Drug delivery and release systems for targeted tumor therapy.

Special Issue Review Received: 9 December 2014

Revised: 19 December 2014

Accepted: 19 December 2014

Published online in Wiley Online Library: 23 February 2015

(wileyonlinelibrary.com) DOI 10.1002/psc.2753

Drug delivery and release systems for targeted tumor therapy‡§ David Böhme and Annette G. Beck-Sickinger* Most toxic agents currently used for chemotherapy show a narrow therapeutic window, because of their inability to distinguish between healthy and cancer cells. Targeted drug delivery offers the possibility to overcome this issue by selectively addressing structures on the surface of cancer cells, therefore reducing undesired side effects. In this broad field, peptide–drug conjugates linked by intracellular cleavable structures have evolved as highly promising agents. They can specifically deliver toxophores to tumor cells by targeting distinct receptors overexpressed in cancer. In this review, we focus on these compounds and describe important factors to develop a highly efficient peptide–drug conjugate. The necessary properties of tumor-targeting peptides are described, and the different options for cleavable linkers used to connect toxic agents and peptides are discussed, and synthetic considerations for the introduction of these structures are reported. Furthermore, recent examples and current developments of peptide–drug conjugates are critically evaluated with a special focus on the applied linker structures and their future use in cancer therapy. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. Keywords: peptide–drug conjugate; cleavable linker; drug delivery; tumor-targeting peptide; solid-phase peptide synthesis; targeted therapy; cancer

Introduction

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The controlled cleavage of chemical bonds between two entities is an important property in order to regulate the release of chemical functionalities for synthesis applications or, in chemical biology, to selectively liberate molecules to exert a certain biological function. Cleavable linker structures have a long history in the synthesis of small molecules on solid support [1] and in combinatorial chemistry [2]. They connect a growing organic molecule to the solid support, thereby enabling the use of an excess of reagents and an easy purification of raw products by filtration, leading to high yields during synthesis. Additionally, they need to be stable during the synthesis process and release the molecule under specific conditions. This concept was applied in the most complex manner for solid-phase peptide synthesis, where the use of different cleavable linkers and manifold protecting groups [3] allows the synthesis of large peptides with multiple modifications for biological applications [4]. Unfortunately, the harsh conditions used to cleave these linkers during solid-phase synthesis do not allow their direct use for biological applications. Therefore, chemical moieties have been worked out that can be cleaved specifically under biocompatible conditions. With specific functional groups, the concept of cleavable linkers has emerged in many different biochemical disciplines like protein purification, proteomics [5], and imaging [6–8], as well as drug discovery and particularly in the development of selective drug delivery systems [9,10]. These systems are the base of a targeted therapy, which is especially desirable in the treatment of cancer. Although potent chemotherapeutic agents are used to treat cancer, their therapeutic window remains small owing to their toxicity, which leads to severe side effects [11]. Therefore, a directed approach needs to distinguish between healthy and cancer cells, which are inherently difficult to discriminate, in order to ensure the specific intracellular delivery of toxic molecules to degenerated

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tissue. The concept of targeted therapy/drug delivery is illustrated in Figure 1 and will be explained in more detail throughout the review. In general, the starting point of this approach is a prodrug that consists of three segments, namely a targeting unit, a linker, and a toxic agent, thereby creating a single highly specific and cytotoxic moiety that is inactive during delivery, but highly potent after reaching the target [12]. The targeting unit ensures, as a selective carrier, the specific delivery and uptake of the toxophore by cancer cells. The linker structure defines an exact breaking point between carrier and drug that permits the controlled intracellular release of the active toxic agent. Thus, this concept led to nanoparticle-based delivery platforms [13,14], antibody–drug conjugates [15], and recently also peptide– drug conjugates [16]. In the present review, we focus on peptide–drug conjugates for tumor targeting, connected by cleavable linker structures. First, the necessary properties of peptides used as selective carriers to target tumor cells are described. In the second part, different available linker structures will be discussed to trigger intracellular release for targeted delivery. Synthetic aspects for the introduction of toxophores by different linker bonds are described, and recent

* Correspondence to: Annette G. Beck-Sickinger, Institute of Biochemistry, Universität Leipzig, Brüderstraße 34, 04103 Leipzig, Germany. E-mail: [email protected] ‡

§

Invited Article for the Anniversary Issue 2015 of the Journal of Peptide Science Dedicated to Dr Anselm Sickinger who passed away too early. Institute of Biochemistry, Universität Leipzig, Brüderstraße 34, 04103, Leipzig, Germany

Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

PEPTIDE–DRUG CONJUGATES FOR TARGETED THERAPY Biography David Böhme was born in 1987 in Riesa, Germany. He studied Chemistry at the University of Leipzig in a bachelor’s–master’s program. During his master studies, he spent a 6-month research internship at the Ohio University (USA). In 2012, he joined the research group of Prof. Dr Annette G. Beck-Sickinger for his PhD, which focuses on the development of peptide–drug conjugates for targeted tumor therapy.

Biography Annette Beck-Sickinger studied Chemistry (diploma) and Biology (diploma) at the University of Tübingen and received her PhD with Günther Jung. She did fellowships with R. A. Houghten (Scripps Clinic & Research Foundation, La Jolla, USA ), E. Carafoli (Laboratory of Biochemistry, ETH Zürich), and T. W. Schwartz (University of Copenhagen, Denmark) and was appointed as an Assistant Professor of Pharmaceutical Biochemistry at ETH Zürich (1997–1999). Since 1999, she has been a Full Professor of Biochemistry and Bioorganic Chemistry at the University of Leipzig. In 2009, she spent a semester as a Visiting Professor at Vanderbilt University, Nashville, TN. Her major interests include peptide–receptor interaction of G-protein coupled receptors, peptide drugs, including peptide–drug conjugates, protein expression and modification, biomedical therapeutic and diagnostic approaches in cancer, obesity, and regenerative medicine as well as novel biomaterials.

examples of peptide–drug conjugates are summarized. Finally, perspectives for the identified complex targeting systems are outlined.

Tumor-targeting Peptides as Selective Carriers

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It has been shown that many tumor cells overexpress peptide receptors on their surface [17], compared with the healthy tissue they are derived from. This makes the peptides that bind to these receptors promising carriers for selective targeting of tumor cells [16]. Because these peptides usually have a very high affinity in the nanomolar range, small amounts are required in the body. After binding and activation, the ligand and receptor might be internalized, and the toxophore is released if a cleavable linker was used (Figure 1). Well-known examples of carrier peptides that are currently studied include somatostatin (SST), bombesin, vasoactive intestinal peptide, neurotensin (NT), neuropeptide Y (NPY), α-melanocyte-stimulating hormone, arginylglycyl-aspartic acid (RGD), and gonadotropin-releasing hormone (GnRH). Peptides based on the amino acid sequence RGD bind to integrin receptors, which play an important role in tumor-induced angiogenesis and metastasis

[18]. All other hormone peptides bind to receptors that belong to the family of G-protein-coupled receptors (GPCRs). Because GPCRs are one of the main targets in drug development [19], their biochemistry is well understood. In addition to the novel targeting concept, a number of classical peptide agents are already existing. They directly address cancer owing to the high number of peptide receptors. This includes goserelin (Zoladex®, AstraZeneca, London, UK) [20], a GnRH receptor agonist, which is used for the therapy of breast and prostate cancer, because of its ability to suppress the expression of testosterone and estrogen. Octreotide (Sandostatin®, Novartis Pharmaceuticals, Basel, Switzerland) [21], an agonist of the SST receptor (SSTR), is furthermore used for the treatment of growth hormone-producing tumors. Additionally, the use of radiolabeled peptide analogues for tumor imaging and peptide receptor radionuclide therapy are promising tools for tumor diagnosis and therapy, which have been reviewed recently [22,23]. In addition to peptide carriers derived from natural hormones, combinatorial approaches have been used to identify peptides that interact with tumor cells in a non-rational manner [24]. These so-called tumor-homing peptides have also been used in nanoparticle-based drug delivery systems, in order to mediate selective delivery [25]. There are distinct requirements for the use of peptides in peptide–drug conjugates. Peptides have to bind with high affinity and selectivity to a receptor, which is overexpressed at the surface of cancer cells, whereas only a low amount of this particular receptor should be present in healthy tissue. Because, for most of these receptors, different subtypes exist with their own ligand specificity and expression levels in various tissues in the human body, it is very important to choose peptide analogues exclusively binding to the receptor subtype, which is homogenously expressed in high densities solely in the neoplastic tissue [17]. An example is the expression of the human Y1 receptor (hY1R) in breast cancer. The hY1R subtype was found to be overexpressed in more than 90% of breast tumors and 100% of breast cancer-derived metastases [26,27]. In contrast, the hY2R subtype is generally found in healthy breast tissue. This switch in receptor expression offers a unique situation for selective tumor targeting using a hY1R-preferring peptide. The natural ligand of the human Y receptor (hYR) subtypes hY1R and hY2R is the 36amino-acid peptide NPY, which binds to both receptors with nanomolar affinity [28]. With detailed knowledge of the structure– activity relationship of the peptide–receptor interaction, it was possible to develop the hY1R-preferring peptide [F7,P34]-NPY [29]. The identification of possible modification sites within this peptide that do not alter its receptor selectivity enabled the derivatization with various molecules for breast cancer imaging or therapy. This concept was impressively demonstrated by the successful imaging of breast cancer cells in tumor-bearing patients with an N-terminally 99m Tc-labeled [F7,P34]-NPY analogue [30]. Furthermore, [F7,P34]NPY was modified at three different positions with carbaborane clusters as a potential application in boron neutron capture therapy (BNCT) [31]. The peptide retained its selective receptor activation and internalization properties and therefore is a well-suited carrier for boron-containing moieties. These studies show nicely that, besides a strong peptide affinity to a receptor, the generation of subtype-specific peptides is essential for selective tumor targeting. The peptide should contain at best multiple modification sites for further derivatization, which is a major advantage of peptides over small molecules. The attached molecules should not influence the binding properties and selectivity profile of the carrier peptide. Because usually not all amino acids in a peptide are necessary for receptor binding, an exchange with amino acids that contain

BÖHME AND BECK-SICKINGER

Figure 1. Schematic representation of tumor targeting with peptide–drug conjugates connected by cleavable linkers.

188

modifiable side chains, including lysine, cysteine, glutamate, or serine, is possible. This extends the chemical options for derivatization of the peptide carrier with the linker and toxophore of choice. Peptides are readily available by solid-phase peptide synthesis, which is the method of choice to produce up to 50-amino-acid peptides in a very time-efficient manner. Moreover, very complex modifications can be introduced at distinct points into the peptide sequence [4]. This allows the selective insertion of linker structures and toxophores at desired positions with the suitable conjugation chemistry. Recently, Zhang et al. were able to modify the cellpenetrating peptide TAT at the C-terminus or N-terminus with the anticancer drug doxorubicin (DOX) using an enzymatically cleavable GFLG linker by a combination of solid-phase and solution phase synthesis [32]. Depending on the modification site, both peptides showed different cellular uptake and therefore different cytotoxic activity, emphasizing the importance of a defined modification of carrier molecules with linker-attached toxophores. Another important aspect of tumor targeting with peptide–drug conjugates is that the carrier peptides need to trigger


internalization after receptor binding in order to deliver the toxic agent inside the cell, as most tumor targets of small-molecule drugs are located inside the cell. After binding, receptor and peptide have to internalize by receptor-mediated endocytosis. Prior to use of a certain system, internalization has to be characterized as some GPCRs, for example, the hY5R [33], are not able to internalize. While the receptor is often recycled and relocated to the cellular surface, the ligand with the attached drug most often ends up in lysosomes. Here, the toxophore can be released by various mechanisms, depending on the linkage type. The internalization process can be visualized in vitro by fluorescently labeled receptors or peptides, which has been studied extensively for the NPY-hY receptor system [34]. The hYR subtype-specific internalization of the already described hY1R-preferring [F7,P34]-NPY and the hY2R-preferring [Ahx(5–24)]-NPY has been confirmed with cells stably or transiently expressing hYR subtypes fused to the enhanced yellow fluorescent protein or enhanced green fluorescent protein [35]. The internalization profile of both peptides was retained after their modification with carbaborane clusters for a potential application in BNCT. This indicates that internalization not only of the carrier peptide itself but also of the peptide–drug conjugate is important and needs to be validated after modification. Furthermore, peptide ligands can be labeled by fluorescent dyes, like carboxytetramethylrhodamine, which visualizes the localization of peptide and fluorescently tagged receptor at the same time. This was impressively shown by monitoring the internalization process of the hY4R subtype and fluorescently labeled analogues of its native ligand pancreatic polypeptide (PP), which are investigated as anti-obesity drugs [36]. While PP analogues modified with palmitic acid led to a fast internalization, PEG-modified analogues showed no internalization, although receptor activation was retained. This illustrates that modification of peptides can significantly influence their properties and may initiate different potential applications. In general, peptide-based drugs are safe, owing to the fact that they do not interact with other drugs and produce no toxic products during metabolism. On the other hand, peptides have a reduced bioavailability, because of their accumulation in organs, like kidney and liver, and low half-lives, owing to their degradation by proteolytic enzymes, and show rapid renal excretion [37]. But the possibilities of engineering therapeutic peptides by introducing various modifications in order to increase their half-lives have especially become evident in the field of glucagon-like peptide receptor (GLP-1R) agonists, which are used for the treatment of diabetes. Since the approval of the first GLP-1R agonist exenatide (Byetta®, Bristol-Myers Squibb, New York, NY, USA) in 2005 with a half-life of around 2.4 h, new modified peptides with half-lives of 4–7 days have been developed and are approved or under clinical investigation [38]. These approaches could also be applied to stabilize peptide carriers in peptide–drug conjugates.

Linker Technologies for Controlled Release The linker structure should enable a specific intracellular release of toxic molecules. So, these moieties should be stable during circulation in order to prevent a premature release of the toxic agent, which will lead to a non-specific killing of cells and subsequently to adverse effects. At the same time, mild and natural changes in biological systems should be able to trigger intracellular cleavage after delivery, in order to release the active toxic agent and effectively target cancer cells. Moreover, these linkers need to be introduced during the synthesis process of the delivery system, requiring


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PEPTIDE–DRUG CONJUGATES FOR TARGETED THERAPY stability during synthesis and purification. The choice of a linker is crucial for the planning of the synthetic strategy, as the necessary functional groups need to be present in the carrier and the toxic molecule. Different linker structures have been identified, which are prone to slight environmental changes during the delivery process and therefore are suitable for an application in drug delivery systems. The most important strategies are summarized in Table 1 and discussed in the following subsections. pH-Responsive Linkers The environment of tumor tissue has a slightly lower pH value compared with that of normal tissue. Cancer cells have high rates of converting glucose to lactate [39], which lowers the pH by about

0.5–1.0 units [40]. This small pH shift could enable the extracellular release of toxic molecules, if the delivery system remains in the tumor environment for longer periods. A much larger difference in the pH value is found in the extracellular system, for example, in blood with a pH value of 7.2–7.4 compared with the pH in intracellular compartments, like endosomes or lysosomes, with 4.5–6.0 [41]. Because peptide ligands are taken up by receptor-mediated endocytosis, they accumulate in these compartments. This difference in pH value creates a unique opportunity to use these changes for triggering the cleavage of chemical bonds with stability/lability profiles fitting in this narrow pH range. The most widely used acid-labile structures for ‘prodrugs’ are hydrazones, which offer higher stability at physiological conditions, compared with the acidic conditions in intracellular compartments

Table 1. Selection of available linker technologies for targeted delivery systems Linkage type

Structure, cleavage conditions, and cleavage products

References

pH dependent Hydrazone

[42–44]

Cis-aconityl

[49,52]

Reductive Disulfide

[57,58]

Azo

[60,61]

Enzymatic

Val-Cit

[64,65]

β-Glucuronide

[67,68]

Ester

[71,72]

Carbamate

[71,73]

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BÖHME AND BECK-SICKINGER [42]. They can be formed by the selective reaction of a carbonylcontaining toxic agent with a hydrazide-containing peptide or vice versa, which can be readily synthesized during solid-phase peptide synthesis [43,44]. This concept has been shown for the synthesis and drug release of hydrazone-linked peptide amphiphiles that remain stable at pH 7.4 [45]. Although hydrazones are relevant structures in the development of antibody–drug conjugates, they were shown to be unstable during circulation causing substantial side effects in vivo. This finally led to the withdrawal of the anti-CD33 calicheamicin conjugate Mylotarg® [46]. On the other hand, it has been shown that the stability of hydrazone linkages in drug conjugates can be tuned by substitutions close to the hydrazone bond [47]. Several other acid-labile structures, like acetal, cis-aconityl, imine, trityl, and β-thiopropionate [48] bonds, have been incorporated into the backbone of polymers used for drug delivery in order to release the toxic agent through the breakdown of the polymeric carrier after cellular uptake [49]. Although it is challenging to introduce these structures during solid-phase peptide synthesis, because acidic conditions are required to release the peptides from the solid support, these linkers bear the potential to be used in peptide–drug conjugates. Acetals are promising acid-sensitive structures, as bond cleavage is expected to occur 10 times faster by each unit of decrease in pH value [50]. Additionally, it was shown that these structures can be incorporated by solid-phase peptide synthesis [51]. Recently, the acid-labile group cis-aconityl was used to link the small-molecule drug DOX to a polymer containing the tumortargeting peptide RGD [52]. A comparison with an amide-linked conjugate showed a superior cytotoxic effect for the cis-aconityllinked compound, while both compounds showed extracellular stability and similar cellular uptake. Reduction-sensitive Linkages

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The concentration of thiols in serum is much lower compared with that in intracellular sites, owing to a high concentration of natural antioxidants like glutathione [53]. Furthermore, redox enzymes are present in intracellular compartments, like endosomes or lysosomes, which facilitate the cleavage of reducible chemical moieties [54]. In contrast, Austin et al. have shown that the redox potential in endosomes and lysosomes is oxidizing, rather than reducing [55]. However, FRET-based studies with double fluorescently labeled cell-penetrating peptides [56] or folate conjugates [57], linked by reducible disulfide bonds, showed cleavage of the disulfide bridge in intracellular sites, which was detected by a change in fluorescence. This indicates that the reductive machinery is indeed active in the intracellular lumen and compartments after cell entry. Therefore, these changing conditions provide a trigger for the release of toxic agents from prodrugs after the delivery inside target cells. Disulfide bridges have been extensively used in targeted drug delivery [58], owing to their reducible nature. These structures can be readily integrated in a peptide–drug conjugate, for example, by reaction of a cysteine-containing peptide with a thiol-including toxic agent or prodrug, which makes them highly desirable linker structures. The extracellular and intracellular stability of these bonds can be further tuned by introducing methyl groups adjacent to the disulfide bridges [59]. Azo linkers are able to undergo reductive cleavage by microsomal azo reductases by an NADPH-dependent mechanism. With azo-linked DOX nanoconjugates [60], it was demonstrated that cleavage is caused by enzymes present in hepatic cancer cells. The stability of these DOX–polymeric dendrimer conjugates was varied by substitutions in ortho and para positions to the azo


benzene linker. This approach could be transferred to peptide–drug conjugates using liver cancer-targeting peptides [61]. Recently, thioesters, which are well-known structures, owing to their use for native chemical ligation, have been proposed as novel thiol-responsive elements in drug delivery [62]. They have a similar stability profile like thiols, are stable under extracellular conditions, but are cleavable under glutathione concentrations present in intracellular sites. Moreover, they can be incorporated into biomolecules in a traceless manner as shown by a thioester-linked protein–PEG conjugate [62]. Enzymatically Cleavable Linkers Different chemical entities, like peptide, ester, and glycosidic bonds, can be cleaved by certain enzymes. The specificity of these enzymes for their substrates enables the use of these structures as linkers in drug conjugates. Peptide bonds have high stability in circulation, as proteolytic enzymes have only very low activities in blood and serum, owing to present inhibitors and the unfavorably high pH [63]. After cell entry by receptor-mediated endocytosis, endosomal and lysosomal proteases, like cathepsins, are able to degrade peptide bonds. Therefore, peptide linkers combine the high systemic stability of amide bonds with the rapid intracellular release by specific enzymatic cleavage. Moreover, these proteases are overexpressed in some types of cancer [64]. A wide range of short peptide sequences has been identified, which are prone to cleavage by these proteases [65]. The amino acid sequence Gly-Phe-Leu-Gly (GFLG) and the shorter Val-Cit linker have been extensively used in antibody–drug conjugates [66]. These linkers can also be easily introduced into peptides by solid-phase peptide synthesis. β-Glucuronide glycosidic bonds can be cleaved specifically by the enzyme β-glucuronidase, which is present in lysosomes and overexpressed in some tumors [67]. The design of β-glucuronidase-responsive antitumor prodrugs has been reviewed recently [68]. Also, a glucuronide-containing DOX prodrug has been connected to the plasma protein albumin by maleimide–cysteine linkage [69]. The albumin–DOX conjugate has shown similar antitumor effects in vivo compared with free DOX, while avoiding severe side effects. Prodrugs like this could also be used for peptide–drug conjugates. Ester and carbamate bonds can be hydrolyzed enzymatically after internalization by esterases and cytochrome P450 [70,71] or by the acidic pH value in lysosomes. These structures can be introduced during peptide synthesis [72,73], but because they are prone to hydrolysis in serum, their extracellular stability needs to be carefully controlled. Carbamate linkers of varying stability have been used to connect the tumor-targeting peptide SST with the toxophore camptothecin (CPT) [73]. The compounds displayed high plasma stability, but low cytotoxic effects on SSTR-expressing neuroblastoma cells, which was attributed to the high linker stability. Self-immolative Linkers The direct attachment of a drug to an enzymatic or chemical cleavage site can result in slow-release profiles, owing to a sterically hindered recognition of the cleavage site. Therefore, self-immolative or self-cleaving spacers were developed, which enlarge the distance between the original stimulus-responsive cleavage site and the drug. This enables the efficient release of a prodrug with a residual spacer structure. To avoid an altered cytotoxic activity of the drug, this structure has to be rapidly and spontaneously removed after linker cleavage to release the free drug in a traceless manner [12].


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PEPTIDE–DRUG CONJUGATES FOR TARGETED THERAPY Furthermore, the chemical functionalities present in the carrier and the drug limit the number of available linker structures that can be introduced to connect them. Therefore, the introduction of new functional groups is often necessary in order to use the cleavable linker of choice. The linker should have a traceless nature in order to prevent changes in the toxicity properties of the toxic agent [10]. Self-immolative linkers can be classified by the reaction mechanism, which causes the self-decomposition of the linker, including elimination-based and cyclization-based systems, which have been reviewed in great detail [74]. Structures based on 1,6-elimination received much attention in linkers containing enzymatic cleavage sites, like short peptides or β-glucuronidase-responsive bonds, in order to enhance the cleavage rate. In this case, hydroxy-containing or amine-containing drugs are linked by a carbonate or carbamate group to aniline or phenol, respectively, at the benzylic position. Enzymatic or chemical triggers initiate an electronic cascade releasing the free drug. The p-aminobenzyl alcohol (PABC) group was used to connect different small-molecule drugs by the cathepsin B-cleavable Phe-Lys linker (Scheme 1a) [75]. All compounds showed high stability in serum, whereas a fast cleavage was observed in the presence of cathepsin B. Prodrugs without a PABC spacer showed only slow-release profiles owing to the sterical hindrance of the enzymatic cleavage site. The release mechanism by cyclization represents an important alternative to the elimination-based self-immolative linkers. These structures release the free drug by rapid intramolecular cyclization. Stimuli-responsive cleavage releases a nucleophile, which intramolecularly attacks a carbonyl group or an electron-deficient C atom, from which the free drug is liberated as a leaving group. Jain et al. have used this concept to produce mutual prodrugs connected by reducible disulfide linkers that contained different selfeliminating spacers, depending on the functional groups present in the toxic agents [76]. These structures consist of ester, carbonate, or carbamate functionalities in the β-position to the disulfide bridge, which causes drug release upon disulfide reduction by thiol-assisted cyclization. One example is the connection of carboxy-containing drugs by ester bonds in the β-position to the disulfide linker (Scheme 1b). Disulfide cleavage releases a thiol group, followed by an intramolecular reaction to release thiirane and the free carboxy-containing drug. These linker systems could also be introduced into peptide–drug conjugates and greatly expand the variety of available structures for traceless drug release.

Toxic Agents and Conjugation Chemistry The third part of a peptide–drug conjugate is the toxic agent that is responsible for the cytotoxic activity of the entire smart delivery

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191

Scheme 1. Schematic representation of cleavage mechanisms of selfimmolative linkers based on 1,6-elimination (a) and thiol-assisted cyclization (b).

system. This molecule should be inactive in the conjugated form, but highly potent after intracellular delivery and release, with a well-defined mechanism of action. Additionally, it should possess functional groups that enable the attachment to the carrier peptide by a cleavable linker of choice. If the site of attachment is a chemical moiety that is important for its biological activity, this group will need to be released in a traceless manner. Because the chemical options present in the toxophore are limited, this is not always easy to achieve. If a functional group, important for the toxicity is modified, the fast and traceless release of the toxophore will have to be guaranteed. The use of chemical crosslinkers and the selective introduction of functionalities with the help of solid-phase peptide synthesis open up manifold possibilities for the incorporation of toxophores into peptides, which is illustrated in the next section by some specific examples. Toxic agents used in peptide–drug conjugates are most often well-established, classical chemotherapeutics, like DOX, daunorubicin (DAU), methotrexate (MTX), CPT, paclitaxel (PCT), and metalbased complexes. The unspecific uptake of these agents by cancer and healthy cells causes the severe side effects related to chemotherapy [77]. The modifiable functional groups present in the toxic molecule determine the possibility of linkage. Some options are exemplarily shown for DOX and MTX in Scheme 2. DOX exerts its biological activity primarily by DNA intercalation, inhibition of the topoisomerase II, and generation of free radicals [78]. It contains three different functional groups for a potential conjugation, which offer multiple possibilities for a chemical modification. The ketone group at position C13 can be used for the generation of a hydrazone linker by a hydrazine-containing crosslinker. This was shown by the derivatization of DOX with p-maleimidophenylacetic acid hydrazide, to afford a hydrazone-linked DOX intermediate (Scheme 2a, red) [79]. The thiol-sensitive maleimide group was coupled to a cysteine residue introduced into the carrier peptide NPY to give the respective hydrazone-linked conjugate. The hydroxy functionality at position C14 in DOX was used to form ester linkages. An example is the derivatization of DOX with glutaric anhydride and the subsequent reaction of the released carboxylic acid with an amino group of a breast cancer cell-selective carrier peptide, derived from phage display (Scheme 2a, blue) [80]. The ester-linked peptide showed a higher toxic effect on breast cancer cells than an amide-linked DOX conjugate. Moreover, in comparison with free DOX, the compound was more toxic on DOX-resistant cells and showed less toxicity on non-cancerous cells, indicating an improved therapeutic index. The primary amine in the sugar moiety is a third conjugation site of DOX. Although this group is important for the biological activity of DOX, small moieties still being attached to DOX after intracellular linker cleavage do not alter the cytotoxic activity [81]. Therefore, DOX was conjugated to the chemical crosslinker dithio-bis(succinimidyl propionate), which contains two N-succinimidyl-activated carboxy groups connected by a disulfide bridge that are prone to nucleophilic attack by the DOX amino group and an amine-containing carrier molecule (Scheme 2a, black) [82]. With this approach, DOX was linked to folic acid, binding to the well-known folate receptor that is overexpressed in different cancers [83]. Although 3mercapto-propionyl was still attached to the amino group of DOX after intracellular disulfide cleavage, the conjugate had a higher activity than free DOX on folate receptor-positive cells. This synthetic strategy could be easily transferred to peptide–DOX conjugates. In contrast to DOX, the primary target of MTX is the enzyme dihydrofolate reductase (DHFR). Binding of MTX results in an inhibition of the metabolism of folic acid, thereby leading to the


Scheme 2. Conjugation strategies used to connect DOX (a) and MTX (b) to peptides by different cleavable linker structures. (a) DOX was linked to a carrier peptide by hydrazone (red) [79], ester (blue) [80], and disulfide bonds [82]. (b) MTX was connected to a targeting peptide by the enzymatic cleavage site GFLG [86].

disruption of thymidylate and purine biosynthesis and the initiation of apoptosis [84]. MTX can be incorporated into peptides through its α-carboxy or γ-carboxy group (Scheme 2b). However, conjugation through the γ-carboxy group is more favorable because the α-carboxylic acid is important for binding to DHFR [85]. The stability of MTX under the conditions used for solid-phase peptide synthesis allows the selective and direct insertion of MTX by this technique. So, MTX was coupled to the N-terminus of the peptide sequence GFLGC, containing an enzymatic cleavage site for intracellular release and a C-terminal cysteine for further modification [86]. The purification with reversed-phase (RP)-HPLC allowed the separation of the γ-linked and α-linked MTX-GFLGC conjugates. Using the free thiol group of the C-terminal cysteine, both molecules were separately conjugated to a chloroacetylated carrier peptide, forming a stable thioether linkage. The cytotoxic effects of both conjugates were comparable with those of free MTX, indicating a fully preserved activity of the carrier-linked MTX. Furthermore, although MTX was connected by the α-carboxy group, its activity was sustained. This indicates that the conjugation of toxophores by functional groups, which are important for their toxic effect, is possible, when linker structures with fast and traceless release are used.

targeting. Table 2 summarizes promising analogues for tumor targeting of each peptide family.

Somatostatin-derived Peptide–Drug Conjugates

Peptide–Drug Conjugates

192

In addition to the role of the individual segments, peptide–drug conjugates as complete delivery systems contain more aspects. Figure 2 summarizes the most important features for all three compartments of a peptide–drug conjugate. Exemplary conjugates are classified depending on the peptide family that is used for tumor


Figure 2. Necessary key properties of each part of a peptide–drug conjugate.

The peptide SST binds to five different SSTR subtypes [87]. The SST2R and SST5R are overexpressed in various kinds of cancer, like breast, lung, and neuroendocrine tumors [88]. Most peptide–drug conjugates from the SST peptide family are octreotide analogues, and various linker structures have been used to connect different toxic agents to octreotide [89].


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PEPTIDE–DRUG CONJUGATES FOR TARGETED THERAPY Table 2. Sequences of tumor-targeting peptides with potential sites for modification or amino acid exchange marked in red and the expression of the corresponding peptide receptor in human tumors Peptide family

Tumor-targeting peptide

Somatostatin

Octreotide (Sandostatin®) H2N-fc[CFwKTC]T(ol) GnRH-III pEHWSHDWKPG-NH2 6 11 13 14 [D-Tyr ,β-Ala ,Phe ,Nle ]-BN(6-14) H2N-yQWAVβHFX-NH2 NT(8-13) H2N-RRPYIL-OH c(RGDfK) HSDAVFTDNYTRLRKQMAVKKYLNSILN-NH2 NAPamide Ac-XDHfRWGK-NH2 7 34 [Phe ,Pro ]-NPY YPSKPDFPGEDAPAEDLARYYSALRHYINLITRPRY-NH2

GnRH/LHRH Bombesin Neurotensin RGD VIP α-MSH NPY

Tumor expression

References

Breast, lung cancer, NETs

[87–89]

Prostate, breast, ovarian cancer

[98,99]

Lung, prostate, breast, colon, pancreatic cancer

[110,113]

Pancreatic, prostate, breast, colon, lung cancer

[118,119]

Glioblastoma, melanoma, breast, prostate cancer Colon, breast cancer, endocrine tumors Melanoma

[18,149] [17,127] [130,131]

Breast cancer, Ewing sarcoma

[27,29]

Ac = acetyl, β = β-alanine, BN = bombesin, c = cyclic, GnRH = gonadotropin-releasing hormone, f = D-Phe, LHRH = luteinizing hormone-releasing hormone, MSH = melanocyte-stimulating hormone, NET = neuroendocrine tumor, NPY = neuropeptide Y, NT = neurotensin, pE = pyroglutamic acid, VIP = vasoactive intestinal peptide, w = D-Trp, X = L-norleucine, y = D-Tyr.

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Gonadotropin-releasing Hormone-derived Peptide–Drug Conjugates Gonadotropin-releasing hormone receptors (GnRH-Rs) are particularly overexpressed in cancer cells, related to the reproductive system, like prostate, breast, and ovarian cancer [98]. Continued stimulation with GnRH agonists desensitizes GnRH-Rs and therefore suppresses their activity, which has antiproliferative effects on tumor cells. These antitumor properties can be enhanced by connecting toxic molecules to these GnRH agonists. Currently, conjugates based on GnRH-III are investigated because of their decreased endocrine effect and their potent antitumor activity [99]. In a comparative study, the anthracyclines DAU and DOX were conjugated to Lys8 of GnRH-III over different linkages [100]. DOX was connected by an ester bond using a glutaric acid spacer, while DAU was linked by a hydrazone-and-oxime bond using the C13 ketone group. Additionally, DAU was coupled to its amino group in the sugar moiety by the cathepsin B cleavage site Phe-Lys, containing a PABC self-immolative spacer. The in vitro cytotoxic activity of all conjugates on human cancer cell lines revealed higher toxicity for the ester-linked, hydrazone-linked, and enzymatic cleavage sitelinked compounds, compared with the conjugate coupled by the oxime bond. These findings were attributed to the lower tumor cell uptake and higher linker stability of the oxime-linked compound, leading to less release of toxic DAU. In general, oxime bonds are chemically [101] and enzymatically [102] stable moieties. Degradation in rat liver homogenate revealed no release of DAU from the oxime linker, whereas toxophore release was detected for all other investigated linkers. In order to increase the DAU release, an enzymatically cleavable GFLG spacer was inserted between the peptide and the oxime–DAU linkage [103]. Interestingly, the GFLG linker did not improve the in vitro cytostatic effect. In a similar approach, DOX was linked to the cell-penetrating peptide octaarginine by an oxime linker with an inserted disulfide linkage to increase the DOX release. This compound displayed an efficient release after disulfide reduction and exerted potent in vitro toxicity, although no comparative results with an only oxime-connected conjugate were presented [104]. However, because of the high stability, combined with a moderate toxic activity, the oxime-linked conjugate [Lys8(DAU = Aoa)]-



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The chemotherapeutic agent PCT blocks mitosis by inhibition of microtubule depolymerization [90] and was linked by an ester bond to the N-terminus of octreotide using a succinic acid spacer [91]. This conjugate retained the SSTR-binding properties of octreotide and showed comparable toxic activity with that of free PCT on SST2R-expressing and SST5R-expressing MCF-7 breast cancer cells. In contrast, no effect was observed on SSTR-negative CHO cells for the conjugate, whereas PCT alone exerted similar toxicities on both cell lines. Furthermore, the conjugate was evaluated in non-small-cell lung cancer A549 xenografts, in comparison with PCT and an octreotide conjugate coupled to two PCT molecules, both linked by ester bonds [92]. Remarkably, doubly PCT-linked octreotide exerted a stronger inhibition of tumor growth than the singly coupled PCT–octreotide and free PCT, whereas its toxicity was lower compared with PCT, showing that the ester linker offers stability and efficient release also in vivo. Furthermore, these studies underline the reduction of side effects by the use of tumor-targeting peptides. Doxorubicin has also been linked to different octreotide analogues by its C14 hydroxy group by an ester bond using glutaric acid [93]. One of these compounds was recently applied for targeting breast tumors xenografted into mice [94]. The growth of the SSTR-positive breast cancers was more strongly inhibited by the conjugate than by an equimolar dose of free DOX. Although the stability of the ester linker in different sera was quite low [95], no toxic effects could be observed. Camptothecin, which is a potent topoisomerase I inhibitor, has been linked to an octreotide derivative with an extended N-terminus by different carbamate linkers [73]. The stability of the CPT conjugates was varied by linking groups of differing nucleophilicity to the nitrogen atom of the carbamate linker. Compounds containing ethylendiamine and N-methylethylenediamine showed remarkable serum stability but exerted lower toxic effects on SST2R-expressing neuroblastoma cells than free CPT. Additional in vitro studies of these conjugates revealed preserved receptor binding [96], internalization, and efficient inhibition of a wide range of different SST2R-expressing tumors in vivo [97], although it was not compared with free CPT. These studies indicate that carbamate linkages are promising alternatives for ester bonds, especially for toxic agents containing modifiable hydroxy groups.

BÖHME AND BECK-SICKINGER GnRH-III (Aoa is aminooxyacetyl) was evaluated in vivo using healthy and colon carcinoma-bearing mice [105]. The conjugate showed significant tumor growth inhibition and no toxicity in healthy mice, in contrast to an unselective effect observed for free DAU. In order to further increase the toxicity and stability of this compound, several approaches have been employed. The replacement of Ser4 in GnRH-III with Lys(Ac) resulted in higher stability and enhanced in vitro toxicity and tumor growth inhibition in vivo [106]. Furthermore, the side chain of Lys4 was modified by the incorporation of fatty acids of different lengths [107]. The conjugate [Lys4(butyryl), Lys8(DAU = Aoa)]-GnRH-III, containing n-butyric acid, was the most potent molecule with an increased in vitro stability, toxicity, and binding affinity to GnRH-Rs. The cytotoxic activity of peptide–drug conjugates can be increased by attaching more than one toxic agent to the peptide carrier. Modifications of the introduced Lys4 and the native Lys8 in GnRH-III enable the coupling of two toxophores to the same carrier peptide. Two DAU molecules were incorporated in GnRH-III by oxime linkage to afford the conjugate [Lys4,8(DAU = Aoa)]-GnRH-III [108]. Furthermore, in a highly innovative approach, a GnRH-III analogue was developed, containing MTX at Lys4, linked by an amide bond, and DAU at Lys8, coupled by an oxime bond ([Lys4(MTX), Lys8(DAU = Aoa)]-GnRH-III) [109] (Figure 3a). Both conjugates showed higher in vitro toxic activity than the singly DAU-modified or MTX-modified analogues. Using a different drug design, another lysine residue was coupled to the ε-amino group of Lys8 in GnRH-III, which generated two amino groups available for the conjugation of

two drug molecules (DAU and/or MTX) [108,109]. In this approach, the compounds [Lys8(MTX-Lys(DAU = Aoa))]-GnRH-III (Figure 3b) and [Lys8(DAU = Aoa-Lys(DAU = Aoa))]-GnRH-III were synthesized and displayed similar toxic effects in vitro compared with the conjugates doubly modified with toxophores at Lys4 and Lys8. Although this approach is very promising, additional in vivo studies are necessary to compare the efficiency of the different doubly toxophorelabeled compounds. Bombesin-derived Peptide–Drug Conjugates The overexpression of bombesin receptors (BBRs) on various kinds of cancer facilitated the development of bombesin–drug conjugates [110]. The peptide [D-Tyr6, β-Ala11, Phe13, Nle14]-BN(6-14), which is a fast internalizing universal ligand with high affinity to all three BBRs [111], has been used for bombesin-derived drug conjugates [112]. In general, many bombesin analogues have been linked to various toxic agents by different cleavable linker structures, similar to the approaches discussed in the previous sections [113–115]. In addition, four tumor-targeting peptides with anticancer properties were linked by enzyme cleavable linkers [116]. Here, no toxophore is added, and the toxic properties of the peptides are used instead: a bombesin antagonist, an SST agonist, a vasoactive intestinal peptide receptor binding inhibitor, and a substance P antagonist were incorporated, all comprising effective antiproliferative properties. The peptides were connected by Lys-Lys linkers, which are prone to enzymatic cleavage by transmembrane proprotein convertases overexpressed in cancer [117]. Indeed, the incubation of the precursor peptide with trypsin revealed the efficient release of all antitumor peptides. Treatment of different cancer cell lines expressing the target receptors revealed the efficient inhibition of cell proliferation in all cases. Moreover, the growth of colon tumor xenografts was inhibited more efficiently by the linked peptides than by the individual peptides. This system could be further advanced by linking various peptide–drug conjugates that target different receptors in order to efficiently address tumors with heterogeneous expression patterns of the targeted receptors. Neurotensin-derived and RGD-derived Peptide–Drug Conjugates

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Figure 3. GnRH-III analogues linked to two toxic agents. (a) Conjugate 4 8 4 [Lys (MTX), Lys (DAU = Aoa)]-GnRH-III containing MTX linked to Lys by an 8 amide bond and DAU linked to Lys by an oxime bond [109]. (b) Compound 8 [Lys (MTX-Lys(DAU = Aoa))]-GnRH-III containing MTX and DAU coupled to the 8 α-amino and ε-amino group of another Lys, introduced at Lys , linked by amide and oxime bonds, respectively [109]. Glp = pyroglutamic acid, Aoa = aminooxyacetyl.


The concept of branched peptides for tumor targeting, which contain more than one peptide of the same kind to achieve multivalent binding and higher stability, was nicely demonstrated with analogues of NT and RGD. The NT1 receptor (NT1R) is overexpressed in various types of cancer like pancreatic, breast, colon, lung, and prostate cancer [118]. NT shows high affinity to NT1R, but low affinity for the other NTR subtypes, and therefore is an ideal peptide for tumor targeting. Because the C-terminal fragment NT(8–13) contains all elements necessary for receptor binding [119], it is most often used as carrier in peptide–drug conjugates [120]. A fluorescently labeled branched peptide containing four repeats of NT(8–13) showed an efficient intracellular delivery on surgical resections of colon and pancreas carcinoma [121]. In contrast, binding of labeled monomeric NT conjugates was not observable. Furthermore, the fluorophore was replaced by the thymidylate synthase inhibitor 5-fluorodeoxyuridine linked by a cleavable ester bond. The ester linker was found to be more labile in serum and cell culture supernatant compared with an MTX conjugate linked by an amide bond. Nevertheless, in toxicity studies avoiding premature toxophore release, the ester-linked conjugate showed higher in vitro toxic activity on different human cancer cell lines, compared


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PEPTIDE–DRUG CONJUGATES FOR TARGETED THERAPY with the free drug. In vivo experiments in xenografted mice also displayed stronger tumor growth inhibition by the conjugate [121]. The short peptide sequence RGD binds to integrins that are involved in the progression and metastasis of tumors and therefore are overexpressed in several types of cancer [18]. It was demonstrated that constrained analogues, embedding the sequence RGD, have a higher affinity for integrins [122]. Therefore, the cyclic sequence c(RGDfK), containing a lysine residue for further derivatization, is most often used for peptide–drug conjugates [123,124]. Scaffolds containing four c(RGDfK) peptides displayed much higher binding and internalization efficacy than monomeric versions and are therefore promising shuttle systems [125]. Recently, four c (RGDfK) monomers were bound to a cyclodecapeptide scaffold, which was linked by an ester bond to a Pt(IV) complex using an axial succinic acid ligand [126]. Comparison with a monomeric c(RGDfK)– Pt(IV) conjugate and the free Pt(IV) complex displayed the highest toxicity and uptake for the tetramer on integrin-expressing melanoma cells. Interestingly, also the tetrameric scaffold without the Pt(IV) complex displayed high cytotoxic effects, emphasizing that the strong antiproliferative properties of cyclic RGD analogues can be enhanced by conjugation to toxic agents. Peptide–Drug Conjugates Based on Other Tumor-targeting Peptides There are numerous additional tumor-targeting peptides that have a high potential as peptide–drug conjugates. Some of them are briefly discussed in the next section. The vasoactive intestinal peptide (VIP) binds to the VIP receptors (VIPRs), which are overexpressed in numerous types of cancer like colon cancer, breast cancer, and endocrine tumors [17]. VIP was especially used for targeted imaging applying radiolabeled VIP conjugates [127]. However, there are also a few examples for its use in peptide–drug conjugates. CPT was linked to an analogue of VIP by a cleavable carbamate bond [128]. The replacement of several amino acid residues by alanine stabilized the peptide, while its high VIPR affinity was retained. The introduction of a lysine at position 28 enabled the selective incorporation of the toxin, and the formed

conjugate displayed antiproliferative activity on breast cancer cells in vitro, although it was not compared with free CPT. Malignant melanoma have a very poor diagnosis owing to their high resistance against traditional chemotherapy [129]. The overexpression of the melanocortin-1 receptor in melanoma opens up the opportunity for directed targeting using toxophore-linked analogues of its natural ligand α-melanocyte-stimulating hormone (α-MSH) [130]. Shortened analogues of α-MSH, like NAPamide, have been applied as diagnostic markers using radiolabeled peptides [131] but also bear the potential for their use in peptide–drug conjugates to overcome melanoma drug resistance. A more recent system is the NPY–hYR system. Because the hY1R is overexpressed in breast cancer and Ewing’s sarcoma, these diseases can be addressed by NPY analogues linked to toxic agents [27]. Therefore, NPY was linked to DOX by a cleavable hydrazone and a stable amide linker [79]. Cell viability assays with hY1R-expressing neuroblastoma cells revealed a comparable toxicity of the hydrazone-linked conjugate to free DOX, whereas the amidelinked DOX–NPY conjugate exerted no toxic effect. The development of the hY1R-preferring peptide [F7,P34]-NPY [29] allows an even more specific delivery to these types of cancer [30,31]. Furthermore, hY2R are overexpressed in neuroblastoma cells, and for colon cancer, hY4R are suspected to be promising targets [132]. Peptide–Drug Conjugates in Clinical Trials Although no peptide–drug conjugate has gained approval yet, there are several compounds in clinical development [38]. Some peptide–drug conjugates containing cleavable linker structures now facing clinical trials are briefly discussed in the next section, and their structures are shown in Figure 4. Zoptarelin DOX (AN152, AEterna Zentaris, Quebec, Canada) [133] is built up by DOX linked to the D-Lys6 side chain of the GnRH-R agonist [D-Lys6]-GnRH-I by an ester bond using a glutaric acid spacer (Figure 4a). Stability assays in mouse and human serum showed that serum carboxylesterases can hydrolyze the ester bond [134], therefore leading to an unspecific DOX release in circulation. The addition of a carboxylesterase inhibitor significantly reduced

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Figure 4. Chemical structures of peptide–drug conjugates now facing clinical trials. (a) Zoptarelin doxorubicin (AN152, AEterna Zentaris) [133]. (b) G202 (GenSpera) [138]. (c) ANG1005 (Angiochem Inc.) [141]. Glp = pyroglutamic acid, Ac = acetyl.

BÖHME AND BECK-SICKINGER the ester cleavage and prolonged the half-life of the compound. Furthermore, the conjugate displayed potent in vitro and in vivo anticancer activity on various tumor types, while being less toxic and more effective than free DOX [135]. A phase II clinical trial for the treatment of GnRH-R-positive, advanced, or recurrent endometrial cancer evaluated the efficiency and safety of AN152 in a group of 44 patients [136]. The conjugate showed significant activity and low toxicity in patients, supporting the principle of peptide–drugtargeted chemotherapy. A phase III study for patients with metastatic prostate cancer was recruited at the time of writing [137]. The agent G202 (GenSpera, San Antonio, TX, USA) contains thapsigargin, which is a potent inhibitor of the sarcoplasmic/ endoplasmic reticulum calcium adenosine triphosphatase pump, linked to a prostate-specific membrane antigen-specific peptide (Figure 4b) [138]. Thapsigargin is specifically released in the vicinity of tumor cells through hydrolysis by the prostate-specific membrane antigen (PSMA), which is a carboxypeptidase overexpressed in prostate cancer and derived metastases [139]. G202 was shown to be completely stable in human serum, and thapsigargin is specifically hydrolyzed by PSMA [138]. Additionally, the conjugate exerted strong inhibition of tumor growth and high circulation stability in human prostate cancer xenografts and was more effective and less toxic than docetaxel, which represents a first-line agent in the chemotherapy of many cancers. The agent is currently assessed in multiple clinical trials, including a phase II study as a second-line agent in hepatocellular carcinoma [137]. The low blood–brain barrier (BBB) permeability for small-molecule drugs prevents the potential treatment of malignant gliomas, for example, with PCT [140]. Therefore, another promising peptide–drug conjugate, called ANG1005 (Angiochem Inc., Montreal, Canada), was developed, which consists of the BBB-penetrating peptide angiopep2 linked to three PCT molecules by cleavable ester linkers (Figure 4c) [141]. ANG1005 crosses the BBB by transcytosis mediated through the low-density lipoprotein receptor-related protein-1 receptors, which are often overexpressed in brain cancer [142]. In vitro studies with ANG1005 demonstrated comparable inhibition of tumor cell proliferation to free PCT and high activity against tumor growth in vivo with glioblastoma-bearing mice [141]. Moreover, the ester linkages were shown to be stable under cell culture conditions, preventing premature PCT release. ANG1005 was well tolerated and showed evidence of anticancer activity in two phase I trials in patients with advanced solid tumors [143] or recurrent malignant glioma [144]. A phase II study in brain metastases from breast and small cell lung cancer is currently running, and several additional phase II trials are recruiting patients [137].

Conclusion

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Peptide–drug conjugates present a novel approach for the selective delivery of drugs to tumor tissue. Peptides have many favorable properties, like a high target affinity, fast internalization, and low immunogenicity, which makes them very promising tumor-targeting agents. A very important and often underestimated factor in this complex delivery system is the choice of an adequate linker structure in order to efficiently release the toxic agents. There is a variety of different chemical moieties available with diverse release mechanisms. Especially ester bonds are commonly used, owing to their facile introduction and fast-release profiles. However, esters are often associated with a low stability in circulation, leading to the necessity of a detailed monitoring of the stability profile. The carbamate linker might be an alternative for hydroxy-containing drugs


owing to its higher serum stability. Hydrazone bonds are often used for carbonyl-containing drugs, although these structures are unstable in vivo when integrated in antibody–drug conjugates. Oxime bonds have been shown to be a stable substitute, which can be combined with enzymatic cleavage sites or disulfide linkages to potentially enhance drug release. Disulfide bridges are quite rarely used in peptide–drug conjugates, probably owing to the absence of thiol groups in most toxic agents. This problem could be overcome by the use of novel traceless linker structures and chemical crosslinking reagents that can be selectively integrated into peptides by solid-phase peptide synthesis [76]. Linker structures based on enzymatic cleavage are often used because of the stability in circulation and fast intracellular proteolytic cleavage. To evolve the choice of the best linker for a particular peptide–drug conjugate from a random pick to a rational selection, more comparative studies between different linker structures are essential in order to evaluate their in vitro and in vivo properties. The detailed knowledge gained for antibody–drug conjugates during their clinical development [145] can fuel and fasten the progress of peptide–drug conjugates. New promising approaches like the development of peptide–drug conjugates containing toxophores with different modes of action or multibranched ligands of one or different tumor-targeting peptides open up new opportunities. The use of already developed methods to efficiently stabilize peptides could improve their pharmacokinetic properties [146]. Furthermore, novel, more potent toxophores like tubulysines [147] or pyrrolobenzodiazepines [148] could greatly enhance their efficiency. With the first peptide–drug conjugates in clinical trials, this novel class of selective anticancer agents has a promising future and will help to pave the way to personalized medicine for tumor therapy. Acknowledgements We gratefully acknowledge the Graduate School ‘Leipzig School of Natural Sciences-Building with Molecules and Nano-objects’ (BuildMoNa) and the financial support from the Europäischer Fonds für regionale Entwicklung, the European Union, and the Free State of Saxony and the Research Academy Leipzig (RAL). Dr Kathrin Bellmann-Sickert is thanked for helpful revision of the manuscript.

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Tumor vasculature targeted photodynamic therapy for enhanced delivery of nanoparticles.

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pH-sensitive drug-delivery systems for tumor targeting.

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Application of chitosan-based nanocarriers in tumor-targeted drug delivery.

Drug delivery systems 5A. Oral drug delivery.

Drug delivery systems and combination therapy by using vinca alkaloids.

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