PAMAM (G3). (F) Ser-PAMAM PAMAM (G4) 1 4.72 21.19 8.21 5.37 (G4). Results are expressed as mean ± SE values for three mice. 〇, plasma; ▲, liver; ■, kidney; ◇, spleen; △, heart; □, lung. AUC, area under the plasma concentration–time curve.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1808168115 Matsuura et al. Downloaded by guest on October 2, 2021 ABC

Liver Kidney Kidney Kidney

Heart Lung Spleen Ser-PAMAM (G3) Ser-PAMAM (G3) Bladder Bladder

Fig. 2. In vivo and ex vivo imaging of NIR fluorescence dye-labeled Ser-PAMAM (G3) and PAMAM (G4) 60 min after i.v. injection in mice. The fluores- CT-SPECT fused SPECT cence intensities were measured in (A) whole mice and (B) tissues (liver, kidney, spleen, heart, and lung). (C) SPECT/CT imaging of 111In-labeled Ser-PAMAM PAMAM(G4) PAMAM(G4) (G3) at 180 min after i.v. injection in a mouse.

kidneys). There were no significant differences in the transport mice. Although the plasma retention of 111In-labeled Ser- of FITC-labeled PAMAM (G4) in the two directions. In con- PAMAM (G3)-CAP was slightly higher than that of 111In- trast, the apical-to-basolateral (absorptive direction) transport of labeled Ser-PAMAM (G3) (Figs. 1 and 6), 111In-labeled Ser- FITC-labeled Ser-PAMAM (G3) was higher than the basolateral- PAMAM (G3)-CAP accumulated predominantly in the kidney. to-apical (secretory direction) transport. These results indicate Approximately 80.9% of the dose was accumulated in the kid- that Ser-PAMAM (G3) was preferentially transported in the ney at 180 min after i.v. injection of 111In-labeled Ser-PAMAM absorptive direction. (G3)-CAP. Fig. 5C shows the cellular uptake of 111In-labeled Ser- Fig. 6 B and C show the pharmacokinetics of CAP after i.v. PAMAM (G3) in the presence of cellular uptake inhibitors. injection of CAP alone and Ser-PAMAM (G3)-CAP. The PHARMACOLOGY Chlorpromazine (a clathrin-mediated endocytosis inhibitor) had plasma concentration of CAP after i.v. injection of Ser-PAMAM no significant effect on the cellular uptake of 111In- labeled Ser- (G3)-CAP was slightly higher than that after injection of CAP PAMAM (G3). In contrast, genistein (a caveolae-mediated en- alone (Fig. 6B). In addition, the renal accumulation of CAP after docytosis inhibitor), 5-(N-ethyl-N-isopropyl)amiloride (a macro- injection of Ser-PAMAM (G3)-CAP was greater than that after pinocytosis inhibitor), and lysozyme (a megalin substrate) injection of CAP alone (Fig. 6C). After incubation of Ser- significantly decreased the cellular uptake of 111In-labeled Ser- PAMAM (G3)-CAP in plasma for up to 4 h, free CAP was al- PAMAM (G3). In addition, cellular uptake of Ser-PAMAM most undetectable in plasma. (G3) was significantly inhibited in the presence of excess un- Fig. 6D shows renal ACE activity after i.v. injection of CAP labeled Ser-PAMAM (G3). alone or Ser-PAMAM (G3)-CAP. CAP alone and Ser-PAMAM Fig. 6A shows the plasma concentration and tissue distribution (G3)-CAP significantly inhibited renal ACE activity 30 at min of 111In-labeled Ser-PAMAM (G3)-CAP after i.v. injection in after i.v. injection in ddY mice. Furthermore, the decrease in the

A B 100 100 80 80 60 60 40 40

(% of dose/ml) of (% 20 20

Plasma concentration 0 0 0 60 120 180 060120180 C Time (min) (% of dose) Renal accumulation D Time (min) 6 300 5 250 4 200

3 μL/min) 150 * 2 100

1 GFR ( 50 0 0 Normal ARF Normal ARF Renal uptake clearance (mL/h) Renal uptake clearance

Fig. 4. Plasma concentration, renal accumulation, renal clearance, and glomerular filtration rate (GFR) of 111In-labeled Ser-PAMAM (G3) after i.v.

Fig. 3. Intrakidney distribution of FITC-labeled Ser-PAMAM (G3) in renal injection at a dose of 1 mg/kg in normal mice and mice with HgCl2-induced tissue sections at 60 min after i.v. injection in mice. (A) Cortex. (Scale bar: acute renal failure (ARF). Time course of (A) plasma concentration and (B)

200 μm.) (B) Medulla. (Scale bar: 200 μm.) (C) Magnified image of the cortex. renal accumulation. 〇, normal mice; ●, HgCl2-induced ARF mice. (C) Renal (Scale bar: 25 μm.) Fluorescence intensity was observed using a confocal clearance and (D) GFR in normal mice (normal) and mice with HgCl2-induced laser-scanning microscope. ARF (ARF). Results are expressed as the mean ± SE for three mice.

Matsuura et al. PNAS Latest Articles | 3of6 Downloaded by guest on October 2, 2021 A B AB by s.c. injection of HgCl2 as a positive control (Fig. 7 and ). In 15 15 addition, infiltration of inflammatory cells and necrotic and/or damaged cells was observed in the histological sections of renal C 10 10 tissue from ddY mice treated with HgCl2 (Fig. 7 ). In contrast, Ser-PAMAM (G3) had no significant effect on creatinine and 5 5 BUN levels, and histological sections of renal tissue from Ser- PAMAM (G3)–treated mice were similar to those from naïve (% of initial dose) initial of (% Permeated amount C 0 0 and PBS-treated mice (Fig. 7 ). 0123401234 Time (h) Time (h) Discussion C In the present study, biodistribution and imaging studies 120 revealed that Ser-PAMAM (G3) was specifically distributed to 100 the kidneys after i.v. injection in mice. Thus, our results dem- 80 * onstrate effective renal targeting using Ser modification. Because 60 Ser is a biomolecule and a biocompatible compound, Ser mod- (% of NT) of (% * 40 * ification represents a safer mode of drug delivery. 20 * Fluorescent microscopic images indicated that Ser-PAMAM Cellular uptake amount uptake Cellular 0 abcde f (G3) was localized predominantly to the renal cortex, especially to the proximal tubule. The proximal tubules are involved in the Fig. 5. Transport and cellular uptake of various amino acid-modified den- pathogenesis of kidney diseases, such as chronic kidney failure drimers in LLC-PK1 cells. Time course of (A) FITC-labeled Ser-PAMAM (G3) and renal cell carcinoma (15–18). Thus, these findings indicate and (B) PAMAM (G4) transport across LLC-PK1 cell monolayers in the ab- that Ser-PAMAM (G3) represents a promising drug carrier sorptive directions and secretory directions. ■,▲, absorptive directions; □, △ 111 for the treatment of kidney diseases. Nanoparticles with a , secretory directions (C) Cellular uptake study of In-labeled Ser- < PAMAM in LLC-PK1 cells in the presence of various inhibitors. a, 111In- diameter 5.5 nm are efficiently filtered in the glomerulus and labeled Ser-PAMAM (G3). b, 111In-labeled Ser-PAMAM (G3) + 100 μg/mL excreted into urine (19). Given the mean diameter of Ser- unlabeled Ser-PAMAM (G3). c, 111In-labeled Ser-PAMAM (G3) + 100 μM PAMAM (G3) of ∼4 nm in the present study, we hypothesized chlorpromazine. d, 111In-labeled Ser-PAMAM (G3) + 370 μM genistein. e, that Ser-PAMAM (G3) is filtered in the glomerulus and reab- 111 In-labeled Ser-PAMAM (G3) + 100 μM5-(N-ethyl-N-isopropyl)amiloride. f, sorbed in the lumen of the proximal tubule. We demonstrated 111 + In-labeled Ser-PAMAM (G3) 1 mM lysozyme. Results are expressed as that the renal clearance of Ser-PAMAM (G3) was proportional mean ± SE for three experiments. *P < 0.05, significantly different from the 111In-labeled Ser-PAMAM (G3) group a. to the glomerular filtration rate in the mice with HgCl2-induced ARF. These results, together with the permeability direction findings in LLC-PK1 cells, indicate that Ser-PAMAM (G3) was ACE activity persisted for a longer duration after injection of delivered to the proximal tubule through glomerular filtration. Ser-PAMAM (G3)-CAP than after injection of CAP alone. In the present study, renal accumulation of Ser-PAMAM (G3) Fig. 7 shows plasma concentrations of creatinine and blood was saturated at high doses (10 mg/kg; SI Appendix, Fig. S2). In urea nitrogen (BUN), along with histological micrographs of the addition, the results of the in vitro cellular uptake study in LLC- kidney after i.v. injection of Ser-PAMAM (G3). Plasma con- PK1 cells suggest the involvement of caveolae-mediated endo- centrations of creatinine and BUN were significantly increased cytosis, macropinocytosis, or megalin in the renal accumulation

A B 100 80 7 6 60 5 4 40 3 20 2 1 0 0 Fig. 6. Plasma concentration and tissue accumula-

Plasma conc. (% of Plasma conc. (% dose/ml) 0 60 120 180 0 306090120 111 Plasma concentration (μg/ml) tion of In-labeled Ser-PAMAM (G3)-CAP and the Tissue accumulation (% accumulation (% of dose) Tissue Time (min) Time (min) renal accumulation and ACE inhibition activity of C D CAP alone and Ser-PAMAM (G3)-CAP. (A) Time courses of plasma concentration and tissue accumulation of 111In-labeled Ser-PAMAM (G3)-CAP after i.v. 120 〇 △ 4 injection in mice at a dose of 1 mg/kg. , plasma; , 100 * liver; ■, kidney. (B and C) Plasma concentration (B) 3 80 and renal accumulation (C) of CAP after i.v. injection of CAP alone and Ser-PAMAM (G3)-CAP in mice at a 60 2 dose of 2 mg CAP/kg. 〇, CAP alone; ▲, Ser-PAMAM 40 (G3)-CAP. (D) Effect of CAP alone and Ser-PAMAM 1 20 # (G3)-CAP on ACE activity in kidney 30 min or 120 min after i.v. injection in mice at a dose of 0.5 mg CAP/kg. 0 0 Results are expressed as mean ± SE for three mice.

ACE activities (% of Naive) (% ACE activities Naive 30 min120 min30 min120 min 0306090120 *P < 0.05, significantly different from the naive group. Renal accumulation (μg/tissue) Time (min) CAP Ser-PAMAM(G3) #P < 0.05, significantly different from the CAP and Ser- -CAP PAMAM (G3)-CAP groups at the same time.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1808168115 Matsuura et al. Downloaded by guest on October 2, 2021 AB 1.5 * 200 * 150 1.0 ns ns 100 0.5 50 ns ns (mg/dL) (mg/dL) 0 0 BUN level in plasma BUN level Creatinine level in level plasma Creatinine

Naive PBS Ser-PAMAM (G3) HgCl2-induced ARF

Fig. 7. Plasma creatinine (A) and BUN (B) levels and histological micrographs of the kidney (C) after i.v. injection of PBS or Ser-PAMAM (G3) once daily for 5 d.

(Scale bar: 200 μm.) Naive, PBS, Ser-PAMAM (G3), HgCl2-induced ARF (positive control). Results are expressed as mean ± SE for five mice. *P < 0.05, significantly different from the naive group. ns, not significant. PHARMACOLOGY of Ser-PAMAM (G3). Although further studies are needed to and that pharmacologically active CAP was released in the cy- elucidate the detailed mechanism of renal accumulation, these toplasm after renal distribution (20–23). results indicate that active transport mechanisms contribute to Our toxicity study results indicate that Ser-PAMAM (G3) the renal uptake of Ser-PAMAMs. showed no acute toxicity after repetitive administration. Al- Pharmacokinetic studies of Ser-PAMAMs showed the effect though a long-term toxicity study is needed before approval for of size and the degree of Ser modification on the renal targeting clinical use, these results indicate that Ser-PAMAM (G3) is a of Ser-PAMAMs. Although the renal clearance of Ser-PAMAM relatively safe drug carrier for kidney-targeted drug delivery. was proportional to the degree of Ser modification on PAMAM, In conclusion, the renal targeting of PAMAM was success- the renal accumulation of a midsized Ser-PAMAM molecule, fully achieved using Ser modification. Approximately 82% of the Ser-PAMAM (G3), was the greatest of all the Ser-PAMAMs. dose accumulated in the kidney at 3 h after i.v. injection of 111In- This is probably because glomerular filtration of Ser-PAMAM labeled Ser-PAMAM (G3) in mice. An intrakidney distribu- decreased as the size of Ser-PAMAM increased. Thus, Ser- tion study showed that FITC-labeled Ser-PAMAM (G3) accu- PAMAM (G3) strikes the best balance between molecular size and affinity for the proximal tubules. mulated predominantly in proximal tubules. The efficient renal We also examined the pharmacokinetics of Thr-PAMAM distribution and ACE inhibition effect of CAP, an ACE in- (G3) and Tyr-PAMAM (G3) because Thr and Tyr are amino hibitor, was observed after i.v. injection of a Ser-PAMAM (G3)- acids with hydroxyl groups, with a similar structure to Ser. Al- CAP conjugate. These results indicate that Ser modification is though Thr-PAMAM accumulated in the kidneys, the renal accumulation of Thr- PAMAM (G3) was much lower than that of Ser-PAMAM (G3) because of the greater urinary excretion (SI Appendix, Fig. S3). This is likely because, compared with Ser, Thr A 1) Boc-Ser(tBu)-OH has one additional methylene group in its side chain, which might HBTU/HOBt play a key role in attaining the optimal conformation for in- 2) Deprotection creased kidney accumulation. In contrast, Tyr-PAMAM (G3) with TFA cocktail and PAMAM (G4) accumulated in both the liver and the kidney. PAMAM (G3) Ser-PAMAM (G3) Tyr has a phenolic hydroxyl group, and PAMAM (G4) contains several amino groups. These findings indicate that efficient renal B targeting requires the presence of an alcoholic hydroxyl group. The increased renal accumulation of CAP after administration 1) CAP of Ser-PAMAM (G3)-CAP is consistent with the pharmacoki- 111 netics of In-Ser-PAMAM (G3). These results indicate that SPDP SPDP-CAP Ser-PAMAM (G3)-CAP released negligible amounts of CAP in the blood circulation, and CAP modification had no significant effect on the affinity of Ser toward the kidney. Prolonged ACE 2) Ser-PAMAM (G3) inhibition with Ser-PAMAM (G3)-CAP was observed because of the greater renal accumulation of CAP after i.v. injection of Ser-PAMAM(G3) -CAP CAP-loaded Ser-PAMAM. Because CAP was bound to Ser- PAMAM (G3) through disulfide linkage, we hypothesize that Fig. 8. Synthesis and structures of Ser-PAMAM (G3) (A) and Ser-PAMAM the linkage was cleaved by reduced thiols, such as glutathione, (G3)-CAP (B). SPDP, N-succinimidyl 3-(2-pyridyldithio)propionate.

Matsuura et al. PNAS Latest Articles | 5of6 Downloaded by guest on October 2, 2021 promising approach for renal targeting using a macromolecular of CAP, CAP solution or Ser-PAMAM (G3)-CAP solution was administered i.v. drug carrier. to ddY mice at a dose of 2 mg CAP/kg. We used a previously described method with slight modifications to analyze CAP (28). Materials and Methods To elucidate the effect of glomerular filtration on the renal distribution of 111 For this study, we selected PAMAM dendrimers with an ethylenediamine core Ser-PAMAM (G3), we performed a pharmacokinetic study of In-Ser- (G2, G3, or G4) (Sigma-Aldrich) as bioinert dendrimer backbones. Ser-PAMAM PAMAM (G3) in mice with HgCl2-induced ARF (11). Ex vivo and in vivo tissue (G3) was synthesized by reacting PAMAM (G3) with Boc-Ser(tBu)-OH den- distribution were evaluated using the PerkinElmer IVIS imaging system or 111 drimers, using the HBTU-HOBt method (Fig. 8A) (24, 25). The reaction mix- SPECT/CT (Bioscan) to image NIR fluorescence dye-labeled or In-labeled tures were incubated at room temperature until the ninhydrin test yielded Ser-PAMAM (G3) after i.v. injection. Intrakidney distribution of FITC-labeled negative results on TLC. Ser-PAMAM (G3) was identified using matrix-assisted Ser-PAMAM (G3) after i.v. injection was observed with a fluorescence mi- laser desorption/ionization time-of-flight mass spectrometry (Bruker) and croscope. Transport and cellular uptake of Ser-PAMAM (G3) were evaluated 1H-NMR spectroscopy (Bruker) in deuterated water. The mass of PAMAM in LLC-PK1 cells (an epithelial cell line derived from proximal tubular cells of (G3) was 6960 Da, and this increased to 9684 kDa, corresponding to ∼32 porcine kidney). To evaluate the toxicity of Ser-PAMAM (G3), we measured molecules of conjugated Ser (SI Appendix,Fig.S1A). The peaks corresponding creatinine and BUN levels and observed kidney sections under a microscope to Ser were observed in the 1H NMR spectrum at δ 3.72–3.85 (m, 2H) of Ser- (KEYENCE) after i.v. injection of Ser-PAMAM (G3) (1 mg/kg) once daily PAMAM, and the integral ratio of the methylene protons of Ser to the for 5 d in ddY mice.

methylene protons (CONHCH2) of PAMAM (G3) indicates that the desired All animal experiments were conducted according to the principles and product was obtained (SI Appendix,Fig.S1B)(26). procedures outlined in the National Institutes of Health’s Guide for the Care To synthesize Ser-PAMAM (G2), Ser-PAMAM (G4), Thr-PAMAM (G3), and and Use of Laboratory Animals (29). The Animal Experimentation Commit- Tyr-PAMAM (G3), we reacted each generation of PAMAM with Boc-Ser(tBu)- tee of the Kyoto Pharmaceutical University and the Institutional Animal Care OH, Boc-Thr(tBu)-OH, or Boc-Tyr(tBu)-OH, using the same method as for Ser- Committee of the National Cerebral and Cardiovascular Center approved all PAMAM (G3) synthesis. To synthesize CAP-conjugated Ser-PAMAM (G3) [Ser- experimental protocols that used animals. PAMAM (G3)-CAP], we conjugated CAP with Ser-PAMAM (G3) through The experimental procedures are described in detail in SI Appendix, ζ disulfide linkages (Fig. 8B). The mean diameter and potential in PBS were Materials and Methods. analyzed using a Zetasizer Nano (Malvern Instruments) at 25 °C. For tissue distribution studies, we radiolabeled PAMAM dendrimer de- 111 ACKNOWLEDGMENTS. We thank Dr. Shugo Yamashita (Kyoto Pharmaceu- rivatives with In using a bifunctional chelating agent, diethylene- tical University) for supporting the synthesis of PAMAM derivatives. This triaminepentaacetic anhydride, according to the method described by work was supported by the Japanese Ministry of Education, Culture, Sports, Hnatowich et al. (27). Each modified PAMAM dendrimer was administered Science and Technology-Supported Program for the Strategic Research i.v. to ddY mice at a dose of 1 or 10 mg/kg. To evaluate the pharmacokinetics Foundation at Private Universities.

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