Drug Conjugation Affects Pharmacokinetics and Specificity of Kidney

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Drug Conjugation Affects Pharmacokinetics and Specificity of Kidney-Targeted Peptide Carriers Maria Janzer, Gregor Larbig, Armin Kübelbeck, Artjom Wischnjow, Uwe Haberkorn, and Walter Mier Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00397 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

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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.

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Bioconjugate Chemistry

ACS Paragon Plus Environment


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active principle

linker

carrier peptide


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Drug Conjugation Affects Pharmacokinetics and Specificity of Kidney-Targeted Peptide Carriers Maria Janzer†,‡, Gregor Larbig‡, Armin Kübelbeck‡, Artjom Wischnjow†, Uwe Haberkorn† and Walter Mier† † ‡

Department of Nuclear Medicine, Heidelberg University Hospital, INF 400, 69120 Heidelberg, Germany Merck KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany

Abstract: Peptides play a crucial role as biological vectors for targeted drug delivery. In particular in cases of specific receptor expression, peptides are highly potent carriers for drug targeting approaches. Kidney-targeted peptides require specific attention because of the necessity of fine tuning their behavior with respect to extraction and retention in the complex architecture of the kidneys. In order to enable optimal carrier capacity and targeting specificity, this study focuses on pharmacokinetic profiles of different kidney-specific peptides and examines the impact of drug conjugation. γ-Scintigraphy was used to compare the pharmacokinetics and specificity prior and post drug conjugation of the model drug α-lipoic acid. The results revealed that drug conjugation dramatically affects the targeting specificity, in the worst case leading to a total loss of kidney specificity. Nevertheless, efficient drug transport was achieved with the novel kidney carrier (KKEEE)3K, even with a multiple drug loading of α-lipoic acid after i.v. and i.p. administration. In contrast to other peptidic molecules (KKEEE)3K demonstrated its significant potential as a promising carrier candidate for kidney-targeted drug delivery to proximal tubule cells, especially for the treatment of severe kidney diseases. 1

Introduction

Approximately 85% of all drug candidates fail in early clinical trials, due to lack of efficacy and insufficient safety.1 To reduce the failure rate, the realization of concepts such as drug targeting are necessary. In the ideal case targeted drugs exclusively act at the site addressed by their carrier moiety. Over 100 years have passed since Paul Ehrlich had the vision of drug targeting.2 By directing drugs specifically to diseased tissue/organs, drugs could obtain improved efficacy with reduced off-target effects against normal tissue.3 To achieve this goal, directed delivery vectors (“carriers”) or drugs with appropriate co-administrative effects are required.4 In this paper we focus on bioconjugates, where a linker connects a therapeutic agent to the delivery vector (Figure 1). To attain conjugate efficacy, several parameters have to be fulfilled, for both linkage sites, especially the chemical bond between drug and linker necessitates tailored properties. It has to be stable during systemic transport but degradable after reaching the target site to ensure availability and efficacy of the drug. Thus, linker design plays a crucial role in the development of drug targeting concepts. Various strategies have been investigated with different vectors, including proteins,5-7 peptides,8, 9 small molecules10 or different polymers.11, 12 In particular, peptides have obtained importance in the design of targeted drug delivery approaches, e.g. to the lymphatic system, various tumors, brain, kidneys, liver, heart, etc.4, 13 The properties (i.e. isoelectric point, hydrophilicity, structure) of peptides are defined by their amino acid sequence. Peptides that target particular cell-



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surface receptors, can be taken up by receptor-mediated endocytosis. Hence, these peptides provide opportunities for targeted delivery especially where specific receptors are expressed. Moreover, peptides have many advantages for drug targeting concepts. They possess high drug loading capabilities (“payload”), can be synthesized relatively straightforward with automated processes and the controlled regioselective conjugation conditions leads to defined products. In addition, most peptides have a high thermal and hydrolytic stability. In comparison to proteins they have a greater storage stability as well as a much lower immunogenicity.14 When compared to small molecule drugs, peptides suffer from a major drawback: the limitation to parenteral administration. Due to this restriction, drug targeting concepts with peptides have to be focused on severe diseases and drug-peptide conjugates still play a minor role in currently used therapies.15 Further facts that impede the clinical use of drug-peptide conjugates are a) the challenging identification of appropriate ligands in the desired tissue/organ. b) insufficient information about their mode of action, including uptake mechanisms, receptor saturation and receptor recycling and c) the lack of understanding how the drug loading affects the pharmacokinetic and pharmacodynamics. Ideally, the receptor affinity of a carrier peptide should not be impaired by drug conjugation. But most often these substantial questions for targeted drug delivery have not been sufficiently examined. Only a limited number of systematic studies comparing chemical methods and properties for drug-peptide conjugates are available. In case of kidney targeting with low-molecular-weight proteins (LMWP), several issues were examined by studies from de Zeeuw et al.7, 16-19 Every small molecule drug can potentially pass the well perfused kidney – but what is the rationale for renal targeting? Glomerular filtration does not necessarily lead to renal uptake by the cells targeted and does not avoid systemic side effects of the circulating drug. Therefore, renal targeting is the basis for improving the therapeutic index of drugs designed for the treatment of renal diseases17. This paper focuses on pharmacokinetic properties of different kidney-specific peptidic carriers, with emphasis on the novel highly kidney-specific carrier (KKEEE)3K (1) which accumulates in the proximal tubular cells of the kidneys. In comparison to other kidney-specific carriers, this peptide shows a non-toxic profile, which is the disadvantage of many LMWPs, is readily available by chemical synthesis and shows rapid renal clearance within a few hours.20 Moreover, defined drug-carrier conjugates can be obtained - this is not possible with LMWPs such as lysozyme. Scintigraphic distribution studies demonstrate that drug conjugation can affect strongly the pharmacokinetic profile of carrier molecules. As a consequence the meaningful assessment of a peptidic renal carrier should be performed in combination with an appropriate drug candidate.

Figure 1. Simplified sketch of a drug-linker-peptide conjugate structure comprising a kidney-specific peptide motif. The pharmacokinetics is defined by the nature of the three components and their linkage.


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Scheme 1. Synthesis of multiple loaded lipoic acid conjugate via Lys-ε-amid linkage. Reagents and conditions: (a) 1.05 eq N-hydroxysuccinimide, 1.02 eq EDC, DMF; rt, 4 h; (b) 1,05 eq Fmoc-Lys-OH, HEPES buffer, pH 7.4/ acetone (v/v, 1/1), rt, 3 h; (c) 4 eq FmocGlu(tBu)-OH, 4 eq Fmoc-Lys(Boc)-OH, 4 eq Fmoc-Lys-ε(LA)-OH, 4 eq HBTU, 10 eq DIEA, automated synthesis; Cleavage conditions: TFA/thioanisole/anisole (v/v/v, 92/8/2) 1 ml/100 mg resin, rt, 1 h.

2 Results 2.1 Synthesis All peptides and conjugates were prepared by solid-phase synthesis using standard Fmoc/tBu chemistry. For multiple drug loading of peptides, a new synthesis route was developed. For this purpose Fmoc-protected lysine-drug building blocks with lipoic acid (Scheme 1) or caffeic acid (not shown) coupled to the ε-amino group were synthesized and afterwards applied in the automated synthesis. Detailed experimental procedures are described in chapter 1.3. To achieve products at high purity, the cleavage cocktails had to be adapted. The peptides containing cysteine (e.g. G3-C12 peptide), as well as all lipoic acid-conjugates were cleaved with TFA/thioanisole/anisole in ratio 92/8/2 (v/v/v). All other peptides were cleaved with a standard cleavage cocktail consisting of TFA/ Triisopropylsilane (TIPS)/H2O (95/2.5/2.5, v/v/v).

2.2

Radiolabeling and Biodistribution

To trace the carrier peptides via γ-scintigraphy, an additional D-tyrosine was coupled at the N-terminus of peptides if no tyrosine was present in the original sequence. Radiolabeling with 125I was performed at the phenolic group of tyrosine using the chloramine-T method. Studies with L-tyrosine labeled peptides frequently show accumulation of radioactivity in the stomach and thyroid (Supplementary Information). This can be caused by deiodination processes via deiodinases. To avoid this enzymatic cleavage “unnatural” D-tyrosine was used for all scintigraphic studies. The technetium labeling was performed with 99mTc pertechnetate eluted from a commercial generator and tin(II) chloride as the reducing agent. As the complexation of technetium requires the free thiols of lipoic acid, it was used in its reduced (dihydrolipoic acid) form.



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2.3

Biodistribution studies of the carrier peptides

Different peptides were synthesized to examine the effect of the peptide characteristics on the specificity of accumulation in the kidneys. Table 1 shows an overview of all peptides synthesized with their molecular mass and their calculated isoelectric point. To achieve a broad structure-property correlation, peptides with various attributes were selected. Figure 2 illustrates the wide range of peptides analyzed regarding their charges at physiological pH of 7.4. The charge conditions of all analyzed peptides were calculated with the protein property calculator version 3.4 by Putman C, Scripps Research Institute (http://protcalc.sourceforge.net/).

Table 1. Sequence and physicochemical properties of the synthesized peptides. Molecular No Peptide IEPi Literature weight [g/mol]

I

(1)

y(KKEEE)3K

2240.1

4.94

20

(2)

y(KKEE)5K

2881.8

8.41

-

(3)

y(RREEE)3R

2436.1

4.94

-

(4)

y(KKQQQ)3K

2231.1

10.8

-

(5)

y(MARIA)3

1808.8

12.3

-

(6)

(APASLyN)2

1614.3

5.5

14

(7)

ANTPCGPyT HDCPVKR

1921.7

8.03

9

8)

yD8

1101.5

3.16

21

(9)

y(DSS)4

1336.7

3.34

22

Calculated isoelectric point using Isoelectric Point Calculator, Kozlowski L.P., 2007-2016; http://isoelectric.ovh.org.

Some of these peptides were already described in previous studies as kidney specific carriers. Here, we focused on the highly specific kidney carrier (KKEEE)3K (1), which had shown outstanding properties as renal carrier (Figure 2): a rapid and almost exclusive accumulation in the kidneys after bolus injection followed by slow excretion via the urine within approximately 4 hours. Histochemical examinations revealed the highly specific accumulation of (KKEEE)3K (1) in proximal tubular cells of the kidneys.20 To study the properties of this carrier, initially selected amino acids of the peptide sequence (KKEEE)3K (1) were replaced. Even though the substitution of lysine (K) with arginine (R) does not lead to a change of the number of positively charged amino acids, (RREEE)3R (3) demonstrated a substantially decreased kidney uptake as well as faster excretion to the bladder. In contrast, the biodistribution of (KKEE)5K (2) and (KKQQQ)3K (4) indicated no change in the pharmacokinetic and renal specificity, even though the charges changed from -2.2 to +6.8 at physiological conditions (Figure 2). A total loss of renal retention was detected for the acidic peptides (DSS)4 (9) and D8 (8). However, closer examination of both peptides indicated a slightly higher uptake of (DSS)4 (9) with 4.2 negative charges in comparison to the very acidic peptide D8 (8) containing 8.2 negative charges. Two other literature-known kidney specific peptides, as well as a random sequence were synthesized to compare their specificity to (1). The peptide G3-C12 (ANTPCGPYTHDCPCKR) (7), a galectin-3 targeting peptide identified by phage display, showed comparable excellent kidney specificity to (1).9 The sequence APASLYN (6) was also identified using phage displaying technologies as a kidney specific peptide.23 For better mass comparability to the other analyzed peptides the sequence of APASLYN was doubled. Contrary to the phage displaying studies of Denby et al. our distribution studies demonstrated no kidney specificity of (APASL125I-yN)2 (6). The random sequence (MARIA)3 (5) also displayed good kidney specificity. However, in comparison to (KKEEE)3K (1) the distribution of this peptide revealed almost no retention in the kidney, resulting a faster excretion via urine into bladder.


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Figure 2. Dependency of kidney accumulation of different 125I-labeled peptides, 60 minutes post injection in female NMRI mice on their number of charges. While all peptides, except for (APASLyN)2 show renal excretion the scintigraphic imaging clearly shows that the peptide motif (KKEEE)3K shows optimal properties with respect to retention and avoidance of unspecific distribution. i(Calculated by Protein Calculator, V.3.4. by Putnam, C., (2013), The Scripps Research Institute, U.S.A; http://protcalc.sourceforge.net/).

Figure 3. Time course of the scintigraphic distribution of 125I-y(KKEEE)3K (1) after intravenous application into an NMRI mouse. The peptide shows a highly specific accumulation in the kidneys for approximately 60 minutes and is subsequently distributed to the bladder.

2.4

Biodistribution studies of α-lipoic acid-peptide conjugates

To assess the impact of drug conjugation on the renal specificity of carriers, the model drug α-lipoic acid was conjugated to different kidney specific carriers. The pharmacokinetic profiles of these peptides were initially examined in detail in section 2.3. The most promising peptides (KKEEE)3K (1), (KKQQQ)3K (4), G3-C12 peptide and the random sequence (MARIA)3 (5) were selected to study their carrier properties. α-Lipoic acid a small molecule drug (206 g/mol), was chosen as model drug for the conjugation studies. Lipoic acid has strong antioxidative properties and its reduced form, dihydrolipoic acid, enables scavenging of free radicals. In various studies lipoic acid was used to prevent nephrotoxicity of different compounds toxic to tubular cells, such as cisplatin, ifosfamide or gentamicin.24-26 Consequently, lipoic acid is a non-toxic model compound used as a radical scavenger to protect cells stressed by chemotherapy-induced low glutathione levels.27, 28 Despite of its low molecular mass its highly lipophilic character (LogP 2.25, calculated by using software of www.molinspiration.com) causes a heavy load for the polar targeting peptides. To avoid premature cleavage of conjugated drug, a stable amide bond was used as linkage between N-terminus of the peptides and lipoic acid.



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Initially, the pharmacokinetics and specificity of free α-lipoic acid was analyzed in scintigraphic distribution studies. For this purpose 99mTc was bound to the sulfhydryl groups of reduced lipoic acid. Figure 4 (upper row) shows fast accumulation of the labelled lipoic acid in the liver, which is in accordance to previous pharmacokinetic studies of lipoic acid.29 Furthermore, it can be seen that only trace amounts of radioactivity were detected in bladder. At the same time, accumulation rises in lower abdominal region after 30 minutes, revealing that lipoic acid is mainly excreted via bile tract into duodenum, as confirmed by studies of Gregus, et al.30

Figure 4. Scintigraphic imaging of α-lipoic acid after bolus injection in NMRI mice. Upper row: 99mTc-labeled α-lipoic acid shows a fast accumulation in liver. Bottom row: 99mTc-α-lipoic acid-(KKEEE)3K shows a complete change of the pharmacokinetics of α-lipoic acid resulting in its specific transport to the kidneys.

After conjugation of this lipophilic drug the pharmacokinetics of different conjugates was analyzed. Figure 5 shows the distribution of the carrier peptides (upper row) in comparison to their LA-peptide conjugate (bottom row). Substantial changes of the specificity and kinetics of some of the peptides was detected. The drug loading did not obstruct the kidney accumulation of (KKEEE)3K (1). Consequently, the transport of lipoic acid into the kidneys was achieved. However slightly higher amounts of the tracer were measured in the bladder 60 minutes post injection in comparison to the free carrier peptide. Similar results were obtained with G3-C12 peptide (ANTPCGPYTHDCPCKR) (7). Only marginal changes of specificity and increased secretion via bladder after drug conjugation were observed.


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Figure 5. Scintigraphic imaging of a selection of carrier peptides (upper row) and their corresponding lipoic acid (LA) peptide conjugates (bottom row) distributions at 60 minutes after intravenous application. Depending on the carrier capacity, the conjugation of lipoic acid does not affect the pharmacokinetic properties.

In contrast, N-terminal drug loading dramatically changed their pharmacokinetic properties of (KKQQQ)3K (4) and (MARIA)3 (5). The scintigraphic distribution of LA-125I-y(KKQQQ)3K indicated a high accumulation in the liver 60 minutes post injection and only minor amounts reached the kidneys. The organ distribution studies of LA-125Iy(MARIA)3 showed high amounts of the tracer in the intestinal area, thyroid and lower amounts in the kidneys and the bladder. Moreover, both conjugates showed only low amounts of radioactivity in bladder, which indicates changed secretion pathways caused by the drug conjugation. Consequently, drug transport to the kidneys was not achieved with the kidney specific peptides (KKQQQ)3K (4) and (MARIA)3 (5).

2.4.1

Biodistribution studies of multiple LA-loaded (KKEEE)3K

To evaluate the capacity of (KKEEE)3K (1) multiple lipoic acid moieties per peptide were incorporated and the pharmacokinetics of the conjugates analyzed via planar γ-scintigraphy. Additionally, a direct drug labeling on sulfhydryl groups of lipoic acid via 99mTc was carried out. Hence, the tracing of drug transport was possible and simultaneously allowed the validation of the D-tyrosine-labeled peptides.



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Figure 6. Scintigraphic imaging of different LA-conjugates 60 minutes after injection into NMRI mice. A: 99mTc-LA-(KKEEE)2K, single LA-loading and direct drug-labeling via 99mTc. B: KKKε-(99mTc-LA)E(EEK)2KKKε-(99mTc-LA)EEEK, double loading of lysine and direct drug labeling with 99mTc. C: 125I-y(KKKε-(LA)EEE)3K, triple loading and labeling of N-terminal D-tyrosine.

At first a shortened carrier (KKEEE)2K was synthesized to increase the drug per mass ratio. The pharmacokinetics of this LA-11-mer conjugate showed no differences when compared to the 16mer-conjugate LA-125I-y(KKEEE)3K (Figure 6). Even a double and triple loading with lipoic acid via ε-linkage of lysine did not affect the carrier properties of (KKEEE)3K. Drug transport was achieved with very high renal specificity. No significant changes were determined in comparison to the single conjugated peptide.

2.4.2

Biodistribution studies of LA-loaded (KKEEE)3K after intraperitoneal application

Peptidic drugs are limited to parenteral application routes. With the background of patients’ compliance and simplified applications, the double LA-loaded carrier (KKKε-(99mTc-LA)E(EEK)2KKKε-(99mTc-LA)EEEK) was administered intraperitoneally. Scintigraphic distribution of the LA-conjugate showed initially a long retention at the injection site (Figure 7). But after 60 minutes a gradually accumulation in both kidneys was detected. 2 hours after intraperitoneal administration a retarded but also very high specific accumulation in kidneys was observed. Thus, drug targeting to the kidneys with (KKEEE)3K is also possible after an intraperitoneal injection.

3

Discussion

The proximal tubule cells (PTC) represent the largest part of the kidney and play an important role in etiology of many renal diseases.36, 37 Therefore PTC’s are an attractive target for site-specific drug delivery. It is well known that low-molecular-weight proteins of less than 20 kDa are filtered in the glomerulus and are actively reabsorbed at the apical site of the proximal tubules. Especially, peptides with less than 5 kDa pass the glomerulus very efficiently.38 Reabsorption is facilitated by different receptors and transporters via endocytosis. After internalization, peptides are transferred to the lysosomes, where they are proteolytically degraded. The most prominent peptide transporter present at the apical side of PTC’s is megalin. This large (~600 kDa) glycoprotein receptor contains a huge extracellular domain, which is responsible for its multispecific properties.39 Together with its associated receptor cubilin, they are mainly responsible for reabsorption of glomerular filtered low-molecular-weight proteins. It has been reported that in general renal uptake of peptides depends on size, structure, net charge and number of positively charged amino acids, which can bind to the negatively charged surface of PTC’s.9, 35


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We have previously shown that (KKEEE)3K (1) is taken up by megalin into proximal tubule cells.20 Further evidence for the specificity of uptake was obtained by a competitive study using (KKEEE)3K as an inhibitor for the uptake of 177LuDOTATOC. The megalin receptor is responsible for significant fraction of the renal uptake.40, 41 Biodistribution studies of 177Lu-DOTATOC indicate that simultaneous application of (KKEEE)3K leads to a decreased renal uptake of 177LuDOTATOC (Supplemental, Figure S 5). These results confirm the previous studies with megalin-deficient mice and demonstrate a specific ligand-receptor interaction of the peptide (KKEEE)3K.20 To study the structure-activity relations for renal specific uptake of peptidic carriers, different peptides were synthesized and analyzed via planar γ-Scintigraphy in mice. Our biodistribution studies (Figure 2) showed that the acidic peptides D8 (8) and (DSS)4 (9), containing 8 and 4 net negative charges were immediately excreted without significant reabsorption. These results may indicate that acidic peptides are not reabsorbed into proximal tubule cells, due to negative charges. Previous studies by Béhé, et al. reported that the megalin-induced uptake of minigastrin is reduced by up to 90% due to co-administration of acidic polyglutamic acid.36 Additionally, polyaspartic acid did not reduce the renal uptake of minigastrin, which confirms our kinetic studies of D8 (8). Another significant examination regarding charge impact on renal uptake and retention of peptides was shown by (RREEE)3R (3). Exchange of only one lysine moiety with arginine, resulting in the same charge conditions causes an essential decrease of reabsorption in comparison to (KKEEE)3K (1). Distribution studies of 111In-DTPA-octreotide, where one amino acid was exchanged at N-terminus showed also significant affections of renal accumulation.37 These findings indicate that net charges play only a minor role for renal uptake. In comparison to all other examined peptides, the lipophilic and neutral charged peptide (APASL125I-yN)2 (6) showed a completely different pharmacokinetic profile. Scintigraphic studies of (APASL125I-yN)2 (6) indicate hepatic uptake of this peptide. Several studies demonstrated an increased hepatic uptake and hepatobiliary excretion, due to high overall lipophilicity of targeting peptides,35, 38 which encourages our assumption. Nevertheless, it can be concluded that net charges of peptides do not constitute the factors that dominate the renal reabsorption processes. Other parameters, e.g. structural features may have higher importance for receptor affinities at the apical site of proximal tubule cells. An overview of megalin ligands with widespread characteristics additionally underlines our assumption.39 To summarize, many peptides with different properties are able to accumulate in proximal tubule cells and consequently are potential carriers for renal targeting.

Figure 7. Scintigraphic imaging of the biodistribution of 99mTc-labeled α-lipoic acid and its carrier conjugate after intraperitoneal injection in NMRI mice. Upper row: The predominantly biliary excretion of α-lipoic acid observed after intravenous application is also observed after intraperitoneal injection. Bottom row: (KKEEE)3K efficiently carries α-lipoic to the kidneys.

In this paper, we further focused on pharmacokinetic changes of kidney targeting peptides after drug loading. α-Lipoic acid, a small molecule drug was used as model drug for all further examinations. Four peptides with promising pharmacokinetic profile were selected for N-terminal drug conjugation via amide linkage. The γ-scintigraphy results showed significant pharmacokinetic changes after drug conjugation, in comparison to the corresponding free carrier peptide (Fig-



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ure 5). Two of four peptides demonstrated a total loss of their kidney specificity. A useful drug delivery to kidneys was not achieved in these cases. Both conjugates LA-125I-y(MARIA)3 and LA-125I-y(KKQQQ)3K immediately accumulate in the upper abdomen and only minor amounts were taken up by the kidneys. The changed excretion route of both peptidedrug conjugates emphasizes high impact of the coupled drug (Figure 5). This phenomenon suggested that drug properties can outperform the carrier characteristics, despite the high difference in molecular weight of both components. In contrast, y(KKEEE)3K (1) and the G3-C12 peptide (ANTPCGPyTHDCPCKR) (7) showed only minor changes of their pharmacokinetic profile after drug conjugation. In general the conjugated drug might have no influence to receptormediated uptake. In the case of LA-125I-y(MARIA)3 and LA-125I-y(KKQQQ)3K, obviously both conjugates do not reach the binding receptors, because of major changes in pharmacokinetics. Therefore, in vitro receptor affinity studies are of minor predictive value for conjugate assessment. Our results show that drug conjugation remarkably affects specificity and pharmacokinetic of peptidic carriers. This might be a reason for the so far, low success of drug-peptide conjugates. Therefore, impact of drug conjugation always has to be considered during development of drug targeting concepts. Every drug conjugate should be analyzed individually, because of different properties of various drug groups. (KKEEE)3K (1) already showed a high potential as a renal drug carrier and efficiently transports lipophilic and hydrophilic compounds into proximal tubule cells (FITC as model drug, a ciprofloxacin analogue, caffeic acid and α-lipoic acid).20 To further challenge this promising kidney carrier, a shortened carrier (KKEEE)2K was synthesized to achieve an increased drug per mass relation (Figure 6). No change of specificity or other pharmacokinetic characteristic were determined in comparison to the free carrier peptide. The pharmacokinetics of lipoic acid, a compound that is mainly excreted by the liver, was completely changed and a specific drug delivery to kidneys was achieved. One major advantage of peptidic carriers is their high drug load capacity as proven for (KKEEE)3K (1) which tolerates even multiple conjugation of lipoic acid without changes in specificity or pharmacokinetic (Figure 6). Drug conjugation at side groups of peptides might have less influence on pharmacokinetic profile of peptides. This assumption was seen with caffeic acid derivatives, too (Supplementary Information). Additionally, it can be concluded, that transport of lipophilic (α-lipoic acid) as well as hydrophilic compounds and high molecular weight pharmaceuticals. Moreover, a highly specific drug delivery of lipoic acid was also shown after intraperitoneal application of the double loaded LA-conjugate (KKKε-(99mTc-LA)E(EEK)2KKKε-(99mTc-LA)EEEK) (Figure 7). The high plasma stability of this peptidic carrier20 and the drug linkages (amide) used lead to an extended retention time in plasma/intraperitoneal area. These results are of high importance as intraperitoneal injection strongly improve patients’ compliance, particularly with regard to long-term treatments, e.g. for chronic kidney diseases. One general question is the fate of the transported drug after uptake in PTC’s. To guarantee drug efficacy, drugs have to be cleaved to its original active structure. Consequently, linker chemistry plays a crucial role for successful drug targeting. Moreover, drugs fate at target site depends on its properties and pharmacokinetics. Figure 8 illustrates an overview of the main drug routes after conjugate reabsorption into proximal tubule cells. Consequently, all aspects mentioned have to be considered individually for every conjugate.

Figure 8. Main possible drug routes after receptor-mediated conjugate uptake into proximal tubule cell. 1. Lysosomal drug degradation. 2. Drug excretion via apical site of proximal tubule into primary urine. 3. Drug excretion into tubolointerstitium via basolateral site of proximal tubular cell and renewed uptake into proximal tubule. 4. Drug excretion into tubolointerstitium and uptake into blood


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stream.

To summarize, the kidney carrier (KKEEE)3K (1) confirmed its excellent drug targeting potential in comparison to other kidney specific peptides. In addition, this carrier showed high drug loading capabilities, good tolerability and high plasma stabilities.20 Thus, (KKEEE)3K (1) is a promising candidate for kidney-targeted drug delivery to proximal tubule cells.

4

Outlook

Peptidic carriers are limited to parental administration. Therefore, treatment hurdles for chronic diseases are quite high and patients’ compliance is an issue. Under these circumstances peptidic renal targeting approaches are particularly suited for use in severe diseases, as e.g. polycystic kidney diseases, acute renal failure or renal cell carcinoma. For these indications renal drug targeting could reduce failure rate in clinical development and opens new treatment opportunities.

Materials and Methods 4.1

Materials

All reagents listed below were obtained from commercial sources and used without further purification. Solvents, trifluoroacetic acid (TFA), thioanisole, anisole, different protected amino acids, α-lipoic acid (LA), 1-Ethyl-3-(3'dimethylaminopropyl)carbodiimide (EDC), N,N-Diisopropylethylamine (DIPEA), 2-(1H-Benzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HBTU) were purchased from Merck KGaA (Darmstadt, Germany), dihydrolipoic acid was obtained from Sigma Aldrich (Darmstadt, Germany). Tenta Gel S Ram resin was obtained from Rapp Polymere (Tübingen, Germany). 4.2

Animal studies

Animal experiments were in compliance with the German Animal Protection Laws. The wildtype NMRI mice for organ distribution studies and imaging were obtained from Janvier Labs.

4.3

Methods

4.3.1 Peptide synthesis Peptides were synthesized by solid-phase synthesis using the fluorenylmethoxycarbonyl/t-butyl (Fmoc/tBu) chemistry on an Applied Biosystems 433A peptide synthesizer. A Rink-amide linker functionalized resin was used as the solid-phase. 4.3.2 Purification and characterization Purification was achieved by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) on a Waters XBridge BEH130 PREP C18 (5 µm, 19 × 150 mm). As eluents, 0.1% TFA in water (eluent A) and 0.1% TFA in acetonitrile (eluent B) were used. A standard gradient from 0 to 40% acetonitrile within 20 minutes was used, flow rate 15 mL/min; UV absorbance λ = 220 nm. Analyses were performed on an Agilent 1100 HPLC system using a Chromolith Performance RP-C18e column (100 × 3 mm). As eluents, eluent A and eluent B were used. Conditions: linear gradient from 0 to 100% B within 2.8 minutes; flow rate 2 mL/min; UV absorbance λ = 220 nm. The identity of the peptides was verified by HPLC-MS analysis (Exactive, Thermo Fisher Scientific). 4.3.3 Synthesis of α-lipoic acid conjugates 4.3.3.1 N-terminal conjugation α-Lipoic acid was conjugated via the N-terminal amide bond. 0.1 mmol of fully protected resin bound peptides were swollen by shaking in methylene chloride (DCM) for 20 min. Fmoc-deprotection was performed by piperidine (20% in NMethyl-2-pyrrolidone (NMP), 5 ml, 3 × 5 min). After a washing step (NMP, 3 × 5 ml), the resin was reacted with 4 eq αlipoic acid (82.5 mg, 0.4 mmol), 4 eq HBTU (152 mg, 0.4 mmol) and 10 eq (162 µL, 0.10 mmol) DIPEA dissolved in 8 ml NMP and shaken for 2 h at room temperature. Five washing steps with NMP and one with DCM were carried out after this coupling step. The resin was dried in vacuo and 4 ml of a cleavage cocktail TFA/thioanisole/anisole (v/v/v, 92/8/2) was



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used for deprotection and cleavage of the conjugate. After incubation for one hour, the resin was removed by filtration and the conjugate was precipitated in cold methyl tert-butyl ether (MTBE). The compounds were resuspended and centrifuged (2000 × g, 15 min) three times with 10 mL MTBE, dried and purified by preparative RP-HPLC. The products were obtained as white powders after lyophilization. 4.3.3.2 Multiple conjugation via Lys-ε-amide linkage To obtain multiple drug conjugates, a core building block was synthesized (Scheme 1). 4.3.3.2.1

Synthesis of lipoic acid-NHS-ester (I)

α-Lipoic acid (2022 mg, 9.8 mmol), 1.05 eq N-hydroxy-succinimide (1150 mg, 10 mmol) and 1.02 eq EDC (1917 mg, 10 mmol) were dissolved in 50 mL DMF and were stirred for 4 h at room temperature under argon. 60 ml ethyl acetate was added and the reaction mixture was extracted with water (3 × 60 mL), followed by saturated sodium hydrogen carbonate solution (3 × 60 mL) and 60 mL saturated NaCl solution. After extraction the ethyl acetate phase was dried over sodium sulfate, filtered and the solvent was removed to obtain the NHS-ester (I) (2230 mg, 73.5 % yield). 4.3.3.2.2 Synthesis of Fmoc-Lys-ε(lipoic acid)-OH (II) Fmoc-Lys-OH (2640 mg, 7.19 mmol) was suspended in 110 mL HEPES buffer (pH 7.4) and mixed with (I) (2139 mg, 7.05 mmol) dissolved 130 mL acetone. After 3 h stirring at room temperature, the reaction mixture was adjusted to pH 7 by addition of NaOH (0.1 N). The reaction was continued for 20 h. The pH was adjusted to pH 9 and the solution was extracted twice with 30 mL ethyl acetate. 1 N HCl was slowly added to the aqueous phase until a pH of 3 was reached. Subsequently, the aqueous phase was extracted with ethyl acetate (3 × 40 mL). The collected organic phases were washed with saturated NaCl, dried over sodium sulfate and filtered. The filtrate was concentrated and dried in vacuo over night to yield (II) (3945 mg, 98% yield).

4.3.3.2.3 Solid phase synthesis of multiple loaded peptides Standard automated solid phase synthesis using (Fmoc/tBu) chemistry with (II). Cleavage conditions and processing were performed as described above (N-terminal conjugation). Lyophilized products were obtained as white powders in quantitative yield. 4.3.4 Labeling with 125I For radiolabeling, the peptides were synthesized with an additional D-tyrosine at the N-terminus and a 1 mM stock solution of the respective peptide in water or if needed in a mixture of water and dimethyl sulfoxide (DMSO) was prepared. Labeling with 125I was performed by the chloramine-T method. The reaction mixture was purified by semi-preparative HPLC using a Chromolith Performance RP-18e-column, 100 × 4.6 mm) to 0.1% TFA in acetonitrile (eluent B) within 10 minutes; flow rate 2 mL/min; UV absorbance λ = 214 nm; γ-detection. 4.3.5 Labeling with 99mTc Tin(II) chloride dihydrate, 80 mg was dissolved in 10 mL concentrated hydrochloric acid and subsequently diluted with water to a final concentration of 10 mg/mL. 10 µ L of a 1 mM stock solution of α-lipoic acid as dihydrolipoic acid or αlipoic acid conjugate was mixed with 10 µl 0.5 M phosphate buffer (pH 9). 4 µL sodium tartrate (100 mg/mL H2O), 2 µL lactose (100 mg/mL H2O) and 1 µL SnCl2 (10 mg/mL SnCl2 × H2O) were added to the peptide solution. The amount of 99m Tc pertechnetate solution required was added and the mixture was stirred for 15 min at 95 °C. The product was purified by semi-preparative HPLC, as described for the iodination procedure, but without using TFA in both eluents. The eluate was dried and dissolved in isotonic saline solution. 4.3.6 In vivo planar γ-Scintigraphy 100 µL of the peptide solution containing 5 to 8 MBq activity of coupled nuclide (125iodine or 99mtechnetium) after radiolabeling was administered as an intravenous bolus injection or intraperitoneal into anesthetized animals (female NMRImice) and scintigraphic images were obtained using a γ-camera (Gamma Imager, Biospace, France). The recording time was 10 minutes.

Associated Content


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Supporting Information The supporting information is available free of charge on the ACS Publication website at DOI:

Author Information Corresponding author *Email: [email protected] Notes: The authors declare the following competing financial interest(s): A patent application regarding renal targeting with specific peptides has been filed.

Acknowledgements We gratefully acknowledge Karin Leotta (Heidelberg University Hospital, Germany) for her excellent support in γScintigraphy.

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Drug Conjugation Affects Pharmacokinetics and Specificity of Kidney

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