Renal Targeting: Peptide-Based Drug Delivery to Proximal Tubule

Subscriber access provided by Washington University | Libraries

Article

Renal targeting: peptide-based drug delivery to proximal tubule cells Artjom Wischnjow, Dikran Sarko, Maria Janzer, Christina Kaufman, Barbro Beijer, Sebastian Brings, Uwe Haberkorn, Gregor Larbig, Armin Kuebelbeck, and Walter Mier Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.6b00057 • Publication Date (Web): 21 Mar 2016 Downloaded from http://pubs.acs.org on March 24, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Bioconjugate Chemistry

carrier conjugate

free drug K

E

E

K

E

K

E

E K

E K

K

K

E E

E

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 2 of 16

(1 1) Polymer with varying numbers and positions of the moieties Y and Z (2 2) FITC derivative of (1 1)

Y

Z


(4 4) R = H (5 5) R = D-Tyr (6 6) R = Cipro. (7 7) R = FITC

Page 3 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Page 4 of 16

Page 5 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

A

B

C

D


Page 6 of 16

Page 7 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 16

Renal targeting: peptide-based drug delivery to proximal tubule cells Artjom Wischnjow†, Dikran Sarko†, Maria Janzer†,‡, Christina Kaufman†, Barbro Beijer†, Sebastian Brings†, Uwe Haberkorn†, Gregor Larbig‡, Armin Kübelbeck‡, Walter Mier† † Department of Nuclear Medicine, Heidelberg University Hospital, INF 400, 69120 Heidelberg, Germany ‡ Merck KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany

Supporting Information Placeholder ABSTRACT: Kidney-specific drug targeting is an attractive strategy to reduce unwanted side effects and to enhance drug efficacy within the renal tissue. For this purpose a novel kidneyspecific drug carrier was developed. The peptide sequence (KKEEE)3K triggers exceptional renal specificity at high accumulation rates. Micro-PET imaging studies of megalin-deficient mice indicate that the cellular endocytosis of this carrier is mediated by megalin. This assumption is supported by immunohistochemical analysis of FITC-labeled carrier peptide, which exclusively accumulated at the apical side of proximal tubule cells within the renal cortex. Scintigraphic studies of modified ciprofloxacin conjugated to (KKEEE)3K confirmed the excellent drug targeting potential of the peptide carrier. The conjugate accumulated entirely in the kidneys, revealing flawless redirection of ciprofloxacin, a compound that is mainly excreted by the liver. In conclusion, these results suggest the potential of (KKEEE)3K as a promising candidate for kidney-targeted drug delivery to proximal tubule cells.

INTRODUCTION Side effects limit the potential of systemically administered therapeutics.(1-3) One approach to improve drug efficacy and to reduce side effects is drug targeting. The aim of drug targeting is to specifically transport drugs directly to their site of action.(4, 5) The prevalence of acute and chronic kidney diseases (CKD) is reaching epidemic proportions. Currently, the only treatment option for CKD is prevention or retardation of the progression of further kidney damage. However, long term therapy of CKD is accompanied by serious side effects and numerous promising drug candidates failed during clinic trials due to safety issues and lack of efficacy, e.g. bardoxolone methyl(6, 7) and paricalcitol in CKD(8-10), avosentan in diabetic nephropathy(11), sirolimus and everolimus in autosomal dominant polycystic kidney disease (ADPKD).(12, 13) Consequently, renal targeted drug delivery would broaden the therapeutic window of drugs acting in the kidneys, reduce systemic side effects and thus enable new treatment opportunities. Several renal drug delivery systems have been described. Examples comprise macromolecular carriers, e.g. polyvinylpyrrolidone (PVP)(14) and chitosan(15) or prodrugs cleaved by kidneyassociated enzymes that trigger the kidney selective release of drugs.(4, 16)

Low-molecular weight proteins (LMWP) such as lysozyme have been studied extensively as selective kidney carriers for various drugs.(17) LMWP are excreted by glomerular filtration and subsequently reabsorbed by receptor-mediated endocytosis into proximal tubular cells. Following internalization and transfer into lysosomes, they are catabolized to small peptides and single amino acids.(18, 19) LMWP were used for the delivery of various small molecule drugs such as doxorubicin(20), triptolide(21), naproxen(17, 22) , indomethacin and captopril.(23) Depending on the drug linkage, the active component can be released from the carrier either enzymatically or by chemical cleavage. Unfortunately, the clinical value of lysozyme as a renal carrier is hampered by significant cardiovascular side effects.(24) Moreover, technical aspects limit the application of LMWP for targeting strategies: conjugation of drugs with a LMWP requires careful design, because LMWP comprising a high number of active groups and are susceptible to self-aggregation.(25) The defined conjugation to lysozyme, which has several linkage sites, is very challenging. Uncontrolled conjugation leads to modification at different sites and multiple conjugations. The resulting mixture of isomers and conjugates, are difficult to characterize and to separate. Modification of LMWP can also yield immunogenic products.(26) Smaller carriers, such as peptides, offer the potential to overcome these disadvantages. Based on the effect of lysine which interacts with receptors on the apical side of proximal tubule cells(27, 28), a development program for a kidney-specific carrier peptide was started. Based on εpolylysine, synthetic peptides were developed resulting in the peptide (KKEEE)3K which shows ideal pharmacokinetic properties with respect to its specificity of accumulation as examined by molecular imaging, biodistribution studies and immunohistochemistry. The optimized peptidic carrier enables the targeting of small molecule drugs to proximal tubule cells and consequently represents a promising candidate for the treatment of kidney diseases.

RESULTS εPLL as a drug delivery system. The native εPLL is prepared by microbial synthesis providing a relatively high homogeneity with a polymerization degree ranging between 25-30 Llysine residues. εPLL shows a high stability at high temperature and under acidic and alkaline conditions. First, εPLL was modified with DOTA at different ratios and the compound with approximately 14 DOTA moieties per 30 lysines (1) was defined as the lead compound (Figure 1). Using PET-imaging it was possible


Page 9 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


to obtain high-resolution images of the biodistribution of this DOTA-εPLL derivative.

specifically accumulate in the kidneys was established by the determination of their biodistribution (Figure S 1). Within this series, substitution with three DOTA moieties εLys7(εLys[DOTA])3 (3) resulted in highest kidney accumulation (71.8 ± 9.6 %ID/g).

Renal uptake of FITC-DOTA-εPLL (2).The localization of (1) in the kidney was examined by immunohistochemical staining of kidney sections (NMRI mice). For this purpose the DOTAmodified εPLL was conjugated to FITC (2).In order to detect the FITC conjugated DOTA-εPLL, an anti-FITC antibody was added to the paraffin sections. The stained sections revealed that (2) is detected in the tubuli of the renal cortex (Figure 2B & 2D), the proximal tubule cells have a brush border (microvilli) at the apical side (Figure 2E). Inside the tubule cells endocytosis vesicles are highlighted (Figure 2F). Glomeruli staining was found to be negative (Figure 2G). Pharmacokinetics of the carrier peptide (KKEEE)3K (4). DOTA-εPLL shows very exclusive accumulation in the

Figure 1: Chemical structures of two generations of kidney tracers consisting of epsilon polylysine modified with DOTA (1), (2) and multimers of the peptide motif KKEEE (4)-(7). The PET images of the 68Ga-labeled compound (1) at 1 h after i.v. injection in female Wistar rats, showed an almost exclusive uptake in the kidneys (Figure 2A). Apparently, several factors, such as the length of the peptide, the amount of DOTA-residues or the charge, may cause the specific accumulation of the DOTA-conjugated εPLL in the kidneys. In order to determine the structural requirements of these factors, a series of DOTA-conjugated deca-εPLL was synthesized by solid phase peptide synthesis (SPPS). The chelator DOTA was attached through one of its carboxylic acid arms to an α-amine of the lysine residues. A homologous series comprising one to ten modifications was synthesized. Radiolabeling was performed using 68 Ga or 111In. The ability of these DOTA-conjugated εPLL to

kidneys. Due to the structure of DOTA-εPLL comprising unnatural peptide bonds, DOTA as additional compound and a long retention in the kidneys, a peptide consisting only of natural amino acids and standard peptide linkages was developed. The ideal peptide not comprising any unnatural residues was found to be (KKEEE)3K (4) (Figure 1). Moreover, (KKEEE)3K shows an optimized ratio between positive and negative charged functional groups. The amino acid sequence (KKEEE)3K was synthesized by SPPS. Biodistribution of (4) was examined using γ-scintigraphy. In order to enable the radioiodination of the peptide, a D-tyrosine was added to the N-terminus. D-tyrosine was used to prevent deiodination by deiodinases.(29, 30) The peptide was iodinated using the chloramine-T method(31) with 125I or 131I. Scintigraphic images of 125I-y(KKEEE)3K (5) showed high carrier enrichment in the kidneys 10 min post injection followed by renal clearance within 24 h (Figure 3B, Figure S 2). A biodistribution study confirmed the results of the γ-scintigraphy (Figure 3A, Figure S 3). The carrier capacity of the peptide (KKEEE)3K (4) was determined by conjugation of ciprofloxacin, an antibiotic used in severe urinary tract infections (Figure 4A).(32)

Figure 2: Confirmation of the specificity of kidney targeting by in vivo micro-PET imaging of 68Ga-labelled (1) in a female Wistar-rat (A). IHC staining of a mouse kidney at 1 h post intravenous injection of the fluorescent compound (2). The microscans B and D demonstrate a general view of the localization of the FITC-labeled DOTA-εPLL in the renal cortex. C. Control: kidney of a NMRI-mouse injected with 0.9% NaCl. 2D, E, F and G show the localization of (2) in the tubule using immunostaining of FITC.

2 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For comparison, D-Tyr-ciprofloxacin was labeled with 125I. Figure 4B shows the changed pharmacokinetics of D-Tyr-ciprofloxacin, after conjugation to (KKEEE)3K (6). While D-Tyr-ciprofloxacin is rapidly excreted via renal and biliary excretion routes, the targeted derivative (6) accumulates specifically in the kidneys and has there a long retention time.

Page 10 of 16

meruli (Figure 5C & D). The same results had been obtained with (2), a FITC labeled derivative of DOTA-εPLL (Figure 2). Megalin is a binding receptor for various ligands. It is localized in the plasma membrane of many absorptive epithelial cells.(33) In order to evaluate the influence of the megalin receptor on renal carrier uptake, male megalin-deficient mice received an injection of 124I-y(KKEEE)3K (5). The reduction of megalin was previously shown by immunohistochemical and western blot investigation with an anti-megalin antibody (Figure S 5). Micro-PET-imaging at 2 h post injection showed that renal retention was lower in megalin-deficient kidneys, compared to kidneys of wild type mice (Figure 6). On the other hand excretion of 124I-y(KKEEE)3K (5) is significantly higher in megalin-deficient mice, leading to a higher amount of radioactivity in the bladder.

Tolerability of (KKEEE)3K (4). To assess the suitability of (KKEEE)3K (4) as a renal targeting drug carrier, tolerability of the carrier peptide was evaluated. Three dose levels of carrier peptide (2 mg, 5 mg and 10 mg) were administered i.v. to CD1 mice (n=3). The animals were monitored for 7 days for clinical findings, behavioral changes, and survival rate. BUN (blood urea nitrogen) and creatinine were measured in order to check kidney function after administration of (KKEEE)3K (4). Mean body weight in all groups increased from day 0 to day 7. Necropsy also showed no abnormalities. Determination of BUN shows no significant increase (Figure S 6). Creatinine was also found to be not increased in all groups (

Renal Targeting: Peptide-Based Drug Delivery to Proximal Tubule

Recommend Documents