Design and Applications of Mono- and Multifunctional

Current Medicinal Chemistry, 2010, 17, 1255-1299

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Targeting v 3 Integrin: Design and Applications of Mono- and Multifunctional RGD-Based Peptides and Semipeptides L. Auzzas*,1, F. Zanardi2, L. Battistini2, P. Burreddu1, P. Carta1, G. Rassu1, C. Curti2 and G. Casiraghi*,2 1

Istituto di Chimica Biomolecolare del CNR, Traversa La Crucca 3, Li Punti, Sassari I-07100, Italy

2

Dipartimento Farmaceutico, Università degli Studi di Parma, Viale G. P. Usberti 27A, Parma I-43100, Italy Abstract: The outstanding physio-pathological role played by integrin receptors in living subjects motivates the enormous interest shown by scientists worldwide for this topic. More than twenty years of research has spanned across the structural and functional elucidation of these proteins and over their antagonism-based biomedical applications. The proof-ofconcept stage, aimed at identifying potent inhibitors, covered a decade of studies, and paved the way for a more advanced era of research where these antagonist molecules were thrown into the deep end of applicative studies. This review intends to summarize the major efforts conducted thus far and focuses on the design, synthesis and biomedical applications of cyclic RGD-containing v3 integrin antagonists, in both their small and macromolecular formats. In particular, Chapters 1 and 2 offer a comprehensive outlook on the rational basis for the design of integrin inhibitors, Chapter 3 chronicles the biological and medical applications of monofunctional RGD integrin ligands both in their monomeric and multimeric asset, and Chapter 4 illustrates the potential of RGD-based multifunctional systems in molecular medicine.

Keywords: Integrins, design of RGD ligands, cancer therapy and imaging, targeted drug delivery, multimodality, integrin targeted nanoparticles. 1. INTRODUCTION Research on integrin receptors undoubtedly stands among the most enthralling chapters of modern drug discovery. Since their identification more than twenty years ago [1], this ample glycoprotein family has captivated the attention of scientists interested in understanding their structure, function, cell biology and in vivo significance [2, 3]. These studies have opened an exciting area of research which continuously discloses novel opportunities for a myriad of medical applications. As their name suggests, these proteins are comprised of an integral transmembrane complex, which modulates the association between the extracellular matrix and the cytoskeleton. More than twenty different / heterodimeric integrins have been recognized so far, which result from the non-covalent combination of eighteen and eight subunits [2]. Both subunits are type I transmembrane proteins containing a large extracellular domain and a short cytoplasmic tail, which are connected together through a single transmembrane region. Most integrins, especially the v3 type, are able to bind to a wide variety of ligands, while only a few, such as 51, can recognize a single ligand. Natural integrin ligands include laminin, fibronectin, vitronectin, fibrinogen and fibrin, trombospondin, MMP-2 and fibroblast growth factor-2. Many of these ligands are also mutually recognized by other extracellular matrix and cell surface adhesion proteins. Some integrins – e.g. 41 – can also bind to cell surface receptors, such as Vascular Cell Adhesion *Address correspondence to these authors at the Istituto di Chimica Biomolecolare del CNR, Traversa La Crucca 3, Li Punti, Sassari I-07100, Italy; Tel: +39-079-2841225; Fax: +39-079-2841299; E-mail: [email protected] Dipartimento Farmaceutico, Università degli Studi di Parma, Viale G. P. Usberti 27A, Parma I-43100, Italy; Tel: +39-0521-905080; Fax: +39-0521905006; E-mail: [email protected] 0929-8673/10 $55.00+.00

Molecule 1 (VCAM1), to promote cell-cell adhesion. In response to extracellular chemicals and/or mechanical stress signals, integrin activation promotes either the intracellular assembly of cytoskeletal polymers and signaling complexes, or the engagement of extracellular matrix macromolecules and counter-receptors on adjacent cell surfaces. Through these events, integrins behave as mechanochemical transducers, and orchestrate a synergic cross-talk with other extracellular matrix constituents, providing anchorage for endothelial cells [3]. Most of the integrins are expressed in a low-affinity ligand-binding state. However, their activation status is regulated by the delicate balance of a bidirectional signaling mechanism which drives reversible changes in integrin affinity and avidity for their ligands [3]. Either extracellular or intracellular signals are responsible for long-range conformational changes in the quaternary structure of integrins which occur in an allosteric fashion, and are propagated from the integrin headpiece to the cytoplasmic domains (and viceversa). Upon extracellular binding (outside-in signaling), a conformational change to the high affinity conformation is induced, which further increases ligand binding and generates intracellular signals via structural modifications to the integrin cytoplasmic domain. In parallel, the lateral assembly and clustering of integrins within the membrane are promoted by biological ligands, which modulate further ligand binding by increasing integrin avidity. The clustering mechanism seems to be required for the outside-in signaling and, in fact, all biological ligands are multivalent. One or several major intracellular pathways may be activated, depending on the ECM context, by subsequently recruiting non-receptor tyrosine kinases, such as the focal adhesion kinases (FAKs) and the Src families. These regulate, in turn, downstream signals via mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways, which are known regulators of cyclin-dependent kinases (CDKs) and cell progression. Loss of integrin ligation inhib© 2010 Bentham Science Publishers Ltd.

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its these events, and unligated integrins can indeed actively initiate apoptosis. This form of cell death is independent of stress response and death-receptor activation, but dependent on caspase-8 activation, and is referred to as IntegrinMediated Death (IMD). Other factors trans-activated by integrins include Met, Platelet-derived Growth Factor Receptor, Vascular Endothelium Growth Factor Receptor, Epidermal Growth Factor Receptor, Integrin Linked Kinase (ILK), Protein Kinase B (PKB/Akt), and Nuclear Factor kappa B (NF-B). The activation by signals within the cell (inside-out signaling) stabilizes integrins in a conformation that increases the affinity for their ligands, hence tuning the degree and kinetics of cell adhesion. Several cytoplasmic proteins (e.g. talin, vinculin, kindlins) have been found that are involved in this mechanism. Interestingly, outside-in and inside-out signaling mechanisms may occur simultaneously and reinforce each other. In such a way, integrin adhesiveness is controlled through the modulation of their affinity and avidity for ECM ligands. Depending on the endothelial cell local environment, the ability of cells to proliferate, migrate or die may therefore be regulated [3-5]. The impressive, multifaceted functional diversity ascribed to integrins has been translated into a wide variety of biological processes, including inflammation, innate and antigen specific immunity, haemostasis, wound healing, tissue morphogenesis, and regulation of cell growth and differentiation [6]. Accordingly, specific integrin dysregulation phenomena have been linked to the pathogenesis of many disease states, incessantly offering opportunities in the development of new drugs for a number of diseases including asthma, cancer, epilepsy, hypercholesterolemia, inflammatory bowel disease, osteoarthritis, osteoporosis, schizophrenia, thrombosis, and vascular diseases. The discovery of the structural basis of the recognition between integrins and their natural ligands by means of short amino acid sequences [7], together with the crystallographic analysis of integrin v3 in complex with a high-affinity RGD ligand [8], have provided a breakthrough for the rational design of class-selective integrin inhibitors [9-11]. The most significant advances in this field have led to the development of agents that have reached clinical applications, such as drugs targeting IIb3 integrin on platelets for inhibiting thrombosis [9], inhibitors of v3 and v5 integrins against angiogenesis, cancer, and bone resorption [10a-c,11], and of 2 and 4 integrins on leukocytes for treating autoimmune diseases and other inflammatory disorders [12]. Advances on the design of integrin antagonists have been systematically described by several reviews and accounts [9,11], mostly dealing with the design of the most popular peptidic or peptidomimetic small molecules targeting IIb3 and v3 integrins. However, continuous efforts are being made to offer novel structural, functional, and biological knowledge for developing more effective and selective antagonists, as well as for improving the use of currently available ones. Several interesting examples of rationally designed RGD-based macrocyclic ligands targeting v3 integrin have been discovered in the recent past by the efforts from academic research. Chapter 2 aims at offering a comprehensive outlook on the rational basis for the design of integrin inhibitors, bringing up to date the existing inventory

Auzzas et al.

of v3 integrin antagonists based on RGD-peptides and semipeptides. Paralleling these advances, mono- and multimeric integrin ligands have served as biochemical tools for understanding the mechanism of integrin targeting and uptake on a cellular level and, they have risen the question if dimeric and multimeric inhibitors may display higher receptor binding affinity and avidity in vivo, improved cell targeting, and cellular endocytosis (Chapter 3) [13]. Capitalizing on the modern concept of polypharmacology [14], these findings have opened the prosperous age of the integrin antagonistcontaining multifunctional systems (Chapter 4) [15]. An exceptional number of recent contributions illustrate the potential of integrin ligand-containing multifunctional systems committed to a broad spectrum of biomedical and pharmaceutical applications, which spans from cell-specific drug and gene delivery, molecular imaging and biomarker detection, across to medical engineering of biologically inductive materials. Multifunctional systems containing an RGD motif are mostly represented, which combine a second bioactive entity under the shape of a conventional bioconjugate (polymers, dendrimers, peptides, lipopeptides), or complex supramolecular particles (polymeric micelles, artificial membranes, particulate systems such as liposomes, non-viral gene vectors, bacteriophage display particles, nanoparticles). 2. STRUCTURE-BASED DESIGN OF RGDCONTAINING V 3 INTEGRIN ANTAGONISTS 2.1. Historical Background Integrins bind to their endogenous ligands by recognizing short amino acid sequences on exposed loops [7,16]. Among these, the Arg-Gly-Asp (RGD) consensus motif is ubiquitous among a wide variety of ligands, and is recognized by cells expressing several v integrins (e.g. 1, 3, 5, 6, and 8), as well as integrins IIb3, 51 and 81 [7]. This epitope is present in a class of potent integrin inhibitor proteins, the snake venom disintegrins, among which the cystein-rich kistrin, echistatin, ornatin, and decorsin are included [17]. Bearing the RGD motif at the tip of a mobile but structurally controlled loop, these proteins show an extremely varied selectivity and potency in targeting RGD-recognizing integrins, and may behave as potent inhibitors of platelet aggregation but also of angiogenesis. A number of disintegrins have been identified and their structure elucidated in solution as well as by X-ray, offering the earliest suggestion that a proper restriction of the RGD flexibility can lead to integrin inhibition [17]. Historically, the search for selective antagonists focused on IIb3 integrin (i.e. the platelet fibrinogen receptor involved in thrombosis) and v3 integrin, a promiscuous receptor involved in the activation of endothelial cells during tumor angiogenesis, and of smooth muscle cells during proliferation. Before the first integrin was available in a crystalline form [8], essential contributions in the design of integrin antagonists came from peptide chemistry, which helped in defining the tridimensional arrangement required by the RGD sequence for achieving selectivity towards integrins IIb3 and v3. Significant results were obtained with constrained peptides embedding the RGD sequence within a

Integrin-Targeting RGD-Based Peptides and Semipeptides

Current Medicinal Chemistry, 2010 Vol. 17, No. 13

cyclic penta- or hexapeptidic template. Several groups contributed to this objective with pioneering achievements, providing detailed investigations by NMR conformational analysis and molecular dynamics simulations. In early studies, McDowell and colleagues at Genetech identified the synthetic chiral sulfoxide G-4120 (1) (Fig. (1)) as an IIb3 inhibitor comparable to the disintegrin kistrin [18]. A detailed NMR structural analysis of (1) revealed a local ‘cupped’ shape geometry of the RGD array, with the lateral chains of both Asp and Arg placed in an extended conformation. An extensive network of hydrogen bonds was found stabilizing the conformation of (1), which presented a type II' turn with D-Tyr and Asp at the i+1 and i+2 positions. This structural model was proposed as a useful ‘blueprint’ for the design of non peptidic IIb3 antagonists. How-

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ever, it was not specified if (1) was selective against this receptor. In parallel, a monumental work started by the group of Kessler led to the identification of the first RGD-based cyclic peptide selectively active against v3 integrin (c(RGDfV) (2), Fig. (1)) through a procedure of spatial screening on a stereoisomeric library of RGD-containing peptides [11b, 11c,19]. The conformational analysis of (2) in solution revealed a II'/ conformation for the peptidic backbone, with Gly in the central position of the -turn, and D-Phe at the i+1 position of the II' turn (Fig. (2)). The overall conformation allowed the lateral chains of Asp and Arg to adopt a unstretched arrangement, with a calculated distance between their C atoms of 6.9 Å. A similar conformation was also found by Burgess and co-workers in the cyclic RGD dimer O

HOOC O

S

HN

O

D

N H

G

NH

R

O

NH

HOOC

HOOC

O

O O

O

NH

NH2 NH

0.69 nm v3 50 nM

(2) c(RGDfV)

COOH

H N

O H N

O NH

N H

H N

O NH

N H

NH

H N

N H

NH2

NH

NH

IIb3 150 nM

HN

O

O H N

O

NH

O

H N

(1) G-4120

H2N

OH

O

O

NH

HOOC

H N

HN

NH2 NH

HOOC

O

O

O

O

N

Me H N

NH

N H

O v3 140 nM

(3) c(RGDRGD)

NH2 NH

v3 500 nM IIb3 2 nM

(4) DMP728

O N HN HOOC

O

O

O

O

NH

O (5)

O H N

NH

N H

O

N

v3 13 nM IIb3 45000 nM

NH2 NH

HOOC

NH O N H

Me O

NH

NH

H N

NH2 NH

v3 0.6 nM (6) IIb3 860 nM c(RGDf[NMe]V) EMD121974 (Cilengitide)

Fig. (1). Relevant RGD-based cyclic hexa- and pentapeptides. Here and hereafter, biological activity data are IC50 values and are given as reported in the original papers.


Auzzas et al.

c(RGDRGD) (3) (Fig. (1)), which turned out to be a good inhibitor of v3 integrin [20]. A kink centred around Gly was found in (3), with an average distance between the C of the Asp and Arg side chains of 5.8 Å. At the same time, Bach and DeGrado at DuPont Merck showed that the selectivity of hexacyclic peptides could be shifted from IIb3 to v3 by forcing the conformation of simple RGD-containing cyclic peptides from II' to I, as in (4) and (5) (Fig. (1)) [21]. As a consequence of the different twists, the N-Me-Arg side chain in (4) adopted a pseudoequatorial orientation, while the Arg chain in (5) raised above the plane of the backbone in a pseudoaxial orientation (Fig. (2)).

bridges with Asp150 and Asp218 in the subunit, together with a strong interaction between the Asp carboxylic group of the ligand and the Mn2+ ion at MIDAS (Metal-IonDependent Adhesion Site) in the subunit. The Gly residue lied at the interface between the and subunits, and was engaged in several hydrophobic interactions.

II'

D

D i i

R

G

G

(2)

Mamb NMeArg (4)

(6)

Arg

Gly

DAbu

R

Asp

Gly

Pro

Mamb

Asp

(5)

Fig. (2). Schematic representation of favored average conformers of peptides (2) and (4)-(6) [11c, 21, 22].

These early findings suggested that structural restrictions within the cyclic RGD backbone can lead to selective v3 antagonists, provided that a proper vectorial and spatial relationship between the Asp and Arg lateral chains is ensured. The discovery of the potent v3 inhibitor EMD121974 (Cilengitide (6), Fig. (1)) by Kessler in collaboration with researchers at Merck definitely pointed this concept out [22]. Selected from a small library of N-methylated analogues of (2), peptide (6) displayed a three-dimensional conformation in solution different from the II'/ array observed with the parent macrocycle. Compound (6) featured three turns, i.e. two inverse -turns with Arg and Asp at i+1 position, and a -turn with Gly at i+1 position (Fig. (2)). The distance between the C of Asp and Arg lateral chains in (6) (8.0-8.5 Å) strongly deviated from the one observed with other ligands [19]. The potential of Cilengitide (6) was soon recognized by various clinical programs (see paragraph 2.3), opening the era of the integrin inhibitor class as investigational agents for antiangiogenic and anticancer therapies [10a, 10b]. The isolation and crystallographic analysis of the extracellular segment of v3 integrin in complex with (6) offered the foremost opportunity to acquire the first clear picture of the RGD binding mode (Fig. (3)) [8]. The examination of the 3D structure of (6) bound to v3 integrin revealed a conformation featuring an inverse -turn centred on Asp, and a distorted II'-turn with Gly and Asp at the i+1 and i+2 positions, respectively. A distance of 8.9 Å between the Asp and Arg C and an almost extended conformation of the RGD motif were observed. The most important interactions between the ligand and the receptor involved the Arg guanidinium group of (6) which was held in place by salt

Fig. (3). Representation of Cilengitide (6) bound on v3 integrin and main interactions between the ligand and integrin (dashed bonds). The active site Mn2+ (MIDAS), together with ADMIDAS (Adjacent to MIDAS) and LIMBS (Ligand-Associated Metal Binding Site) are shown as white spheres. Data derived from the Protein Data Bank of the Research Collaboratory for Structural Bioinformatics (PDB code = IL5G), using MOE 2010.3, Chemical Computing Group Inc., for graphic elaboration [8a].

The X-ray analysis gave confirmation to the structureactivity relationships previously observed for cyclic peptide inhibitors of v3 integrin such as (2) and (6). The pharmacophore shared by v3 inhibitors was refined, and models of the preferential binding of peptidic and non peptidic ligands to v3 integrin over IIb3 were developed [23, 24]. In parallel, more knowledge concerning the structural biology of other integrins was acquired. The binding mode of RGDbased IIb3 antagonists was established through mutagenesis experiments [25] and crystallographic analysis of the platelet fibrinogen receptor [26]. The high degree of sequence identity between v3 and 51 integrins led to the development of homology models for the 51 receptor [27], suggesting the rationale for the design of selective 51 antagonists [28]. Despite their high v3/IIb3 selectivity, c(RGDfV) (2) and Cilengitide (6) inhibit another closely related integrin, i.e. v5 protein [29]. A homology model for this receptor was developed, together with some useful biostructural information to achieve selectivity over v3 integrin [29]. According to this theoretical model, dual RGD ligands share a common binding mode on the two v receptors. The two integrins were found to mostly differ in the region comprising residues 159-188 in the 3 subunit. A ‘roof’ was described for v5 integrin featuring Tyr and Lys residues, which would hamper the binding of compounds containing bulky substituents nearby their Asp-mimicking group. Because of this difference, a few inhibitors of v3 integrin displaying selectivity over v5 have actually been found [10d, 29], but specific inhibitors of v5 integrins have not



been described yet. These integrins play a strategic role in the angiogenesis process [30], activating different cellular pathways [31]. In addition, v3 and v5 integrin subfamilies, together with 51, are not generally expressed by epithelial cells nor by quiescent endothelial cells, but may become highly overexpressed in activated endothelial and metastatic cancer cells [32]. Until further evidence with highly selective inhibitors becomes available, the development of bispecific v3/v5 (and v3/51) inhibitors might be regarded as beneficial [33]. Since the discovery of Cilengitide (6), the search for inhibitors with improved pharmacokinetic properties compared to the peptidic lead has been a major concern [10a,10d,10e, 10i]. Following a ligand-based approach, peptidomimetic small molecules were designed by ‘delinking’ known RGDbased peptides and by bridging bioisosteric equivalents of Asp and Arg with non peptidic scaffolds [34]. This has led to the identification of selective low-molecular weight inhibitors of v3 integrin, and a few are under evaluation in advanced clinical trials. For details on this topic, the reader can refer to leading reviews [10a,10e,10i].

most popular constrained mimetics of natural amino acids, and their use as rigid scaffolds for inducing preferred conformations has been validated by successful examples of peptide mimicry [35]. In an early work, Kessler exploited traditional -turn inducers to forge macrocycles based on azabicycloalkane and spyrocyclic systems ((7)-(9), Fig. (4)) [36]. By replacing the D-Phe-Val sequence of c(RGDfV) (2), these scaffolds were expected to occupy the i+1 and 1+2 positions of a II'-turn in (7) and (9), thus affecting the flexibility of the -turn within the RGD array. While the incorporation of the spyro moiety produced the desired II'/ arrangement, a distorted II' arrangement with Gly in the i+2 position was observed with (7). Semipeptide (8) displayed the / arrangement typical of cyclic hexapeptides. The scaffold occupied the i+1 and i+2 positions of a II'-turn, in front of a second II'-turn with Gly at the i+2 position. Compound (9) was completely inactive, while (7) displayed a poor activity, albeit with some v3/IIb3 selectivity. The most active and less constrained compound (8) was a fully promiscuous antagonist. H

S

In the following examples, the inhibitory activity presented is compared with the reference compound (RC) used as a positive control. For a deeper insight concerning the biological assays, the reader is invited to refer to the original works. 2.2.1. RGD-Based Semipeptides Turn-Inducing Motifs

Containing

Bicyclic

In the structure-based design of peptidomimetics, bicyclic heterocycles (azacycles in primis) stand among the

O

H

S

N

2.2. RGD-Based Semipeptidic Macrocycles Inhibiting v 3 Integrins In the search for drug-like inhibitors of v3 integrin, peptidomimetic small molecules might not be an exclusive option. Representative examples of semipeptidic v3 antagonists are nicely represented in the literature, where the RGD motif is appropriately constrained to match the v3 targeting by means of a non-peptidic and rigid turn-inducing motif, usually mimetic of natural amino acids or dipeptides. In principle, these hybrids are expected to benefit from better physicochemical properties compared to ‘more’ peptidic counterparts, especially in terms of drug-like (ADME) properties. In addition, a wide range of chemically diverse structures are supposed to be tolerated by the constraining motifs, in view of a limited involvement in the binding. Classic bicyclic, but also monocyclic scaffolds and simple linear tethers have been used to properly fold the RGD sequence within a macrocyclic template. Among the examples, turn inducing systems have often been designed to permit the incorporation of additional anchoring points tolerated upon binding to v3 integrin, anticipating their utility for grafting mono- and multi-RGD-based conjugated systems. In this section, a survey of the most relevant examples of this class is offered together with a description of the rationale that inspired their design and the key biostructural features accounting for their activity. A highlight of the synthetic strategies used for obtaining original turn inducing motifs is also provided.

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O N

Val

Arg O HN

O

Gly HN

Asp v3 280 nM IIb3 4.8.103 nM

(7)

(8)

Asp

Arg Gly

v3 43 nM IIb3 550 nM

N N Asp

O Gly (9)

O Arg v3 na IIb3 na

RC: c(RGDfV) v3 2.2 nM IIb3 800 nM

Fig. (4). Molecular structure of semipeptides (7)-(9) (RC = reference compound, na = not active) [36].

In a program started in the early 2000, Scolastico and colleagues used 5,6- and 5,7-fused azabicycloalkane amino acids to generate a stereoisomeric library of RGD pentapeptide mimetics (compounds (10)-(16), Fig. (5)) [37]. High affinity ligands were found within the collection. Particularly, macrocycles (14) and (15) emerged as low nanomolar binders of v3 and v5 integrins. The selectivity of the most active compound ST1646 (15) was also evaluated, and a complete lack of activity against IIb3 and 51 integrins was observed. In additional experiments (15) inhibited the proliferation of endothelial cells, displaying a significant antiangiogenic activity in vitro [38]. Furthermore, ST1646 (15) inhibited the growth of tumor xenografts induced by human ovarian carcinoma A2780, a form of tumor expressing both v3 and v5 integrins. It did not affect the proliferation of non-adherent v3/v5-expressing NB4 and HL60 human leukemia cells, this accounting for its selectivity for anchorage-dependent cells. Macrocycles (10)-(16) were assembled by conventional solid phase synthesis starting from the corresponding stereoi-


Auzzas et al.

O n

O

N

n

n

Arg

O Gly

HN

N

Arg

O

Asp

O

N

n

Arg HN

O

N

Arg

O Gly

HN

Asp

O Gly

HN

Asp

(10) n = 1 v3 154 nM v5 >103 nM

(12) n = 1 v3 202 nM v5 >103 nM

(14) n = 1 v3 14.3 nM v5 >103 nM

(11) n = 2 v3 491 nM v5 >103 nM

(13) n = 2 v3 461 nM v5 1.3 nM

(15) n = 2 v3 3.8 nM (ST1646) v5 1.39 nM

Gly Asp

(16) n = 2 v3 3343 nM v5 >103 nM v3 196 nM v5 0.11 nM Cilengitide v3 18.9 nM v5 0.13 nM

RC: c(RGDfV)

Fig. (5). Molecular structure and activity of RGD-macrocycles (10)-(16) [37].

(22) by reaction with benzaldehyde. The stereoselectivity and yield of the alkylation reaction could be modulated and optimized by varying the base and/or using coordinating metals as additives, efficiently delivering both isomers (25)/(27) and (26)/(28) as a mixture of easily separable epimers. In the case of imine (29), isomer (30) was preferentially obtained.

someric collection of azabicycloalkane scaffolds [39, 40]. Conformational analysis of macrocycles (10)-(16) revealed a strong dependence of the overall conformation of the cyclic peptides on the lactam ring size and stereochemistry. Particularly, compounds (14) and (15) (ST1646) showed preferred geometries featuring an almost extended conformation of the RGD sequence, and an orientation of the Asp NH group suitable to maintain almost the same interactions observed in the crystalline complex of Cilengitide (6) with v3. An equilibrium of two main conformations was detected for both macrocycles. In the first, an inverse -turn at Asp and a distorted II'-turn at Gly-Asp were detected. The second conformation displayed a I-turn at Pro-Arg together with an inverse turn at Asp. The average distance observed between Arg and Asp C was 8.8 Å in the case of (14), and 8.5 Å in (15). Docking of ST1646 (15) on the binding site of v3 integrin revealed ligand-receptor distances and interactions very close to those observed with Cilengitide (6) [38].

Cyclopeptide (20) emerged as a nanomolar antagonist of both v3 and v5 integrins comparable to ST1646 (15). NMR analysis revealed a rigid conformation of the scaffold embedded in (20), which displayed the carboxylic and benzylic substituents in a pseudo-axial orientation. Two preferred arrangements highly stabilized by H bonds were found within the RGD motifs. In the first, an extended RGD conformation was observed, and the cyclopeptide geometry was stabilized by a -turn centered on Asp, and a -turn on ProArg. In the second, this arrangement was shifted, and Gly was detected at the center of the -turn, combined with a turn displaying Gly and Arg at the i+1 and i+2 positions. Docking of (20) at the v3 binding site was complicated by the presence of three different conformations, (Gly)/ (GlyAsp), Inv(Asp)(Gly-Asp), and Inv(Asp)/(ProArg). While docking of the first structure resulted in the loss of all interactions between ligand and subunit, the conformations containing an inverse -turn on Asp produced topranked poses conserving the main contacts observed in the X-ray crystal structure with Cilengitide (6). In these poses,

In order to broaden these findings, the same group prepared new analogues featuring a quaternary stereogenic center and a hydrophobic benzyl group within the turn-inducing scaffold, as in compounds (17)-(20) (Fig. (6)) [41]. The new macrocycles (17)-(20) were assembled in solution by incorporating the corresponding scaffolds, (25)-(28) and (30)-(31), in the peptide sequence (Scheme 1) [42]. These were prepared by benzylation of imines (23)-(24), in turn easily obtained from the corresponding lactams (21)O n Ph

N

N O

O Gly

Asp

(17) n = 1 v3 v5 (18) n = 2 v3 v5

Arg

Arg

Arg O

HN

O

O N

75.7 nM 325.6 nM 190.4 nM 221.9 nM

Gly

Ph HN

HN

Asp

(19) v3 154.2 nM v5 242.6 nM

Fig. (6). Molecular structure and activity of RGD-macrocycles (17)-(20) [41].

Gly

Ph Asp

(20) v3 6.4 nM v5 7.7 nM

RC: c(RGDfV) v3 195.9 nM v5 0.11 nM Cilengitide v3 18.9 nM v5 0.13 nM


CO2But

N

PhCHO

CO2But

N

n

(eq 1)


n O

BnBr, (a) or (b); then NaBH4

(21) n = 1 (22) n = 2

(23) n = 1 (24) n = 2

CO2But

N

BnBr, LiHMDS or LiHMDS/Mg2+;

(eq 2) O

then NaBH4

(25) n = 1 (26) n = 2

(27) n = 1 (28) n = 2

+

[(26)/(28) 4:6] [(26)/(28) 1:9]

CO2But O

Ph NHBn

NHBn (29)

O NHBn

N

Ph

N

Ph

O

(b) n = 2: LiHMDS, -50 °C NaHMDS, -78 °C to rt

O

CO2But

N

NHBn

CO2But

N

n

+

Ph

N

(a) n = 1: LiHMDS, DMPU, -78 ° C to rt [(25)/(27) 8:2] LiHMDS, -50 °C [(25)/(27) 9:1]

Ph

n

O Ph

NH2

CO2But

N

1261

(30)

(9:1)

(31)

Scheme 1. Synthesis of the turn-inducing motifs (25)-(28) and (30)-(31) [42].

the complex was stabilized by an interaction between the aromatic ring of (20) and the Tyr122 residue.

the cis-configured prolines (41) and (42) to aldehydes (46) and (47), reaction with benzylhydroxyl amine led to the cycloaddition products (48) and (49) with high regio- and stereoselectivity. The same protocol led to isoxazolidines (50) and (51) starting from the corresponding transconfigured prolines (43) and (45). Cleavage of the oxazolidine ring with H2/Pd-C led to intermediates (52), from which cyclic peptides (32)-(39) were obtained.

In pursuing this program, a small set of macrocycles containing a hydroxymethyl side chain were also prepared (Fig. (7), (32), (34), (36), and (38)). This moiety was envisaged as a potential anchoring group to be exploited in the design of multifunctional agents for diagnostic and therapeutic purposes [43] and alkyl amide analogues (33), (35), (37), and (39) were thus prepared in parallel as probes.

Compound (36) and its corresponding valeroyl amide (37) emerged as promising ligands. Additional experiments on cellular models showed that (36) was an efficient v3/v5 antagonist affecting both cell adhesion and migration without displaying evident cytotoxicity. However, the detected activity was significantly lower than the reference compound ST1646 (15). NMR and computational-derived conformational analyses revealed strong similarities between (36) (Fig. (7)) and its unsubstituted relative (14) (Fig. (5)), which both showed an almost rigid arrangement. Preferentially, a Inv-(Asp)/(Pro-Arg) conformation of the peptide backbone was observed, featuring the RGD array in an extended geometry. The average distances between the C of Arg and Asp were 8.99 Å and 9.2 Å, respectively. Docking (36) on v3 integrin produced top-ranked poses consistent with the interactions between Cilengitide (6) and v3 binding site. However, the presence of lesser populated conformations detected by NMR were deemed to affect negatively

The homoSer-Pro dipeptide mimetic scaffolds embedded in (32)-(39) were prepared through a validated synthetic procedure based on a highly regio- and stereoselective 1,3dipolar nitrone cycloaddition, as depicted in Scheme 2 [44]. Starting from the protected lactol (40a), cis-allyl and transhomoallyl prolines (41) and (43) were obtained by a BF3·OEt2-promoted reaction of the corresponding alkyl stannane and cuprate, respectively. One-carbon homologation of allyl proline (41) by oxidative cleavage of the double bond, followed by a Wittig reaction with methyltriphenylphosphonium bromide and BuLi, led to the corresponding cishomoallylproline (42). trans-Allyl proline (45) was prepared by two sequential Wittig reactions starting from lactol (40b). Two-carbon homologation with triethyl phosphonoacetate and KH led to (44), which was reduced to aldehyde and then methylenated to the desired proline (45). After conversion of O n

N Arg

R O

Gly

HN Asp

(32) n = 1, R = OH

v3 1816 nM v5 na

(33) n = 1, R = C5H11CONH

v3 nd v5 nd

(34) n = 2, R = OH

v3 88 nM v5 929 nM

(35) n = 2, R = C5H11CONH

v3 nd v5 nd

O n

N Arg

R O

Gly

HN

RC: c(RGDfV) v3 3.2 nM v5 7.5 nM

Fig. (7). Molecular structure of macrocycles (32)-(39) (nd = not determined) [44].

Asp

(36) n = 1, R = OH

v3 53.7 nM v5 205 nM

(37) n = 1, R = C5H11CONH

v3 114 nM v5 234 nM

(38) n = 2, R = OH

v3 460 nM v5 334 nM

(39) n = 2, R = C5H11CONH

v3 na v5 na


Auzzas et al. 1. 9-BBN 2. Swern

SnBu3 CO2But

N BF3 (eq 1)

RO

N

.OEt

2

Boc

Boc

(41) cis/trans 66:34

CO2But

Boc

CO2But

N

3. Ph3P=CH2

(42)

MgBr

(40a): R = Me

CO2But

N

CuBr.Me2S, BF3.OEt2

Boc (43) trans/cis 95:5 O K

EtO EtO (eq 2)

RO

N

CO2Et

P

EtO2C

CO2But

1. LAH 2. Swern

CO2But

N Boc

Boc

(eq 3) (41) or (42)

N

n

BnNHOH

CO2But

OHC

n

(45)

CO2But

N

(43) or (45)

(46) n = 1 (47) n = 2

n

CO-Arg(Pmc)

N

O

Bn (48) n = 1 (49) n = 2

Bn (50) n = 1 (51) n = 2

Gly(OMe)

H2, Pd-C

n

N

O

(32)-(39) O

N Bn

N

CO-Arg(Pmc)

HO O

CO2But

N O

N

Gly(OMe) (48)-(51)

n

O

O O

(eq 4)

Boc

(44) trans/cis (9:1)

(40b): R = H

CO2But

N 3. Ph3P=CH2

NH2

(52)

(53)

Scheme 2. Synthesis of scaffolds (48)-(51) and their incorporation into macrocycles (32)-(39) [44].

the binding of (36) and were consequently held accountable for its lower activity compared to reference compound ST1646 (15). 2.2.2. RGD-Based Semipeptides Containing Monocyclic Turn-Inducing Motifs In the search for non peptidic RGD-containing cyclic systems based on II'/ arrangement with the -turn centred on Gly, classic dipeptide mimetics based on amino pyrrolidinone motifs were used by Kessler to graft macrocycles (54) and (55) (Fig. (8)) [36].

N

N

HN Asp (54)

O Gly

O Arg

v3 40 nM IIb3 2.8 μM

HN Asp

O Gly

O Arg

(55) v3 0.8 nM IIb3 8.5 nM

RC: c(RGDfV) v3 2.2 nM IIb3 0.8 μM

Fig. (8). Molecular structure of RGD-semipeptides (54) and (55) [36].

Unexpectedly, the conformation experimentally observed for (54) and (55) contained a II' turn with Gly at the i+1 position. Macrocycle (54) proved to be a moderate and selective antagonist of v3 integrin, while the more flexible (55) was a potent v3 inhibitor, albeit aspecific. The main difference between the two analogues was the orientation of the lactam bond in the turn-motif, which was found to be rotated by 180° in the two isomers, and involved in a H-bond with the receptor in the case of (55). This was further confirmed by molecular modeling of (55) on v3 integrin [23b], which revealed a H-bond between the guanidinium group of Arg214 and the carbonyl oxygen of the lactam. In docking (54), this productive interaction was missing, thus explaining its weaker activity in targeting v3 integrin. Gennari and Piarulli recently reported on the synthesis and binding evaluation of two 17-membered cyclic RGD macrocycles embedding Asp/diaminopropionic acid-derived bifunctional diketopiperazine moieties (compounds (56) and (57), Fig. (9)) [45, 46]. While the trans-disposed derivative (57) showed a nanomolar v3-affinity comparable to c(RGDfV) (2) and ST1646 (15), the cis-analogue (56) was less effective. A high structural pre-organization was observed in the conformational analysis of (57), which dispalyed a pseudo -turn with Arg at the i+1 postion that ensured



an extended RGD conformation (C(Arg)-C(Asp) = 9.3 Å). In (56), a non-extended RGD conformation was found (C(Arg)-C(Asp) = 7.4 Å), thus explaining the lower activity on v3 integrin. H N

O

O

N H

O

N H

O Arg

HN

HN

Gly

Asp

H N

Asp

O

O Arg Gly

(60)-(62) together with (63), the latter containing an additional Val residue (Fig. (11)) [51]. Given their flexibility, these compounds displayed an aspecific activity against v3 and IIb3 receptors, which was in the nanomolar range when a -amino acid sugar was incorporated within the cycle (compounds (60) and (61)). This result was supported by the analysis of the 3D arrangement of the most active compound (61), which was found to lie between the typical kinked conformation of v3-selective antagonists and the extended one required for targeting IIb3 integrin. OBn

OBn

(57) v3 3.2 nM v5 114 nM

(56) v3 3.9 μM v5 >104 nM

BnO

RC: c(RGDfV) v3 3.2 nM v5 7.5 nM ST1646 v3 1.0 nM v5 1.4 nM

BnO

OBn

HN

Guarna and co-workers exploited carboxy-substituted Dand L-morpholines to replace the N-Me-Val motif of Cilengitide (6) (Fig. (10)) [47]. Macrocycles (58) and (59) were thus synthesized by exploiting conventional solid phase synthesis [48].

HO

HN

OH

O

N

HN

HO

O Asp-Gly-Arg (58)

O O

v3 157 nM v5 15.1 nM

Asp-Gly-Arg (59)

RC: Cilengitide v3 18.9 nM v5 0.13 nM

Fig. (10). Molecular structure of RGD-containing semipeptides (58) and (59) [47].

While both macrocycles showed a similar activity against the v5 integrin, (58) was less active on v3 integrin compared to its isomer (59). Interestingly, the binding of (59) to v3 followed a two-site binding model, which was justified as being due to either a shift of the integrin conformation from a low- to a high-affinity conformation produced by the ligand [8a, 10e, 49], or the different binding of (59) to both active and inactive integrin forms [50]. Conformational analysis revealed distinct structural arrangements for (58) and (59), as a consequence of the different conformation of the peptide bond between D-Phe and the morpholine scaffold. Particularly, in the docking analysis the semipeptide (59) adopted a cis conformation, providing the RGD sequence with an arrangement similar to that observed in the v3-Cilengitide complex. Sugar-based scaffolds could serve as flexible turninducing motifs to replace the D-Phe-Val motif of c(RGDfV) (2). Pyranoid and furanoid sugar - and -amino acids were used by Kessler and Overhand in the design of compounds

OH

O

O

Val-Asp-Gly-Arg (63) v3 98.1 μM IIb3 32.2 μM

RC: c(RGDfV) v3 2.5 nM v5 320 nM IIb3 8.103 nM

O

v3 32.6 nM v5 21.0 nM

HN

O

(62) v3 1.49 μM IIb3 0.38 μM

N

HN

Gly

(61) v3 25 nM v5 >104 nM IIb3 13.4 nM

Asp-Gly-Arg O

Arg Asp

Gly

(60) v3 150 nM v5 935 nM IIb3 720 nM

Fig. (9). Molecular structure of diketopiperazine-based RGDmacrocycles (56) and (57) [45].

O

O

Arg

Asp

OBn

O

O

HN

O

1263

Fig. (11). Molecular structure of sugar-containing RGD macrocycles (60)-(63) [51].

Following an analogous inspiration, -amino acid equivalents of furanoid carbasugars were exploited by Casiraghi and colleagues as tight RGD turn-inducers to generate four stereoisomeric variants of macrocycles (64) (Fig. (12)) [52]. The restriction of the macrocycle flexibility produced by the -amino acid motif was expected to force the RGD sequence to adopt a 3D arrangement strongly dependent on the stereochemistry pattern. At the outset, 1,3-cis disposed amino acidcontaining carbafuranoses were incorporated in the peptidic sequence leading to macrocycles (65)-(68) (Fig. (12)). OH

HO

OH

HO

5 3 1 O HN

HN

OH 5

O

Asp-Gly-Arg

Asp-Gly-Arg

(64)

(65):(5S) v3 132.9 nM v5 411.5 nM

HN

O

Asp-Gly-Arg (67):(5S) v3 51.4 nM v5 165.5 nM

(66):(5R) v3 712.7 nM (68):(5R) v3 44.7 nM v5 1.3.103 nM v5 125.4 nM RC: c(RGDfV) v3 195.9 nM v5 0.11 nM

Fig. (12). Molecular structure of carbasugar-containing macrocycles (65)-(68) [52].

1264 Current Medicinal Chemistry, 2010 Vol. 17, No. 13 (eq 1)

O (70)

H

O

HO H

OTBS

N Bn

O

(71)

N Bn

O

FmocHN

(74)

CO2H

O

(74)

(80:20) OH

FmocHN

CO2H (76)

(75) (eq 2)

HO

O (69)

H N Bn

HO

OH

OTBS

H

O

(73)

(72) HO

+

H

H

H N Boc

(73)

TBSOTf DIEA

H

TBSO

OTBS

TBSO

TBSO

SnCl4 -90°

N Boc (69)

O

O

O

O

Auzzas et al.

HO

OH

OH

O

+ O

FmocHN H ent-(70)

FmocHN

CO2H ent-(75)

CO2H ent-(76)

Scheme 3. Synthesis of N-Fmoc-protected turn-inducing motifs (75), (76), ent-(75), and ent-(76) [52].

The enantiopure amino acid scaffolds (75)-(76) and ent(75)/ent-(76) were prepared as N-Fmoc protected derivatives following an efficient protocol based on two pivotal C-C bond forming reactions (Scheme 3). An initial diastereoselective vinylogous aldol coupling between silyloxypyrrole (69) and (R)-glyceraldehyde (70) under the promotion of SnCl4 led to the unsaturated adduct (71). After simple manipulations of (71), aldehyde (72) was the substrate for the creation of the cyclopentane scaffold, which was performed by a highly productive silylative aldol reaction promoted by the Lewis acid/Lewis base system TBSOTf/DIPEA. The bicyclic isomers (73) and (74) were obtained in a diastereomeric ratio of 8:2, and were easily transformed into the requisite -amino acids (75) and (76). The enantiomeric couple ent-(75) and ent-(76) were prepared following the same protocol starting from silyloxydiene (69) and (S)-glyceraldehyde ent-(70). The four scaffolds (75), (76), ent-(75), and ent-(76) were incorporated as free alcohols in the RGD sequence by conventional solid phase synthesis, and then cyclized in solution to provide compounds (65)-(68). HO

HO

O HN Asp-Gly-Arg (77)

O

HN

HN Asp-Gly-Arg

(80)

v3 5.6 nM v5 4.6 nM

(78)

v3 7.7 nM v5 6.6 nM

A general improvement of the activity was observed against both v3 and v5 integrins, which was surprisingly almost irrespective of the configuration at the carbons bearing the amino acid functions. The NMR-derived solution structures of the most active compounds revealed highly organized arrangements of the peptide backbones, which displayed close kinks, such as inverted -turns, centered on Asp. These arrangements ensured the distance between the C of Asp and Arg in the range of 8.0-8.4 Å. Docking analyses predicted binding modes closely resembling the one observed for Cilengitide (6) in the co-crystal with v3. However, when one or two hydroxyl groups were present on the HO

O HN Asp-Gly-Arg

v3 7.2 nM v5 13.1 nM

A nanomolar activity was detected for isomers (67) and (68), bearing the hydroxy groups in 4,5-cis and 4,5-trans relationship, respectively. In order to understand the hydroxy groups and stereochemistry influence on the activity, simplified deoxy- and dideoxy analogues (77)-(83) (Fig. (13)) were prepared by inserting readily available scaffolds and featuring the amino and carboxy groups either in a 1,3-cis or 1,3trans relationship.

O

Asp-Gly-Arg (81) v3 4.6 nM v5 3.4 nM

O HN Asp-Gly-Arg

RC: c(RGDfV) v3 195.9 nM v5 0.11 nM

v3 35.6 nM v5 29.6 nM

(79)

HN

O


Fig. (13). Molecular structure of cyclopentane-based -amino acid turn promoters (77)-(83) [52].

HN

O




1265

(87), (88), and (89), which showed a picomolar activity for v3 integrin in the high affinity status. The NMR-derived conformational analysis detected a preferential conformation featuring an inverse -turn motif around Asp for the macrocycles containing a cis-disposed -amino acid motif (compounds (86)-(89), and (92)). Conversely, when a transconfigured -amino acid motif was present, the macrocycles were more flexible, and non-preferential orientations were detected (compounds (90)-(91), and (93)). It was speculated that for this subfamily of analogues, particularly in the case of (91) and (93), the loss of entropy upon binding might increase compared to ligands in a predefined arrangement and this, in turn, would lead to a significant loss of binding ability. All the macrocycles showed a distance between the C atoms of Asp and Arg residues in the 7.8-8.2 Å range, with the only exception of (90) (8.8 Å).

cyclopentane ring as in (65)-(68) and (79) (Fig. (12) and Fig. (13)), alternative high scoring binding poses were observed, which differed in the position of the ligand in the binding site and involved the OH groups at C4 and C5. In these poses, less active compounds such as (77) and (79) missed several of the stabilizing interactions observed with Cilengitide (6). Extending these findings, the same group considered the 4-amino proline (Amp) nucleus ((84), Fig. (14)) as an alternatively locked GABA-mimetic to forge RGD peptides of general formula (85) [53]. The presence of the nitrogen atom of the Amp unit was expected to be beneficial, offering a convenient anchorage point for the covalent binding of additional bioactive functions by means of diverse tethers. To verify this potential, a panel of Amp-containing macrocycles (86)-(93) was designed and synthesized featuring all the amino acid stereo-options. Next, the N-alkyl and N-acyl derivatives (87)-(89) and (91) were prepared as probes for predicting the binding affinity of potential c(RGD-Amp)-based bioconjugate systems.

The binding mode of the most active analogues was investigated by docking the representative compounds (86)(88) into the X-ray-derived v3 binding site (Fig. (15)). The results from the simulation revealed peptide backbone arrangements substantially consistent with the 3D in-solution structures observed by NMR analysis. Importantly, the topranking poses of (86)-(88) were found to almost maintain the relevant key interactions of Cilengitide (6) co-crystallyzed with the protein. Compound (86) was stabilized by a strong H-bonding contact between the NH in the aminoproline motif and Tyr178. Quite interestingly, the alkyl and acyl chains of (87) and (88) provided additional contacts for binding, pointing towards a large hydrophobic hollow rich with aro-

The Amp modules were synthesized from readily available 4-hydroxy L- and D-prolines, and subsequently used to forge the RGD cyclic motifs by conventional solid phase synthesis. The whole series of macrocycles (86)-(93) displayed a low nanomolar activity against both the v receptors (Fig. (14)). Low- and high affinity IC50 values were detected against v3, and often against v5, according to a classic two-site binding model [8a, 49]. In particular, exceptionally high affinity levels were observed with compounds -amino acid module constrained pyrrolidine ring

R

R

N

H2N

N CO2H

O

HN Asp-Gly-Arg

GABA module (84)

(85)

R

R

N HN Asp-Gly-Arg

H

N O

N O

HN Asp-Gly-Arg

O

HN IC50

Asp-Gly-Arg

(IC50h, IC50l)

IC50

(IC50h, IC50l)

(86) R = H

v3 4.4 nM v5 80.2 nM

(0.47, 23.7 nM) (30, 390 nM)

(90) R = H

v3 7.01 nM v5 22.8 nM

(5.12, 550 nM) (1.87, 53.70 nM)

(87) R = n-Heptyl

v3 2.84 nMa v5 36.1 nM

(0.08, 178 nM) (0.88, 62.3 nM)

(91) R = Bz

v3 174.0 nM v5 89.2 nM

(0.91, 327 nM)

IC50 v3 75.14 nMa v5 154 nM

(92)

(IC50h, IC50l) H

(0.18, 1350 nM)

N

(88) R = Propionyl

v3 5.60 nMa v5 94.0 nM

(0.03, 251 nM)

(89) R = Bz

v3 5.15 nMa v5 86.6 nM

(0.16, 600 nM)

O

HN Asp-Gly-Arg RC: c(RGDfV) v3 18.9 nM v5 0.13 nM

(93)

IC50

v3 530.0 nM v5 128.0 nM (IC50h, IC50l) (1.1, 840 nM)

Fig. (14). Molecular structure of proline-containing RGD-macrocycles (86)-(93). IC50h and IC50l are the IC50 values for high affinity and low affinity state of the integrin, respectively. a Average value estimated by extrapolation of the two-site model values [53].


Tyr166

Asp150 O

H

Arg214 H

O

O Tyr178

N

HN

O

H H2N

Auzzas et al.

H

NH

N NH

Asn215 H2N

O

NH2

O

HN NH

O

H

O O

O

O

O

Ser123

Mn2+ Asp218

(86)

Tyr166

Asp150 O

Arg214

Tyr178 O N

H

HN

O

NH

H

NH

HN

N

H

H

NH O

O

Asn215 H2N

O HN

O

O

O

NH

H

O

Mn2+ Tyr166

Asp150

Arg214 OH

O

O

Tyr178

N

H

HN

O

NH

H

NH

HN

N

H

H

NH O

O

Ser123

(87)

Asp218

O

O

Asn215 H2N

O HN

O

O

O

NH

H

O

Mn2+ Asp218

O Ser123

(88)

Fig. (15). Schematic 2D representation showing the extensive interactions between ligands (86)-(88) and the surrounding residues of the v3 receptor [53].

matic residues (Tyr178, Trp179, Phe 154 on the subunit, and Tyr166 in the subunit). Major binding poses were detected wherein their aliphatic tails formed favourable interactions with Tyr166 and Tyr178. Furthermore, (88) gained extra-stabilization by the presence of a H-bond between the propanoyl carbonyl and Tyr178. Overall, these additional stabilizations did not affect the binding on v5 integrin. Sewald, Reiser and co-workers considered cis--aminocyclopropanecarboxylic acid (-Acc) for grafting pseudopentapeptides containing both enantiomers of -Acc ((94) and (95), Fig. (16)) [54]. The synthetic strategy for the preparation of both enantiomers of -Acc relied on the cyclopropanation of N-Bocpyrrole (96) with methyl diazoacetate, under phenylhydra-

zine-activated catalytic copper(II) triflate promotion (Scheme 4) [55]. Both enantiomers of (97) were isolated after kinetic resolution by enzymatic hydrolysis with lipase L2. Following the oxidative cleavage of the double bond and a further oxidation to carboxylic acid (98), an Ndeformylation was performed with 2-diethylaminoethylamine (DEAEA), leading to the orthogonally protected Acc (99). Macrocycle (94) displayed a nanomolar v3 activity superior to the reference c(RGDfV) (2). Interestingly, these macrocycles were more active than c(RGDfV) on 51 integrin. Restrained molecular dynamics calculations were performed on (94) and (95) to explain the difference in their v3 activity (right side, Fig. (16)). The distance between the C atoms of Asp and Arg was found considerably shorter in



1267

2.3. Biomedical Applications of RGD-Based Peptides and Semipeptides

Fig. (16). Molecular structure of cyclopropyl-based macrocycles (94) and (95) [54].

Boc N

(96)

CO2Me, N2 Cu(OTf)2, PhNHNH2 then kinetic resolution (lipase L2) CO2Me

Boc N

H

CO2Me

Boc

1. O3, Me2S

N

H (97)

CO2Me

1. DEAEA 2. BnBr, NaHCO3

CO2H

2. NaClO2, H2O2

HN

CO2Bn

Boc

CHO (98)

(99)

Scheme 4. Synthesis of scaffold (99) [54].

(94) than in (95) (7.06 Å vs 8.26 Å). In the structure of (94), a -turn was observed with Gly in the central position, together with a pseudo -turn wherein (+)--Acc occupied the i+1 position. In this conformation, the - and -turns were at the same position as in c(RGDfV), although the types of turn were different between the two peptides. Compound (95) showed an inverted -turn, with Asp occupying the central position. This forced the RGD motif to adopt a more stretched conformation than in (94). Only a small number of additional examples have been reported to complete the scenario of RGD-based semipeptides [46, 56-62]. Interestingly, modern trends in organic chemistry are significantly affecting the search for innovative synthetic protocols to efficiently forge macrocyclic RGD-based structures. Intriguing examples are reported, in which the critical macrocyclyzation step was successfully performed by click chemistry [56, 63], ring closing metathesis [58], carbonylative macrolactamization [59], Heck reaction [60], and by means of metal coordination [61, 62], either in solution or by solid phase-supported synthesis.

Despite the impressive work dedicated to the identification of semipeptide analogues, Cilengitide (6) is the only investigational agent of this class that has been developed for clinical testing on cancer patients. In advanced clinical models, compound (6) holds a prominent position against certain brain cancer types that are usually treated with poor outcomes, demonstrating to be effective either as a single agent, or in combination with a second drug or radiotherapy [10a-c, 64]. However, there is still little evidence that (6) is effective in other forms of cancer. The growth inhibitory activity of Cilengitide observed in the clinic is likely due to a combination of multifaceted mechanisms. These might depend on whether the drug is administered alone or in combination, and include inhibition of angiogenesis, direct cytotoxic activity on tumor cells, increase of endothelial cell permeability, and inhibition of cell adhesion, migration and invasion [65]. In consideration of the pleiotropic nature of cancer, Cilengitide effects on molecularly selected human patients are still poorly understood, or even contradictory if compared with results from preclinical models [65]. Recent evidences in this context have been disclosed, which are drawing attention to a paradoxical proangiogenic activity of low doses of Cilengitide that can be observed in certain preclinical studies [66] The assessment of the potential benefits from integrin inhibitors since the early trials is of fundamental importance in the drug discovery process, provided that preclinical models are appropriately designed to decode the multiple effects of these agents and to understand the adaptation mechanisms of angiogenesis and cancer [65, 66]. The availability of novel therapeutic options may contribute to this issue. Among the Cilengitide analogues reviewed in this chapter, examples have been illustrated that display a similar or even improved v3-targeting compared to Cilengitide, often with clear-cut information concerning their selectivity. However, advanced preclinical investigations are not available to demonstrate whether these analogues might offer advantages pertaining to their in vivo properties, leaving an important question unanswered. 3. MONOFUNCTIONAL SYSTEMS: ‘(ONLY)RGD’CONTAINING DISPLAYS Since the early discovery of c(RDGfV) (2) and Cilengitide (6), an impressive deal of work has been devoted to the design and synthesis of monomeric and multimeric systems embedding the cyclic RGD motif. This flourishing field of research has found its expression in a vast scenario of applications, which encompass chemical biology and medicinal chemistry, pharmaceutical technology, material science, biomedical engineering, and nanotechnology. Among the monofunctional systems, a number of assemblies have been developed and based exclusively on a single copy (monomers) or replicates (multimers) of v3 ligands, among which cyclic RGD peptides, which have been modified for covalent linkage by conjugation, are mostly represented. Mono- and multi-RGD-presenting systems have been prepared based on simple conjugates with proteins, lipids, polymers, and even inert, biocompatible matrixes and sur-


faces. Together with these, more elaborated supramolecular assemblies have been manufactured as well, such as liposomes, polymeric micelles, and nanoparticles. All these arrangements have been useful for understanding the molecular basis of integrin targeting, inkeeping with the principles of multivalency [13, 67]. They also served as biological tools to clarify the mechanisms underlying integrin adhesion, clustering, internalization and endocytosis; in generating models of cell adhesion, spreading and proliferation; in developing alternative binding assays for systematic studies; and, finally, in elaborating biomaterials for cell and tissue engineering. Comprehensive review articles, book chapters and accounts exist covering this subject area, discussing the use of anti-angiogenic ‘only(RGD)’ integrin inhibitors in preclinical and clinical programmes. For in-depth view of this panorama, readers may refer to these dedicated contributions [10a, 10c, 10e, 68]. In this section, a survey of the most relevant examples and applications concerning monofunctional systems based on mono- and multimeric presentations of v3 ligands are illustrated, together with the principles that drove the research of monomeric constructs towards the development of multivalent presentations. 3.1. From Monomeric to Multimeric RGD-Based Systems Since the early studies, RGD-peptide polymer conjugates have served to demonstrate the ‘enhanced permeability and retention’ (EPR) effect [69, 70]. According to this phenomenon, biocompatible macromolecules larger than 40 kDa accumulate in tumors in higher concentrations than in normal tissues, organs and plasma. This is a consequence of the high vascular density in tumors, as well as the increased permeability and defective architecture of tumor vessels. Polymers, peptides, lipids, and particulate systems (e.g. liposomes, nanoparticles, viral gene vectors), have been developed as carrier systems for enhancing the affinity of the RGDmediated binding according to this principle, anticipating their utility as intelligent systems enabling the targeting of diagnostics and therapeutics [10d, 15]. In parallel with these findings, the objective of enhancing the affinity of RGDligands to their binding site has been furthermore pursued by designing multimeric RGD-containing systems, according to the general concepts of multivalency [13, 67]. Embedding multiple copies of the RGD recognition motif on a central scaffold, multimeric RGD systems have served to increase integrin affinity and avidity, facilitate their clustering [71], and induce an active integrin-mediated internalization [72]. For instance, a progressive increased affinity was observed by comparing the v3 inhibitory activity of a series of c(RGDfK)- and c(RGDfE)-containing monomers, dimers, tetramers, and octamers. Within the series, the octamers displayed the highest affinity [71]. Exploiting this concept, Kok and collaborators prepared multivalent derivatives of c(RGDfK) by covalent attachment of a short side chain of the peptide to the amino groups of HuMab, an irrelevant human monoclonal antibody that served as a protein backbone [73]. The peptide-based multivalent presentation (100) was obtained (Fig. (17)), which was expected to enhance the affinity and avidity of v3/v5

Auzzas et al.

integrins, while benefiting from the EPR effect. Radiobinding and displacement studies with human umbilical vein endothelial cells (HUVECs) showed an increased affinity of (100) as compared to the free peptide, with IC50 values ranging from 23 to 0.6 nM, depending on the amount of coupled RGD peptide per protein (an average of 23 coupled RGD peptides per HuMab antibody was determined by titration). Furthermore, (100) displayed a considerably higher affinity than c(RGDfV) (158 nM). Importantly, the RGD peptideprotein conjugate proved to be internalized and degraded by HUVECs, demonstrating for the first time that multivalent presentations can be internalized by endothelial cells, and suggesting the use of (100) as a selective carrier molecule for the delivery of therapeutic agents to angiogenic endothelial cells. Monomeric and multimeric RGD ligands linked together via aminohexanoic acid spacers were developed by the group of Kessler, who used them as probes for developing a cellfree binding assay for v3 and v5 receptors [74]. In the assay, integrin-functionalized artificial phospholipide bilayers tethered to biosensor surfaces were monitored by surface plasmon-enhanced fluorescence spectroscopy (SPFS). Among the examples, the dimeric compound (101) is depicted in Fig. (17). Surprisingly, dimer (101) showed higher binding affinity than its corresponding tetramer, albeit both resulted more active than the corresponding RGD monomer. It was reasoned that the dimer could bind two integrins at a time, while the steric hindrance of the tetramer kept it from binding more than two receptors. On the other hand, a prominent difference in the affinity of the RGD dimer and tetramer was not found in classic in vitro cell adhesion assays, and both multivalent probes showed higher affinity than the RGD monomer. Surely, the difference in the spatial organization of integrins on natural cells versus that on integrin-functionalized bilayers may certainly be taken into account to explain the discrepancy between the assays. In a more recent example, a regioselectively addressable functionalized template presenting four RGD pentapeptide motifs (RAFT, (102)) was prepared by the group of Coll, and compared with its monomeric counterpart [72]. It was demonstrated that (102) decreased the lateral mobility of v3 receptors, thus suggesting that four RGD motifs within a single template allowed integrin clustering. An active internalization of the tetrameric system (102) was observed, together with the natural, chlatrin-mediated endocytosis of the integrin. In addition to (102), biotinylated and fluoresceincontaining derivatives were designed as useful bifunctional reagents for integrin clustering characterization, and for applications in imaging and drug delivery (vide infra). As a further confirmation, Casiraghi and colleagues assembled the triazole-linked conjugate (103) by click chemistry starting from proline-based cyclopeptides featuring a Nalkylazide anchor [75]. This multivalent presentation was comprised of three proline-based RGD-cyclopeptide repeats built on a phenolic core, and showed higher binding affinity than its monomeric azide counterpart (IC50(v3) 0.2 nM, IC50(v5) 5.0 nM vs IC50(v3) 6.0 nM, IC50(v5) 1210 nM). Also, high affinity levels were found with hyperbranched and dendrimer-based integrin ligands targeting IIb3 or 41 receptors [76], demonstrating that the multivalent effect



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Fig. (17). Molecular structure of multimeric RGD-based displays (100)-(103) and fluorescent-labelled conjugate (104) [72-75,77].

might be extended to different subclasses of the integrin family. As a natural evolution of these studies, mono- and multimeric integrin ligand-based bioconjugates and macromolecules have been exploited for the generation of intelligent ‘integrin-hunting’ systems for various applications in imaging, diagnosis, drug delivery, and even gene transfection, which are the topics of the next section. Despite these advances, the complexity of integrin biology still leaves the response to monomeric ligands poorly understood. Basic aspects such as the endocytosis which follows the binding of RGD ligands to integrins are still unclear, and contributions are sometimes contradictory [77]. To date, the internalization of RGD-containing monomeric ligands is thought to be v3independent, and likely occurring via a fluid-phase pathway. During this process, integrin internalization is thought to be usually unaffected [72]. For instance, simple fluorescent-

labelled cRGD-based conjugates such as (104) (Fig. (17)) were recently found to bind v3 integrin, and subsequently internalized by endocytosis in cells expressing very high levels of this integrin [77]. It is still unclear whether the specific type of RGD peptide affects the level of endocytosis, or if RGD ligands have the same endocytotic index. 3.2. Monofunctional RGD-based Systems as Tools in Chemical Biology RGD-based peptides have been exploited as chemical probes for understanding the biological structure and function of integrins, either at a molecular or supramolecular level. For instance, RGD ligand-containing systems covalently linked to reactive functional groups and detection tags were used for addressing photoaffinity cross-linking studies leading to the identification of the main binding site for RGD ligands on v3 integrin [78]. Models have been developed


Auzzas et al.

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Fig. (18). Molecular structure of representative monofunctional RGD-based systems (105a,b)-(107) [79, 80, 82].

to shed light on the process by which ECM proteins or anchored RGD-containing peptides trigger signaling events leading to cytoskeleton remodelling, cell spreading and stabilization of focal adhesions. Given their purpose, these models are based on multimeric presentations of RGD ligands. Biotinylated cyclic-RGD peptides such as (105a), (Fig. (18)), were used by Kessler as probes to establish the optimal spacer length for the generation of models of integrin cell adhesion, clustering and focal adhesion selforganization [79]. From (105a), the lipid anchor-containing derivative (105b) was developed (Fig. (18)), which proved to be sequestered almost irreversibly by fluid membranes, mimicking the dynamic aspects of the self-organization of celladhesion. Integrins, reconstituted into phospholipide vesicles, bound to vesicles decorated with (105b), and formed regularly spaced bridges between the two kinds of vesicles. In a similar work, lipopeptide (106) (Fig. (18)) was exploited to functionalize supported membranes presenting accessible RGD sites [80]. Upon selective recognition by v3 and v5 integrins, a rapid spreading of endothelial cells occurred. Lipopeptide (106) was also incorporated into giant vesicles which were found to tether endothelial cells, promoting their clustering. Highly defined nanopatterns based on cyclic RGD peptides were developed by the same author, to understand the role of the distance between integrin ligands in the control of cell adhesion, and modulate the clustering of the associated integrins [81]. These studies revealed that a critical RGD density was essential for the organization of stable integrin adhesions, thus enabling an efficient cell spreading and formation of focal adhesions.

Polyamidoamine (PAMAM) dendrimers are watersoluble and biocompatible macromolecules that can be coupled to many effector molecules, including v3-selective ligands. In this context, Hill et al. [84] reported on the synthesis and characterization of a generation-5 PAMAM dendrimer conjugated to c(RGDyK) and labelled it with fluorescein ((108), Fig. (19)). The authors examined the binding properties and cellular uptake of (108) in endothelial cells and evaluated the binding to predentin of human tooth organs and dental pulp-like MDPC-23 cells.

Recently, an efficient method was developed to covalently immobilize RGD peptides to structured and unstructured surfaces in order to investigate the spreading behaviour of fibroblasts, and the formation of focal contacts in these cells [82]. RGD peptides were linked through an isothiocyanate anchor to amino-functionalized surfaces (e.g. (107), Fig. (18)), yielding biocompatible and functionalized sur-

In particular, the synthesis involved: (1) an initial partial coupling of several exposed amino groups in the dendron core, followed by (2) loading with fluorescein isothiocyanate (ca 4 dye molecules per dendrimer) to provide the construct with a detectable fluorescent probe, and (3) attachment of the c(RGDyK) to dress the nanoparticle with a v3-directing unit (ca 12-13 peptides per dendrimer). The cellular uptake

faces that proved to stimulate cell adhesion, focal contact formation and fibroblast normal growth. Similarly, a bioadhesive platform was recently developed by Vancso based on poly(methacrylic acid) brush layers functionalized with RGD sequences (not shown), and used to evaluate the effect of their chemical composition on the response of human osteoblasts [83]. All together, the above described cell-specific interaction models may be useful in the study of additional phenomena, such as cancerogenesis, in vitro growth of tissues, and mode of action of drug delivery systems. 3.3. Mono- and Multimeric RGD-Based Biomaterials Single and multiple ligand-containing polymer conjugates have been designed to generate biocompatible materials for applications in medical engineering, especially in the field of bone tissue regeneration. A comprehensive review on RGD-containing biomaterials was published a few years ago by Kessler [15d]. Therefore, only recent examples within this topic will be illustrated here.



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dependent binding. These results were confirmed by confocal microscopy analysis, which also revealed a timedependent internalization of (108) in the cytoplasm region of the cells. Finally, dendrimer conjugate (108) was incubated in an acellular tooth organ culture, showing that the binding was indeed clear and robust; and these experiments strongly support the notion that (108) selectively binds to the v3 integrin present in the predentin. One key point for improving osseous integration of metal-based implants is to render them osteopromotive by favoring the adhesion of osteoblasts on the surface of the specific metal involved. In 2005, Kessler et al. [85] introduced an easy and practical coating technique for titanium implants based on the use of a highly branched conjugate system carrying the c(RGDfK) v3-recognizing motif and four phosphonopropionic acid arms useful for Ti-attachment ((109), Fig. (20)). After preliminary assays on isolated v3 receptor which certified that conjugation was not detrimental for selective integrin binding, the macromolecule (109) was attached to titanium surfaces and the resulting coated material was evaluated in adhesion assays on MC3T3-E1 mouse osteoblast cells that express the v3 and v5 integrins. In this assay, the cell adhesion with Ti-coated (109) was about 62%, as compared to 16% for uncoated Ti-surfaces. The stability of coated Ti disks – a key issue for implanting materials in vivo – was also ascertained via -ray sterilization, thermal treatment and washing procedures, proving exceptional stability in all cases. Fig. (19). Schematic representation PAMAM-RGD conjugate (108) [84].

of

In a highlighting work, Ding and Spatz [86] exploited novel ordered and disordered gold nanopatterns supported on a bioinert PEG background and carrying c(RGDfK) integrin recognizers to study whether and how the nanoscopic order of spatial patterning of the integrin ligands influences osteoblast integrin focal adhesion (FA) and clustering. By a number of sophisticated measurements and assays, including atomic force microscopy, imaging and fluorescence micrographic detection, it was concluded that cell adhesion

fluorescein-labelled

of (108) was examined in MDPC-23 odontoblast-like cells via flow cytometric analysis, revealing a time-dependent uptake with a maximum reached after 6 h. Then, the specific binding of (108) toward HDMEC, HUVEC, and MDPC-23 v3-positive cell lines was examined, exhibiting RGDO

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Fig. (20). Molecular structure of phosphonate anchored c(RGDfK) peptide (109) [85].

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Scheme 5. Construction of alkenyl RGD-PEGDA macromer (112) [87].

strongly depended upon the average interligand spacing on the surface, as well as on particle density. In highly ordered systems, when local ligand-ligand spacing is 70 nM results in neither clustering nor FA formation. On the other hand, in a disordered environment, even at >70 nM spacing, some integrin clustering is observed, thus pointing that a disordered pattern may tolerate a much wider range of variation in local interligand spacing, culminating in a still positive cell adhesion scenario. Among the most attractive biomaterials for tissue engineering, hydrogels have been often exploited in consideration of their ability to retain water and soluble proteins, while maintaining chemical and osmotic equilibria with the surrounding media. Furthermore, their viscosity, elasticity, and stability properties can be controlled by design. A recent example is reported on design and synthesis of a biomimetic hydrogel based on c(RGDfE)-containing poly(ethylene glycol) diacrylate (PEGDA) macromers. As detailed in Scheme 5, Zhu and Marchant [87] fabricated the RGD-PEGDA hydrogel by photopolymerization of alkenyl macromer (112), in turn obtained by condensation of lysine-terminating cyclic peptide (110) with acryloyl-PEG-NHS (111). Cell culturing on hydrogels using human pulmonary artery endothelial cells showed that RGD-PEGDA hydrogels facilitate endothelial cell (EC) adhesion and spreading on the hydrogel surfaces, exhibiting significantly higher EC population in comparison with linear RGD counterparts. These studies established cyclic RGD hydrogels as attractive candidates for development of tissue engineering scaffolds with optimum adhesive strenght and ligand density. A more advanced study on the use of RGD-based hydrogels in tissue repair and regeneration was communicated by Mooney and colleagues [88], where a G4CRGDSPC peptide, cyclized through a cystine disulfide bond, was covalently conjugated to an alginate polymer to provide highly structured hygrogel matrices. The authors investigated the effects of the RGD ligand conformation (cyclic vs linear) on osteogenic differentiation of primary human bone marrow stromal cells (hBMSC) and D1 stem cells in 3D cultures, and

compared their response with that of committed MC3T3-E1 preosteoblasts. It was found that cyclic RGD-based matrices enhanced osteoprogenitor differentiation in three dimensions of hBMSC or D1 stem cells, whereas linear RGD counterparts did not; and this may be due to the superior integrin ligand binding affinity of cyclic RGD forms. Overall, these results emphasized the decisive role of designed, artificial RGD-based extracellular matrices in promoting stem cell differentiation and enhancing bone regeneration by transplanted cells. 4. MULTIFUNCTIONAL SYSTEMS: TAINING CONJUGATE DISPLAYS

RGD-CON-

A part from the prospective use of mono- and multimeric RGD-embedded v3/v5 integrin antagonists as therapeutically useful agents per se, such motifs may be exploited with even more success towards the construction of molecular or supramolecular conjugate vectors for drug and imaging targeting purposes. For example, RGD ligand-guided vectorization of therapeutic and diagnostic molecules to tumor tissues and metastases represents a valuable paradigm for improving current cancer diagnosis and therapy, enabling the specific delivery of imaging units and chemotherapeutics to malignant tissues thus increasing local efficacy, while limiting peripheral damages. In this Chapter the focus will be put on conjugate molecules – be they small molecules, macromolecules, or nanosized systems – wherein the RGD motif provides for the homing function of the vector, and the effector moiety is a toxic molecule (Section 4.1), an imaging probe (4.2), or combinations thereof (4.3). 4.1. Targeted Therapy Tumor-targeted medicines are drug delivery systems developed in contemporary molecular medicine to improve drug performance by overcoming classical therapy limitations, such as peripheral toxicity, unfavorable biodistribution, and rapid clearance. Peptide ligands containing the RGD triad can be conveniently coupled to conventional chemotherapeutics to provide drug delivery systems possessing tumor-targeting ability and efficacy against various can-



Several other RGD-4C-based conjugates have been developed [15b], including RGD-targeted prodrugs that require activation by tumor-secreted enzymes. Thus, de Groot et al. [91] developed a prodrug (not shown) consisting of bicyclic RGD-4C as the tumor homing motif, D-Ala-Phe-Lys tripeptide sequence as the plasmin specific cleavage site, and doxorubicin as the drug. Although the prodrug showed a decreased binding affinity as compared to the unconjugated RGD-4C peptide, it was still a potent v3/v5 integrin ligand in vitro. Also, the construct possessed plasminsubstrate properties, as demonstrated by the release of the drug upon incubation with plasmin. This prodrug showed plasmin-dependent cytotoxicity for endothelial cells and HT1080 fibrosarcoma cells in vitro. However, possibly due to its low solubility, this prodrug candidate was not advanced to in vivo experimentation.

cer types and multidrug-resistant tumors. RGD-based carriers can also pave the way for the use of other emerging classes of effector moieties such as therapeutic peptides and proteins, antisense or antigen oligonucleotides, siRNAs or entire genes [10b, 15b, 89]. In spite of the ever increasing progress of research in this field, a few validated RGD ligands and cytotoxic drugs have been exploited so far to furnish the conjugated constructs, the major effort being devoted to design and implementation of the assembled chemical platforms. A selection of seminal contributions are herein discussed, covering small molecule conjugates, macromolecular assemblies, and nanosystems and particles. 4.1.1. Small Molecules In a pioneering work, Arap and colleagues [90] conjugated, via a conventional peptide coupling procedure, the venerable chemotherapeutic agent doxorubicin (Adriamycin) to a small RGD-containing cyclic peptide – CDCRGDCFC (alias RGD-4C) –, giving rise to the covalent construct (113) (RGD-4C-DOX, Fig. (21)). The therapeutic efficacy of (113) was assessed against human MDA-MB-435 breast cancer xenografts in nude mice. Mice treated with doxo-conjugate exhibited smaller tumors, less spreading to regional lymph nodes, and fewer pulmonary metastases than did the mice treated with free doxorubicin. Furthermore, histopathological analysis revealed pronounced destruction of the tumor architecture and widespread cell death. Compound (113) proved to be less toxic to the liver and heart than free doxorubicin, while an unconjugated RGD-4C/doxorubicin combination was no more effective than free doxo. Since the MDA-MB435 breast carcinoma expresses v integrins, it is conceivable that the RGD-4C-DOX conjugate is able to deliver the drug into both the tumor vasculature, and the tumor cells themselves. O

OH

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Poor water solubility of RGD-4C-DOXO-type prodrugs, as well as significant problems associated with the chemical stability of the RGD-4C construct, wherein the reversible disulfide bridge connecting the cysteine components may lead to differently shaped acyclic or cyclic arrangements, all may have contributed to diminishing their appeal as RGDbased platforms. On the contrary, structurally homogeneous and well defined cyclic pentapeptide scaffolds such as Cilengitide c(RGDf[NMe]V) (6), or c(RGDf[NMe]K), proved to be optimal homing ligands, featuring good chemical and metabolic resistance, and water solubility as well. In a comparative study, Burkart et al. [92] designed and synthesized covalent constructs wherein doxorubicinformaldehyde – an active metabolite of doxorubicin – was conjugated to either RGD-4C or ([NMe]VRGDf-NH) ligands via a cleavable hydroxylamine ether tether. Whilst in its bicyclic forms the RGD-4C-doxorubicin conjugate showed a poor affinity for the v3 receptor and a significantly low water solubility, the cyclic c([NMe]VRGDf-NH)O

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Fig. (21). Molecular structure of doxorubicin-containing covalent RGD conjugates (113) and (114) [90,92].

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DOXSF conjugate (114) (Fig. (21)) displayed a low nanomolar IC50 of MDA-MB-453 tumor cell binding to vitronectin, thus demonstrating its superior v3 binding capability. At physiological pH, compound (114) spontaneously released the doxorubicin-formaldehyde active species by hydrolysis of the salicylamide linker; however, experiments suggested that the entire drug construct did not penetrate the plasma membrane, in contrast to native doxorubicin and doxorubicin-formaldehyde. Thus, a likely mechanism of action implies an initial binding to v3 integrin, followed by local release of the drug which can then penetrate the cell membrane. Besides doxorubicin, several reports exist where cyclic RGD-based vehicles were exploited to carry other outstanding consignments to tumor vasculature and cells. A remarkable contribution by Chen and Neamati [93] dealt with a paclitaxel prodrug (115), which was covalently assembled by joining the antimicrotubule drug to a dimeric cyclic RGD platform via a cleavable succinyl linker (Fig. (22)). This assembly was designed to embody two D-Tyr residues to be exploited for 125I-radiolabelling purposes, and two Lys-based anchoring points prone to conjugation. Conjugate (115) accumulated in the estrogen-independent breast cancer cell line MDA-MB-435 in an v3-specific manner, and the efficacy was comparable to that of the dimeric RGD construct per se. Stability was not assessed for the succinate bond, while premature release of paclitaxel during in vitro assays could not be completely ruled out. In a more recent article, the same research group [94] succeeded in completing the experimental work on this dimeric RGD-PTX conjugate, by performing extensive biodistribution studies in vivo and ex vivo. Thus, by comparing the biodistribution of 3H-labelled RGD2-PTX (115) and 3HPTX, it was found that the conjugate had higher tumor uptake and prolonged tumor retention than the tritiated drug itself. Metronomic low-dose treatment of breast cancer indicated that (115) was significantly more effective than the combination of pure ligand/drug. Positron emission tomography (PET) imaging showed reduced tumor metabolism in

RGD2-PTX-treated mice versus those treated with a drug/ligand combination. Finally, fluorescent TUNEL assay also showed that treatment by (115) promoted higher cell apoptosis as compared to drug/ligand combination and solvent controls. Nonetheless, the absolute tumor uptake value of conjugate (115) still remained rather low, due to the lipophilic character of the construct and its small size, and this translated into a short circulation half-life and rapid clearance. Almost the same conjugate (e.g. E-[c(RGDfK)2]paclitaxel) was extensively evaluated in a recent study by Ryppa et al. [95] using several, well-established in vitro and in vivo assays. The following results emerged: (1) in a standard 72 h assay, both the dimeric paclitaxel conjugate and monomeric c(RADfK)-paclitaxel control (not shown) inhibited the proliferation of HUVECs in a similar manner as paclitaxel, suggesting that hydrolysis of the succinate ester linker occurs under physiological conditions; (2) in a 30 min exposure assay, the conjugate showed slightly diminished efficacy as compared to free paclitaxel, but a quite improved efficacy with respect to the control peptide. These differences could, very likely, be due to diverse modalities of cellular uptake, with paclitaxel rapidly diffusing through the cellular membrane and the charged conjugate entering cells via v3 integrin-mediated endocytosis. Decisive in vivo studies in an OVCAR-3 xenograft model demonstrated no antitumor efficacy for either E-[c(RGDfK)2]-paclitaxel or free dimeric RGD scaffold E-[c(RGDfK)2], as compared to the moderate efficacy for paclitaxel. Taken together, these investigations allow one to conclude that this type of RGDpaclitaxel construct necessitates structural re-design of the linkers and drugs, in order to overcome the several drawbacks plaguing these conjugates. With the intent of developing RGD-based conjugated prodrugs to target tumors with an over-expression of v3, and the intention of performing activity profiles both in vitro and in vivo, Ryppa and Kratz [96] synthesized and evaluated two dimeric conjugates, compounds (116) and (117), wherein the E-[c(RGDfK)2] moiety was anchored to doxorubicin via either non-cleavable 6-maleimidocaproyl amide, or



enzymatically cleavable maleimidotriethylene glycol peptide spacers (Fig. (23)). OH O

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NH

R

O O

N O

H N

S

O NH.TFA

NH 3 Ph

(119) (Fig. (24)), where the cytotoxic moiety embraces one or two cyclic c(RGDfK) tumor targeting devices via a succinate linker. Concentration/response curves of these complexes along with those of several control compounds (e.g. non-targeted Pt(IV) drugs, free acyclic and cyclic RGDbased moieties) were examined against different endothelial and human cancer cells in vitro. It was found that RGDtethered Pt(IV) complexes are potent inhibitors of cellular proliferation when compared to both non-targeted Pt(IV) compounds and to the unconjugated targeting RGD derivatives. However, only for about half of the cells tested, the mono- and bis-conjugated compounds (118) and (119) proved slightly superior to the corresponding compounds bearing linear RGD-moieties. The authors hypothesized that, upon conjugation to the succinate group in Pt(IV), the linear RGD tripeptide and the cyclic RGD pentapeptide become comparable at recognizing the v3/v5 integrins. The Pt(IV) environment could possibly sterically hinder the binding of these cyclic peptides, thus decreasing their intrinsic targeting ability.

H N

O

O O

HN

R

O

Arg Asp Gly

O O O

O

R1 O

Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln 3

Ph

O

Fig. (23). Molecular structure of dimeric doxorubicin-containing RGD covalent conjugates (116) and (117) [96].

Whilst compound (117) was proven to be cleaved by matrix metalloproteases MMP-2 in OVCAR-3 tumor homogenates thus releasing the free drug, compound (116) did not exhibit any release of doxorubicin. The ability of doxorubicin conjugates (116) and (117) to inhibit HUVEC proliferation was assessed using a standard 72 h assay, which showed 6/10-fold increased inhibition of (117) (3 nM) as compared to the non-cleavable peptide (116) (30 nM). In a second assay, inhibition of HUVEC sprouting during a 24 h exposure was ~3.5-fold stronger for (117) than for the free drug alone (470 nM vs 1700 nM); in contrast, stable derivative (116) showed no activity at the higher 10,000 nM concentration. Unfortunately enough, in vivo studies in an OVCAR-3 xenografts model demonstrated that (116) was inactive and toxic, while compound (117) was only moderately active. In contrast, free doxorubicin resulted in a modest, but statistically significant antitumor effect. Overall, doxorubicin covalent conjugates of type (116) and (117) seem to be burdened with several limitations – e.g. insufficient receptor density on endothelial cells, possible endocytosis before protease cleavage, rapid clearance leading to low amounts of doxorubicin reaching endothelial cells –, all of which may probably be overcome by passing to nanostructured RGD-based drugloaded vehicles (vide infra). In 2008, Lippard and colleagues [97] designed and synthesized platinum(IV)-RGD conjugates of type (118) and

O

H3N Cl Pt H3N Cl

(116): R=

(117): R=

1275

N H

O

(118): R = OH; R1 =

O

N H HN

Arg Gly

Asp Ph

O

(119): R = R1 =

N H O

N H HN

Arg Asp

Gly

Fig. (24). Molecular structure of conjugated platinum-RGD peptide complexes (118) and (119) [97].

4.1.2. Macromolecules and Particles The rationale for using macromolecules and nanostructured particles as efficient carriers for the delivery of antitumor agents (and imaging probes, vide infra) is based on the pioneering work of Maeda and co-workers, as well as Jain et al. [98]. These studies highlighted striking differences in the biochemical and physiological characteristics of healthy and malignant tissues, which are responsible for the EPR effect (vide supra) [69, 70]. Also, such polymer and nanoparticle therapeutics may benefit from other advantages such as increased active drug targeting, decreased drug localization in non-target tissues, minimal drug leakage during transit to the target tissues, minimal drug degradation and premature clearance, prolonged retention, and survival of the active drug at the target loci. Furthermore, the versatile physicochemical architecture of macromolecules and particles en-


Auzzas et al.

sures the modulation of the pharmacokinetic and pharmacodynamic properties of these materials [99].

bumin-c(RGD) drug conjugates, and to address specific delivery of either p38MAP kinase inhibitors or antitubulin agents to competent v3-positive endothelial tumor cells. In a first contribution [101], platinum-coordination chemistry was employed to bring together the p38MAP kinase inhibitor SB202190 and an albumin carrier containing a PEGylated high affinity v3-binder c(RGDfK). The conjugate construct (121), shown in Fig. (26), contained an average of nine SB202190 units and six c(RGDfK)-PEG groups per albumin macromolecules. Compound (121) bound to v3 integrinexpressing endothelial HUVECs with low nanomolar affinity (71.4 nM, corresponding to a 5.4-fold increase with respect to the free c(RGDfK) ligand), and was subsequently internalized. Furthermore, (121) partially inhibited the TNFinduced activation of HUVECs, both at gene expression levels and when considering the secretion of proinflammatory cytokines. Thus, conjugates of type (121) provide a novel means to combat inflammation disorders such as rheumatoid arthritis.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers have been widely used as polymeric delivery systems to improve the biodistribution of cytotoxic drugs. These copolymers benefit from the passive tumor accumulation via EPR effect and possess a multifunctionalizable backbone suitable for anchorage of multiple copies of active units, such as peptide targeting moieties, anticancer drugs, and solubilizing portions. Along this line, Borgman et al. [100] reported on the synthesis, characterization and biological evaluation of a novel progeny of HPMA copolymers - e.g. compound (120) (Fig. (25)). These constructs embodied cyclic RGDfK moieties and properly activated aminohexylamino geldanamycin derivatives (AH-GDM), and were assayed for their ability to inhibit growth of model prostate and endothelial cell lines in vitro, as well as their toxicity in vivo. The drug moiety, a benzoquinoid ansamycin exhibiting high affinity to the chaperon protein HSP90, was grafted onto the polymer chain via the degradable sequence Gly-Phe-LeuGly, which allowed for specific intracellular release of the drug by lysosomal proteases such as cathepsin B. Competitive binding of compound (120) to v3 integrin was evaluated in prostate cancer and endothelial HUVEC lines, and in vitro growth inhibition was assessed. HPMA copolymer (120) showed active binding to the v3 integrin similar to that of drug-free peptide. Similarly, growth inhibition of cells was comparable to that of the free drug. In vivo tolerability experiments evidenced no signs of toxicity in mice receiving the polymer conjugate (120) over the 14-day evolution period, whilst the free drug caused significant morbidity. These experiments hold promise for the utility of HPMA copolymer-geldanamycin conjugates for treatment of prostate cancer with great efficacy and tolerability.

Also, the same research group explored a similar chemistry to construct albumin-RGD conjugates carrying the known antitumor drugs auristatin E [102] or auristatin F [103] (compounds (122) and (123), Fig. (26)). For compound (122), high binding affinity and specificity for HUVECs was demonstrated, with the conjugate internalized by endothelial cells and killing the targeted cells at low nanomolar concentrations. Furthermore, administration of this prodrug to C26 carcinoma-bearing mice proved excellent tumor-homing properties, thus demonstrating that such RGD-carrying albumins are suitable carriers for cell-selective intracellular delivery of cytotoxic compounds. On the other hand, compound (123), equipped with a C-terminal carboxylate group, proved more potent than its parent congener (122) in killing both v3-positive tumor cells and proliferating endothelial cells. Efficacy increased more in tumor cells than in endothelial cells, suggesting different drug redistribution behaviour depending on the two cell types.

Human serum albumin, a stable 67 KDa protein, was exploited by Temming et al. [101-103] to develop certain al-

x Me HN

O Me HN

z

y O

Me O NH2

HO

m

O Me Gly Gly Gly

Phe

NH

Leu

O

Gly 3 HO

HN D-Phe

NH O 5

Arg

O

HN

Asp Gly

O N H O

Me

OH

MeO

MeO (120)

Me OCONH2

Fig. (25). Molecular structure of HPMA-geldanamycin RGD conjugate (120) [100].



1277

D f

O

K

S

G R

S

O

O

O H

Albumin N H 6

O n (121)

H N

Cl

SB-ULS =

[SB-ULS]q

SB202190

Pt N H

H N

H

OH N F D G R

f

O

K

S

S

O H N

N H

O N H O

Me

Me

H N

Albumin N H 5

OH H N

N OMe

O

OMe

Me

O

Me

Me H N

N

N Me

[X]4

Me

N

O

(123): X =

O n

O

O

(122): X =

O

OMe

O

OMe

O

CO2H

Fig. (26). Molecular structure of albumin-based RGD conjugates (121)-(123) [101-103].

The increasing need for drug delivery systems that improve activity and specificity while overcoming problems associated with local toxicity, has led to the design and development of novel technologies based on soft threedimensional materials such as nanoparticles, liposomes, polymeric micelles, polymersomes, and vesicles. Such platforms enable a highly integrated design that incorporates multiple functions in a sole system such as tumor homing devices, cytotoxic effectors, and solubilizing systems, if necessary. A cyclic 5mer RGD-targeting peptide [104], c[RGDf(thioacetyl)K], exposing a reactive thiol end-group, was coupled to the distal terminus of poly(ethylene glycol)-coated long circulating liposomes (LCL) to obtain a stable drug delivery system acting as a platform for multivalent interaction with v3 integrin. This supramolecular matrix (RGDLCL) was charged with doxorubicin to give the construct (124), as displayed in Fig. (27). RGD-LCL-DOXO (124) inhibited tumor growth in a doxorubicin-insensitive murine C26 colon carcinoma model, whereas LCL-DOXO control failed to decelerate tumor growth. Indeed, coupling of the v3-addressing agent redirected the liposome constructs (124) to angiogenic endothelial cells both in vitro and in vivo. Likely, the superior therapeutic efficacy of (124) was the result of inhibition of tumor progression via inhibition of angiogenesis rather than via direct cytotoxic effects on tumor cells.

Similar V3-targeted liposome-based nanoparticles encapsulating the cytotoxic drug doxorubicin (not shown) were implemented by Murphy et al. [105] aiming at evaluating the antimetastatic effect in clinically relevant pancreatic and renal cell orthotopic models of spontaneous metastases. After having established good targeting properties in vitro on HUVECs, the doxo-free RGD nanoparticles were tested in vivo, showing that the particles indeed targeted the newly formed tips of the tumor neovasculature. Next, the impact of v3-targeted nanoparticle drug delivery on primary tumor growth and spontaneous metastasis on the two orthotopic cancer models was evaluated. Although a modest effect on primary tumor growth was observed, it was noted a substantial impact on metastatic disease. In fact, v3-mediated delivery of doxorubicin to the tumor vasculature resulted in a 15-fold increase in antimetastatic activity, without producing drug-associated weight loss in treated animals, as observed with systemic administration of the free drug. Along this line, Hölig et al. [106] designed and manufactured RGD liposomes based on lipopeptides of type (125) (Fig. (28)) consisting of a constrained monocyclic c(CRGDC) pentapeptide moiety covalently linked to 1,2-dipalmitoylglycero-3-succinyl lysine via a dioxaoctanoic acid spacer. The liposomes were loaded with doxorubicin, and analyzed in vivo for their ability to target C26 colon carcinoma mouse model. These experiments revealed an improved antitumor effect of doxorubicin-loaded liposomes as compared to free


Auzzas et al.

OH

PEG

PEG

O

OH O OH

MeO

D G R

O

f

OH

Me

K

S

H O O

Mal-PEG DSPE

O

HO NH2 PEG

(124) PEG RGD-LCL-DOXO

Fig. (27). Schematic representation of RGD-based, doxorubicin-loaded long circulating liposomes (LCL) (124) and magnification showing the structural formulae of the RGD ligand and doxorubicin drug [104].

Gly Arg

Asp S

O

S

NH O

HN Tyr

Phe

Arg

Asp

Ala

Gly

O

O NH O

O

N nH

Gly Ac

CONH2

O O

3N H

H2C

O

HC

O

CH2

13

O

13

O (125)

Fig. (28). Molecular structure of RGD-containing lipopeptide (125) consisting of a cyclic CRGDC peptide, a dipalmitoyl-glycerosuccinyllysine moiety and a dioxaoctanoic spacer [106].

doxo and untargeted liposomes, indicating that such structurally defined particles could be clinically useful carrier systems for targeting tumor vasculature. Among the various supramolecular drug delivery systems, polymeric micelles have emerged as very important tools to combat cancer and related diseases. The hydrophobic core of the micelles offers a carrier compartment that can host many hydrophobic antitumor drugs such as doxorubicin and paclitaxel. The outer shell consists of a protective corona that stabilizes the nanoparticles in aqueous solutions. In 2004 Gao et al. [107] first developed polymeric micelles by attaching the cyclic pentapeptide c(RGDfK) to the surface of doxorubicin-loaded poly(-caprolactone)-poly(ethyleneglycol) (PCL-PEG) micelles. The uptake of the resulting nanoparticles, structure (126) in Fig. (29), into SLK tumor endothelial cells derived from human Kaposi’s sarcoma, was studied using flow cytometry and confocal laser scanning microscopy. The cellular internalization efficacy was assessed, with a maximum 30-fold enhancement achieved with RGD-doxo micelles (126) with respect to non-targeted doxoloaded micelles. Intracellular distribution studies indicated

that functionalized micelles (126) were internalized by RGD v3-mediated endocytosis, and localized in the endosomal compartments of the cytoplasm rather than the nuclei, where free doxo accumulated quickly after membrane diffusion. Nanoscale coordination polymers are a class of soft materials constructed from metal ion connectors and polydentate bridging ligands. Owing to a limitless choice of building blocks, they have the potential to be engineered for a number of applications including v3/v5-mediated anticancer drug delivery. In a smart report, Lin and colleagues [108] implemented a general strategy for the delivery of Pt-based drugs to cancer cells via their inclusion into nanoscale coordination polymers. The Pt-based nanoparticles (not shown) are stabilized with a corona of amorphous silica to prevent rapid dissolution, and to control the release of the Pt-drug. Specifically, the nanoparticles were forged by interaction between Tb3+ ions and c,c,t-(diaminedichlorodisuccinato)Pt(IV) (DSCP) bridging ligands, followed by silica coating and grafting to silyl-derivatized c(RGDfK). The resulting superstructures were evaluated by in vitro cytotoxicity assay for HT29 angiogenic cancer cells using free DSCP or non-



1279

PEG

D G R

f

PEG

K

S

Mal-PEG PCL

O

OH O

OH O OH

MeO

O Me

PEG

H O

OH

O

PEG HO NH2

(126)

Fig. (29). Schematic structure of doxo-loaded polymeric micelles (126) functionalized with cyclic RGD targeting moieties [107].

targeted DSCP nanoparticles as controls. The results suggest that, while the control molecules did not lead to any appreciable internalization, the targeted particles were effectively internalized, presumably via receptor-mediated endocytosis. Once inside the cells, the anticancer platinum(IV) species released from the silica corona could then be reduced to the active Pt(II) species by intracellular reducing agents that are present in high concentrations. The RGD peptide-mediated delivery of therapeutic nucleic acids, small interfering RNA agents (siRNAs), and non-viral and viral gene macromolecules represents a flourishing area of research, which has been documented by a number of excellent reviews [15b]. The discussion here is restricted to few, emblematic achievements to catch a glimpse into this promising, wide landscape. A cyclic RGDpeptide conjugate block copolymer, namely c[(RGDfK)PEG-polylysine] (127) (Fig. (30)) was assembled by Kataoka et al. [109] by connecting a PEG-polylysine carboxaldehyde to cysteine-terminated cyclic RGD-peptide via a thiazolidine ligation procedure. Then, (127) was associated with plasmid DNA to form a polyplex micelle for the cultured HeLa cells overexpressing v3 and v5 integrins. In contrast, in cultured 293T cells deprived of v3/v5 receptors, the transfection efficiency of RGD-targeted polyplex micelles was similar to that of non targeted systems. Furthermore, flow cytometric analysis revealed a superior uptake of the RGD micelles as compared to

a non-RGD counterpart, consistent with the transfection results. A confocal laser scanning microscopic observation revealed that the plasmid DNA in the RGD-micelles accumulated preferentially in the perinuclear region of the cells, whereas RGD-free micelles did not. Overall, these results indicate that the superior transfection efficiency induced by the RGD ligand within the micelle was attributed to an increase in cellular uptake and to accelerated intracellular trafficking of the micelle toward the perinuclear region of the cell via v3/v5-mediated endocytosis; and this paves the way for a promising use of such micellar formulations in site-specific targeting gene-delivery intervention. Albumin-based intermediate-sized delivery agents for antisense oligonucleotides were very recently developed by the Juliano group at Chapel Hill [110] exploiting a cyclic RGD pentapeptide as the tumor addressing vector. As shown in Fig. (31), the conjugate constructs (128) were engineered by adorning the human serum albumin (HSA) nanomolecules with green fluorophores Alexa-488, c(RGDfK)-terminated PEG moieties, and a selected set of splice switching oligonucleotide residues. As a test system, the authors utilized human melanoma cells that express the v3 integrin and also contain a luciferase reporter gene that can be induced by delivery of splice switching oligonucleotides to the cell nucleus. The conjugates were effectively endocytosed by the cells via v3-mediated internalization so that the oligonucleotides could accumulate in the nucleus.

Asp Gly

D-Phe

O

Arg Lys N H O

O

H N O S (127)

Fig. (30). Molecular structure of c(RGDfK)-polylysine (127) [109].

O n

N H

H N

H m

NH3+


Auzzas et al.

Alexa-488

D f

G R

K

Mal-PEG

HSA

S

S

O

Oligo

(128):

S

S

S

Oligo

S

Oligo = 5'-S-(CH2)6-GTTATTCTTTAGAATGGTGC-3' O N

N

H N

O

O

H N

2

G

K O

4 O

R

S

D f

O

(129): ODNFAM = 5'-(fluorescein)-GTC-CCT-TCG-TCA-ACA-CTA-3'(NH2)

O ODNFAM

Fig. (31). Schematic representation of RGD-containing albumin- and SWNT-based oligonucleotide conjugates (128) and (129) [110,111].

In this line, oligonucleotide-functionalized tumortargetable cyclic RGD-bearing, modified single walled carbon nanotubes (SWNT) (129) (Fig. (31)) were designed and constructed by Scheinberg and colleagues [111], and consequently tested in vivo. Biodistribution studies in mice via flow cytometric assay showed that the presence of the RGDtargeting moiety within (129) allowed for specific binding to tumor cells, as compared to a RGD-free isotype control. Novel, supramolecular v3-targeted delivery systems of type (130) carrying therapeutic small interfering RNA molecules (siRNAs) were assembled by Lu et al. [112] via complexation of a multifunctional carrier (EHCO) with siRNAs to form SH-exposing nanoparticles, followed by chemical conjugation to maleimide-terminated PEG-c(RGDfK) integrin binder (Scheme 6). Systemic administration of a therapeutic anti-HIF-1 (anti-hypoxia inducible factor-1) siRNA via nanoparticles (130) resulted in a tumor growth inhibition significantly higher than a non-targeted analogous system or free siRNA in nude mice bearing human glioma U87 xenografts. These results hold promise for the use of EHCO-based cyclic RGD-guided carriers for targeted delivery of therapeutic siRNAs for effective cancer treatment. 4.2. Targeted Imaging Medical imaging, viz the ability to see within a living species without damage, has been one of the most important tools for disease diagnosis, and represents an exciting and

blooming area of contemporary research [113]. Depending on the imaging technique used, anatomical and/or molecular information can be reached, making abnormal processes visible, quantifiable, and traceable. Because the v3 integrin is an attractive biomarker for cancer treatment, high affinity RGD-based v3-antagonists may offer a valuable resource for the implementation of different types of imaging constructs for selective signaling of tumors and tumor-related angiogenesis. The different imaging technologies possess their own intrinsic strength and weakness, and can be grouped, for clarity, into subcategories depending on the basic principles employed. Such RGD-based strategies have recently been reviewed by different authors including Haubner et al. [114], Temming et al. [15b], Wester et al. [15a], and Liu [115]. We will therefore address this topic focusing upon recent, skilled contributions dealing with those “beacon” molecular arrays which bear cyclic RGDs as tumor addressing navigators. 4.2.1. Radionuclide Imaging The majority of RGD-based imaging conjugates have been developed exploiting relatively stable c(RGDfV), c(RGDfK), and c(RGDyK) pentapeptide templates, which were adapted to optimize the pharmacokinetic properties of the tracer, and improve the chemical ligation issues. Several, seminal investigations by Haubner [116,117] and Conti [118-120] were performed, where monomeric RGD cyclopeptides of type c(RGDyV), c(RGDyK) and c(RGDfK) were labelled by simple chemistry with either 125I, 18F or



1281

Scheme 6. Chemical construction of ligand directed siRNA delivery system (130) [112]. 64

Cu radionuclides to originate useful radiotracer displays [e.g. compounds (131)-(134) in Fig. (32)). As an emblematic example, [18F]-galacto-RGD (132) was developed for PET of v3 integrin expression, aiming at indepth analysis of the kinetics and biodistribution in cancer patients [121]. Due to its highly favourable biodistribution in humans, specific receptor binding, and high-contrast visuali-

zation of the v3 expression in humans, this tracer may offer a viable strategy for non invasive mapping of v3 integrin expression in vivo. After this outstanding imaging active ligand, other [18F]labelled RGD analogues were synthesized and developed by researchers at Siemens Medical Solutions, and advanced in in vitro and in vivo preclinical trials [63]. In particular,

Fig. (32). Representative monomeric RGD-based radiotracers (131)-(134) [116,117,119,120].


Auzzas et al.

Fig. (33). Molecular structure of [18F]-labelled cyclic RGD pseudopeptides (135) and (136) [63].

ies confirmed that such [18F]-radiolabelled compounds are stable and very promising tracer candidates to be advanced to clinical opportunities.

monomeric and dimeric fluorinated RGD-based cyclic ligands (135) and (136) were assembled, which incorporated a bioisosteric triazole moiety within the cycle (Fig. (33)). The synthesis capitalized upon the efficient Huisgen azidealkyne 1,3-dipolar cycloaddition reaction (click chemistry) to effect the macrocyclization process and to attach the sensing moiety. In vitro binding assays via surface plasmon resonance and cell-based v3 binding competition experiments showed (135) and (136) to be competent v3 ligands, very similar in potency to the RGDfK reference compound. In vivo microPET imaging of a tumor-bearing mouse (U87MG human glioblastoma or A431 human squamous cell carcinoma) showed that the compounds were excellent tracers, with good tumor uptake and retention, favourable renal clearance, very little liver uptake, and fast wash-out rate from healthy tissues. Biodistribution metabolic stability stud-

NH2

3 HN

N O

+ O

Asp Gly

N

OH

N

O ButO

N

O

Ph HN

O HN

HN O

Asp Gly

H N

3

Arg

OH

O O

N O

O (138)

(137)

1. DMF 2. TFA 3. 64CuCl2 O

N

Arg

In 2008, Cuthbertson [123] reported a study where three F-labelled analogues (140)-(142) carrying a bicyclic KCRGDCFC-based addressing moiety (NC100717) [124]

18

O

HN

O HN

ButO

O

Ph

Brechbiel and co-workers [122] exploited cyclam monocycle derivative (138) to implement kinetically stable 64CuRGD-radiotracer complex (139), as shown in Scheme 7. In vitro evaluation of (139) in human serum demonstrated a stable complexed radiocopper ion with no evidence of transchelation of 64Cu into serum proteins for up to 48 h. However, in vivo kinetic stability studies were not reported, which could have shed light onto the metabolic fate of the radiometal construct.

O

(139)

64Cu

O N O

64

Scheme 7. Synthesis of RGD peptide- Cu chelate radiotracer (139) [122].

N N


were assembled via known oxime ligation chemistry, and comparatively evaluated in biodistribution and imaging assays (Fig. (34)). Hydrophilic PEGylated conjugate (140) was found to possess superior in vivo pharmacokinetics with higher tumor-to-blood, tumor-to-liver, tumor-to-muscle, and tumor-to-lung ratios with respect to more lipophilic counterparts (141) and (142). This demonstrated that the chemical nature of the attached beacon moiety can be rationally tuned to tailor the overall biodistribution profile of the radiotracer.

Fig. (34). Molecular structure of radiolabelled cyclic RGD peptides (140)-(142) [123].

The same bicyclic RGD pharmacophore was advanced to F-AH111585 for phase I clinical trials in breast cancer patients [125], as well as for biodistribution and radiation dosymetry in healthy volunteers [126] and the evaluation of changes in tumor vascularity after antitumor therapy [127]. 18

In the first report [125], the biodistribution of 18FAH111585 was assessed in 18 tumor lesions from 7 patients with metastatic breast cancer by PET, while the metabolic stability of the radiotracer was evaluated by chromatography of plasma samples. These trials nicely demonstrated the ability of 18F-AH111585 to be retained in tumor tissues and to detect breast cancer lesions by PET in most anatomic sites, while maintaining metabolic stability. In a subsequent study [126], the same research group reported on the safety, biodistribution, and internal radiation dosymetry of 18F-AH111585 by acquisition of PET scans of 8 healthy volunteers after a bolus injection of the drug. 18F-AH111585 proved a safe PET tracer with a dosimetry profile comparable to other common 18F PET tracers.


1283

In a further, very recent contribution [127], a preclinical study was reported, which aimed to assess the potential of the same 18F-molecule as an effective tool to measure changes in tumor vascularity after antitumor therapy. In particular, mice bearing LLC tumors (Lewis lung carcinoma) or Calu-6 non small cell lung tumor xenografts were used for in vivo biodistribution and small animal PET imaging studies. In addition, some animals were treated with either low-dose paclitaxel, or VEGF receptor-2 tyrosine kinase inhibitor ZD4190. Tumor uptake of 18F-AH111585 and microvessel density were then assessed. Biodistribution of the radiotracer demonstrated rapid clearance from the blood and the key background organs, and good tumor accumulation in LLC tumors. Small-animal PET imaging of Calu-6 tumors allowed the visualization of tumors above background tissue. Paclitaxel therapy reduced the microvessel density in LLC tumor-bearing mice, while significantly reducing 18F-drug tumor uptake. Similarly, ZD4190 therapy induced marked decrease of the radiotracer uptake in Calu-6 tumors, as compared to control-treated tumors. Taken together, these evidences highlight a great potential offered by 18F-AH111585 integrin radioligand in detection of cancer lesions and in the assessment of the impact of antitumor therapies, in particular those targeting tumor blood vessels. One of the most used imaging biomarker is the well accepted, easily available 2-[18F]fluoro-2-deoxyglucose ([18F]FGD). Glucose metabolism is deeply connected to many of the metabolic processes involved in the development of malignancy and, consequently, glucose transport and utilization are elevated in most types of cancers [128]. A recent study coming from Gambhir et al. at Stanford [129] dealt with chemical conjugation of [18F]-FDG to a c(RGDyK)-ONH2 aminooxy v3 integrin ligand via well established oxime ligation chemistry [130], to afford radiolabelled peptide (143) (E/Z oxime mixture) in a 41% radiochemical yield (Fig. (35)). The v3 receptor binding affinity of (143) was evaluated using v3-positive U87MG cells resulting in a IC50 value of 0.67 μM in a competitive displacement assay with 125I-echistatin. Biodistribution and microPET imaging of U87MG tumor-bearing nude mice using (143) revealed relatively moderate tumor uptake after 60 min of injection. Although limitation exists regarding the overall in vivo imaging performance of conjugate (143), as well as the rather forcing experimental conditions during its synthesis (the 18F labeling methodology required 100 °C and 1.5-2.5 pH value), the study holds promise for the direct, one-step chemoselective conjugation to unprotected tumor homing peptides in large-scale production of 18F-labelled radiotracers for clinical routine PET applications. A dual receptor-targeting hybrid conjugate (144) (Fig. (35)) embodying a cyclic RGDyK v3-targeting moiety, a GRPR-directed (Gastrin-Releasing Peptide Receptor) linear octapeptide bombesin, and a 18F-labelled PEGylated tail, was manufactured by Chen et al. [131] by exploiting the trivalent nature of a glutamate residue for tripartite amide ligation. Cell binding, cell uptake, and microPET imaging studies were performed on (144) to evaluate its dual targeting properties and biodistribution in vitro and in vivo. In particular, the dual conjugate proved to possess comparable GRPR- and v3-receptor binding affinities as the corresponding singlereceptor targeting counterparts. Compound (144) effectively


showed dual receptor targeting properties both in vitro and in vivo. When assayed in PC3 tumor cells and in PC3-bearing nude mice, high tumor uptake, good tumor-to-background contrast, and favourable pharmacokinetics were certified, warranting for this dual receptor tracer a secure place in the repertoire of chemically relevant imaging candidates.

Fig. (35). Chemical structure of RGD-containing radioligands (143) and (144) [129,131].

As shown in the previous sections, the evolution from mono RGD-containing molecules to RGD-multimers was a fundamental, yet unavoidable passage in both the only-RGD and RGD-conjugate domains. Thus, the design and construction of v3 integrin-directed radiotracers did not escape this

Auzzas et al.

fate, with many scientific contributions being focused on cyclic RGD-embedded dimers, tetramers, polymers, liposomes, and nanoparticles endowed with imaging units. In a leading report, Line et al. [69] studied the uptake of a 99m tecnetium-labelled macromolecular polymethacrylamideRGD4C conjugate (not shown) in PC3 DU145 prostate tumor xenografts and compared the results with the corresponding non-RGD polymers and RGD-monomeric counterparts. Higher tumor localization and reduced extravasation in normal tissues of the HPMA-RGD4C-copolymer elected this compound as a superior v3-integrin directed radiotracer. In a series of papers by Chen et al., where a small repertoire of 64Cu-radiolabelled DOTA-bearing cyclic RGDmonomer, dimer and tetramer conjugates were synthesized [132,133] (not shown), it was pointed out that tetrameric RGD tracers had about twice as much tumor uptake as the corresponding dimers; also, the dimers had significantly higher uptake than the monomers. These results, once more, emphasize the beneficial evolution towards RGDmultipresentation as a powerful tool in cancer therapy and imaging. Along this line, Chen again [134] succeeded in synthesizing a dimeric 18F-fluoroPEG covalent click conjugate (145) embodying the known E[c(RGDyK)]2 framework (Fig. (36)). As expected, this tracer exhibited tumor targeting efficacy, relatively good metabolic stability, as well as favourable in vivo pharmacokinetics. The quick 18F-labelling method developed in this investigation stands as a viable procedure in labelling azido-containing biomolecules in high radiochemical yield for medical applications in vitro and in vivo. Using the same Cu(I)-catalyzed Huisgen cycloaddition procedure between the azido-terminus of cycloRGDfK units,

Fig. (36). Molecular formulae of dimeric and tetrameric RGD radioligands (145) and (146) [134,135].



glioma and MDA-MB-435 breast tumor models. In such models, planar imaging studies pointed out that very smallsized tumors of ca. 5 mm in diameter could be readily visualized with an excellent contrast.

and the alkyne-function of monomeric, dimeric, or tetrameric DOTA-grafted scaffolds, Liskamp et al. [135] synthesized and tested a series of v3-targeted displays. As an example, Fig. (36) shows dendrimer (146) wherein one 111In-labelled DOTA-core structure is covalently linked to four RGDrecognizing units via four stable triazole heterocycles. The v3 binding characteristics of dendrimer (146) and its low cognate derivatives were determined in vitro, and their in vivo v3-targeting properties assessed in nude mice with subcutaneously growing human SK-RC52 tumors. There, it was found that multimeric presentation as in (146) showed specifically enhanced uptake in v3 integrin-expressing tumors in vivo, as compared to monomeric and dimeric counterparts.

Very recently, similar G3- and PEG4-containing covalent DOTA-conjugates were assembled by the same research group [139], and complexed to 64Cu radionuclide. Also in this case, the addition of G3 and PEG4 spacer groups between the two cyclic RGD motifs renders possible for them to achieve a simultaneous integrin binding in a divalent fashion. Biodistribution and imaging studies performed in athymic nude mice bearing U87MG human glioma xenografts revealed an improved tumor uptake and clearance kinetics, comparable to the results obtained for the above (147) and (148) complexes.

During the 2000-2009 decade, an impressive body of work appeared by the S. Liu research team at Purdue dealing with the preparation and biomedical evaluation of a number of 99mTc radiotracers, all based on the popular c(RGDfK) recognizing cyclic motif, either in monomeric version or, better, in covalent multipresentation displays [136-138]. The structure of such Tc-complexes was designed to embody a high level of malleability in order for the radioconjugates to reach those optimal characteristics for a noninvasive monitoring of tumor growth or shrinkage during anti-angiogenic treatments. Two emblematic, dimeric Tc-complexes, (147) [137] and (148) [138], are displayed in Fig. (37), in which variable linkers – triglycine G3 or polyethyleneglycol PEG 4 chains – connect the c(RGDfK) and (hydrazono)nicotinyl (HYNIC) modules. In particular, the integrin v3 binding affinities of tecnetium-free (147) and (148) were determined by competitive displacement of 125I-c(RGDyK) on U87MG glioma cells. It was found that the two linker moieties between the RGD motifs are responsible for their higher integrin binding affinity, as compared to non-spaced counterparts. Biodistribution studies clearly demonstrated that both G3 and PEG4 spacers are particularly useful for improving tumor uptake and clearance kinetics of 99m Tc-dimers (147) and (148). Also, a linear relationship was found between the tumor size and radiotracer tumor uptake in both U87MGAsp

The effects of size, architecture, and topology of multivalent carriers have emerged as decisive factors in their ability to perform as non invasive imaging tools. In this line, a biodegradable dendritic positron-emitting nanoprobe for imaging of angiogenesis was implemented by Almutairi, Fréchet, and colleagues based on the monocyclic CRGDC recognizing peptide [140]. The nanoscale carrier was engineered by adorning a dendritic pentaerythritol core with four flexible divalent arms terminated with RGD moieties. The resulting eight dendritic branches in (149) were functionalized with tyrosine groups to enable attaching radiohalogens such as 76 Br or 125I, useful for imaging and therapy (Fig. (38)). The polyethylene oxide spacers within (149) were designed to impart biological stealth to the particle, while allowing for a modulation of the pharmacokinetics. This nanoscale construction enabled a 50-fold enhancement of the binding affinity to v3 integrin receptor, as compared to the monovalent RGD peptide control (0.18 nM vs 10.40 nM). Cell-based assays of 125I-labelled (149) using v3-positive cells showed a 6-fold increase in v3-mediated endocytosis, as compared to the non-targeted counterpart, whereas v3-negative cells showed no enhancement. In vivo studies in a murine hindlimb ischemia model for angiogenesis revealed high specific accumulation of 76Br-(149) in angiogenic muscles,

D-Phe

Arg

NH

Gly

O

O

O

Gly Asp

Linker

N H

Arg

N H

Linker

N H

HN

O

Linker

O

N O (147): Linker =

H N

N H

N

(G3) O

O

O (148): Linker =

H N

O

Fig. (37). Chemical structure of

P 99mTc

O

(PEG4)

H

O

N

SO3Na

3 O

99m

SO3Na

N

O H N

1285

HO

OH

SO3Na

Tc-labelled dimeric cyclic RGD radiotracers (147) and (148) [137,138].

D-Phe


Auzzas et al.

Fig. (38). Chemical structure of dendritic positron-emitting nanoprobes 76Br-(149) and 125I-(149) [140].

allowing for the highly selective imaging of this critically important process. 4.2.2. Optical and Magnetic Resonance Imaging Optical imaging, in particular fluorescence imaging and magnetic resonance imaging, nicely complements the above mentioned radionuclide-based techniques in “seeing” within the living body and understanding the biological complexities for disease treatment [141]. In the last few years, several research groups have demonstrated the utility of coupling v3-directing cyclic RGD peptides to optical- and MRactive molecular beacons for use in noninvasive in vitro, in vivo, and ex vivo imaging of tumor-related angiogenesis. In this field, Dumy, Coll and colleagues [142,143] developed a regioselectively addressable functionalized template (RAFT, vide supra), that is the cyclic decapeptide c(KKKPGK AKPG), onto which four c(RGDfK) moieties were anchored to four non contiguous lysine residues via oxime ligation chemistry. The remaining vacant K residue was then used to append proper fluorescent labels such as biotin, fluorescein, or cyanine moieties. As an example, conjugate (150) (Fig. (39)) very efficiently prevents v3-mediated cell adhesion to vitronectin and is actively endocytosed due to its multimeric presentation. In vivo experiments in nude mice revealed that administration of low doses of (150) reduces tu-

mor growth of pre-established A549 tumors, whereas neither monomeric c(RGDfK), nor the non-RGD conjugate control display any significant activity. Furthermore, (150) significantly improved the target specificity of subcutaneous v3negative tumor masses as well as that of disseminated metastases after injection; and this testified that (150) is able to target tumor angiogenesis. Fluorescent covalent conjugate probes (151), (152), and (153) (Fig. (40)) were independently constructed by the Casiraghi [75] and Scolastico groups [144] utilizing high affinity cyclic RGD ligands of type (86)-(89) and (36), (37), respectively (vide supra). These conjugates were evaluated in vitro for their ability to inhibit the binding of biotinylated vitronectin to the isolated v3 and/or v5 receptors. The three candidates showed good to excellent nanomolar affinity to v3, ranging from 8.0 nM for aminoproline-based ligand (151) to 142 nM and 207 nM for azabicycles (152) and (153), respectively. In addition, a striking v3/v5 selectivity was assessed for candidate (151). Fluorescent probes (152) and (153) were tested on v3-positive HUVECs, as well as on a panel of cell lines deriving from solid tumors overexpressing this protein (ECV304 bladder cancer cells, Y98G glioblastoma multiforme cells, PC3 prostate cancer cells, Caki-1 renal clear cell carcinoma cells). v3-


Gly Gly

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O

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Lys

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Ala

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Lys Pro

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Gly O

Over the past couple of years we have witnessed a blooming development of a variety of nanomedicine platforms for cancer diagnosis and therapy. Single-walled carbon nanotubes (SWNTs) – nanomaterials that exhibit intrinsic optical properties including photoluminescence in NIR range and strong resonant RAMAN scattering – constitute a promising class of polyvalent scaffolds, whose highly integrated design permits the incorporation of multiple functions in a sole homogeneous system. In a brilliant contribution by Dai and co-workers [146] it was shown that SWNTs with different isotopic 12C/13C compositions displayed well shifted Raman G-band peaks, and could serve for multicolor Raman imaging of live cells. 12C-SWNT, 13C-SWNT, and 12 13 C/ C-SWNT were conjugated to Herceptin (anti-Her2), Erbitux (anti-Her1), and cyclic RGDfK (anti-v3) units,

3 N

N

KO3S

SO3 (150)

Fig. (39). Molecular structure of RAFT-RGD optical imaging vector (150) [143].

Negative MDA-MB-231 cells were used as a control. Both compounds displayed fluorescence distributed at the cell surface and in putative cytosolic vesicles, suggesting that the candidates were internalized upon binding to v3 integrin.

O N O

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2 N H

N H

S

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v3 8.0 nM v5 3751 nM O O

3

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HN (153)

HO

O

v3 142 nM

(152)

O N H

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Monomeric, dimeric, and tetrameric cyanine-ligated c(RGDfK) displays based on a glutamate backbone were introduced by the Chen group [145], and evaluated for optical imaging of integrin v3 expression in living mice. Such systems were tested in a subcutaneous U87MG glioblastoma xenograft model in order to investigate the effect of RGD multipresentation on integrin binding and tumor targeting efficacy. As expected, the binding affinities moved from 42.9 nM for the monomer to 27.5 nM for the dimer, and 12.1 nM for the tetramer, in agreement with the multivalency phenomenon. The subcutaneous tumor could be clearly visualized with the fluorescent probes, with the tetramer displaying the highest tumor uptake and tumor-to-normal tissue ratio.

Asp

Arg

Lys

D-Phe

Arg

Asp D-Phe


v3 207 nM

OH

RC: c(RGDfV) v3 3.2 nM ST1646 v3 1.0 nM

Fig. (40). Molecular structure of fluorescent RGD conjugates (151)-(153) [75,144].

Asp

Gly

Gly


Auzzas et al.

O

Asp Gly Arg

O NH

D-Tyr N

Lys S O

PEG

3

O

R R

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CdTe

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Gly

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ZnS

R (155)

R (154) R =

CdTe

O PEG

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O

Scheme 8. Construction of RGD peptide-labelled near-infrared quantum dots (156) [148].

respectively. Raman spectra of the three compositions were recorded under a near-infrared 785 nM laser excitation, displaying well shifted Raman G bands for each species. Three types of cells including BT474 (Her-2 positive), U87MG (v3 positive), and MDA-MB-468 (Her-1 positive), were incubated with the three-color SWNT mixture, and then subjected to confocal Raman spectroscopy imaging. The resulting images showed specific labeling of cells by SWNTs, with minimal non-specific binding. Also, a mixture of the three cell lines was incubated with the three color SWNT mixture. The Raman images clearly identified the existence of three cell types, each labelled by a distinct Raman color, and this demonstrated the ability of multiplexed cell identification and imaging by SWNTs with different isotope composition. Similar RGD-conjugated SWNTs were also investigated by Gambhir et al. [147] to non-invasively localize targeting in a U87MG tumor model in mice via Raman spectroscopy. The images were acquired in two groups of tumorbearing mice: the control group received non-targeted SWNTs, whilst the experimental group received tumor targeting RGD-SWNTs. Raman imaging revealed increased accumulation of RGD-SWNTs in tumor as opposed to the control, setting the foundation for improved non-invasive diagnostic strategies in tumoral diseases. Quantum dots (QDs) are single nanocrystal colloidal semiconductors with exceptional fluorescent properties. Due to their high photostability, brightness, monodispersity, and multicolor emission, QDs can act as unique markers to trace cancer cells in vivo during metastases, and identify sentinel lymph nodes in cancer – critical issues in development of effective cancer therapies. For example, cyclic RGD-labelled Cd-Te QD-based probes were implemented by Chen and Gambhir [148] to non-invasively visualize integrin v3expression in tumor vasculature and living subjects. As shown in Scheme 8, QD-RGD nanoparticles (156) were assembled by starting with commercially available PEGylated amine-modified QD 705, which were first conju-

gated to activated 4-maleimidobutyric acid yielding maleimide-nanocrystal surfaces (154). Finally, the thiolated RGD peptide (155) was appended to the maleimide terminals of (154) via a Michael addition reaction to afford the targeted QD-RGD particles (156) embodying an estimated 3050 RGD units per QD. After in vitro and ex vivo assessment of the ability of (156) to specifically target integrin v3, athymic nude mice bearing subcutaneous U87MG human glioblastoma tumors were administered (156) intravenously. The tumor fluorescence intensity reached a maximum at 6 h post-injection with good contrast, suggesting that the QDRGD structures like (156) are very promising candidates for integrin-targeted NIR optical imaging in cancer detection and management, including imaging-guided surgery. Using very similar RGD-adorned QDs, Gambhir and coworkers [149] exploited intravital microscopy with subcellular resolution to observe and record live the binding of the nanoparticles to tumor blood vessels in living subjects. This futuristic methodology showed that, in the model, QDs do not extravasate, and solely bind as aggregates. This level of knowledge paves the way towards ensuing regulatory approval of this kind of nanoparticles in humans for both disease diagnosis and therapy. Despite the numerous exciting and fascinating demonstrations of the potential biomedical applications of heavy metal-containing QDs, their intrinsic toxicity and teratogenicity still shadow on the clinical application of these materials; to solve such a critical issue, replacement of toxic metals by more benign elements or substantial surface stabilization are desirable. Highly luminescent Cd-Se/Cd-S/Zn-S one-dimensional QDs coated with PEGylated phospholipids and conjugated to thiolated RGD cyclopeptide c(RGDfC) were developed by Prasad et al. [150], and used for tumor targeting and imaging in live animals. In vivo optical imaging studies in nude mice carrying subcutaneous and orthotopic pancreatic cancer xenografts indicated that QDs accumulate at tumor sites by RGD-mediation following systemic injection. Furthermore,



in vivo studies showed no adverse effects even at high dosages at both cellular and tissue levels. Magnetic resonance imaging (MRI) is a diagnostic technique especially used in the medical fields to produce highquality anatomical images of the living body. In clinical practice, MRI can distinguish pathological tissues (e.g. tumors) from normal tissues, mostly by anatomical differences. The recent development of molecular and cellular imaging, aimed at visualizing the disease-specific biomarkers at the molecular and cellular levels, has led to the recognition of magnetic nanoparticles as effective MRI-targeted contrast agents. To be concise, a couple of recent examples are here discussed, with an emphasis on ultrasmall superparamegnetic iron oxide nanoparticles (USPIOs). For example, novel v3 integrin-directed USPIOs were designed and constructed by Kiessling et al. [151], and their specific uptake by endothelial cells assessed in vitro and in vivo. The USPIOs were firstly coated with 3-aminopropyltrimethoxysilane (APTMS), and then conjugated with c(RGDyE) peptides. The uptake of the resulting RGD-USPIOs by HUVECs was significantly increased when compared to non-RGD counterparts, and could be competitively inhibited by addition of unbound RGD. Furthermore, these MR imaging probes were able to non-invasively distinguish between tumors differing in the degree of v3 integrin expression (HaCat-ros-A-5RT3 vs A431) and in their angiogenesis profile, even when using a clinical 1.5-T magnetic resonance scanner. A novel way of synthesizing and functionalizing ultrasmall Fe3O4 NPs as contrast agents for in vivo tumor detection using MRI was presented by Chen and Sun [152], which was based on Mannich ligation between the iron chelated cathecol unit (157) and the -amino group of the lysine within the c(RGDyK) integrin binder. The schematic illustration of this chemistry is shown in Scheme 9. The resulting 8.4 nM-sized particles (158) proved to be stable in physiological conditions and, when administered intravenously in mice bearing U87MG tumors, they accumulated preferentially in tumor cells, which were readily tracked by MRI. To further investigate the particle distribution in various organs, the animals were sacrificed and visualized. While NPs (158) were rarely seen in kidneys and muscles, they mostly localized in the integrin expressing tumor vasculature and tumor cells with little, if any, macrophage uptake.

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ety co-exists with one therapeutic or imaging entity are discussed above. When instead a single entity of the RGDtargeting moiety is cooperatively or synergistically integrated with two or more functional units (e.g. magnetic resonance plus optical imaging probes; radionuclide plus optical imaging probes; therapeutic drug plus MRI probes; etc.), multimodal displays are generated, which ensure a more advanced performance in detection and/or treatment of a given pathology at a molecular level [153]. In this chapter we briefly outline several examples of such integrated dual or multimodal technologies pertaining to the RGD nanobiocomposite world. In a highlighting example, Mulder and co-workers [154] investigated a dual-mode optical/MRI nanoparticle probe in which a QD core, covered by a micellar PEGylated shell and Gd-DTPA lipids, was conjugated to a cyclic RGD vector to provide specificity for the v3 integrin. A schematic display of this supramolecular architecture, (159), is shown in Fig. (41). To demonstrate molecular imaging of tumor angiogenesis using paramagnetic QD46 at the cellular and macroscopic levels, mice inoculated with B16F10 melanoma were intravenously injected with a contrast agent, and studied with parallel intravital microscopy and MR imaging in vivo. Intravital microscopy data showed widespread angiogenic activity, mainly localized within the rim of the tumor and up 1 cm to the tumor boundary, whereas in vivo MRI proved useful in macroscopic localization of sites of high angiogenic activity, which also were mainly found at the rim of the tumor.

4.3. Multimodal Display Systems

Well defined dendrimeric platforms are desirable because their numerous, often orthogonal conjugation sites permit varied combinations of imaging active units to be covalently implemented, providing valuable and robust nanoscale multimodal diagnostic tools. A clever example is provided by Brechbiel [155], who pursued a nanoscale approach to imaging of angiogenesis using rationally designed polyamidoamine (PAMAM) dendrimers covalently adorned with v3-targeting c(RGDfK) peptides. The synthesis of (160) (Fig. (42)) involved orthogonal aminoxy/aldehyde coupling for peptide conjugation and succinimidyl ester and isothiocyanate chemistry for the dye and nuclide-chelate unit ligation, respectively. As a whole, nanostructures (160) embody an inner PAMAM multivalent core surrounded by a corona of three different active units, the RGD cyclopeptides for directing the construct at the target, the Alexa Fluor 594 dye for CFM and NIR optical studies, and 1B4M-DTPA chelating moiety to host the Gd and 111In ions for in vitro and in vivo MR imaging and biodistribution studies. This work demonstrated the capability of these macromolecular constructs to act as dual MR contrast agents and optical probes. Through fluorescence microscopy, the superior activity of nuclide-free dendrimer in selectively binding to cultured v3-expressing M21 cells was assessed, as compared to non-targeted counterparts. In vivo tissue distribution of Gd/111In mixed-compound (160) in M21 melanoma tumorbearing mice showed mostly renal and reticuloendothelial accumulation with 3.30 tumor/blood ratio at 2 h postinjection.

The biomedical applications of those chemical platforms where one (or multiple copies of) RGD-based targeting moi-

To overcome toxicity issues of leached Gd(III) ions upon in vivo administration of metal-organic frameworks, Lin et

O Fe3O4 O O

HN 1. formaldehyde 2. c(RGDyK)

O Fe3O4

2

Gly HN

O (157)

Arg

Asp D-Tyr

(158)

Scheme 9. Schematic illustration of the construction of c(RGDyK)coated Fe3O4 nanoparticles (158) [152].


Auzzas et al. O

Arg

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Fig. (41). Schematic depiction of RGD-based multimodal optical/MRI quantum dots (159) [154].

al. [156] designed and developed manganese-based nanoscale dual MRI/optical imaging particles of type (161) (Fig. (43)). Nanoparticles (161) were assembled by coating particles of MRI-active Mn3(BTC)2(H2O)6 (BTC = trimesic acid) with a thin silica gel corona, and subsequently grafted to rhodamine B and c(RGDfK) to provide the fluorescence and targeting functionalities. In vitro MR imaging of HT-29 cells showed selective uptake of (161) and higher Mn2+ intracellular delivery, as compared to non-targeted particles and neat Mn2+ ions; and this result was confirmed by CMI studies.

jected with SWNT-PEG5400-RGD-64Cu-DOTA (162) (Fig. (44)), followed by microPET scans at multiple time points. Conjugate (162) exhibited a high tumor uptake of ca 10-15%

A superparamagnetic iron oxide nanoparticle-based (SPION) probe for dual-modality magnetic biology characterization/imaging with dual-targeting capability, was recently introduced by Petri-Fink et al. [157] by exploiting the polyvalent nature of a SPION corona. To this purpose, the APS-coated SPIONs (APS = aminopropyltriethoxysilane) were manufactured by appending a fluorescent 7hydroxycoumarin label, a mitochondrial targeting 22-mer peptide (MTP), and a v3-targeting c(RGDfK) moiety. HeLa cells were cultured and incubated in the presence of the resulting superparamagnetic nanoparticles, and their internalization monitored by confocal microscopy imaging (CMI). The nanoparticles were nicely co-localized at mitochondria, whereas the MTP-free RGD SPION and RGD-free MTP SPION controls were not, and this supported that both the appended MTP and RGD directing peptides cooperate in the active transport of the particles to the mitochondria. Furthermore, the magnetic properties of this material crucially served as effective separation/characterization tool in the physiological context, though no diagnostic MRI experimentation was pursued. The biodistribution and tumor targeting stability of SWNTs in mice using Hipco nanotubes non-covalently functionalized with phospholipidic PEG and carrying c(RGDyK) directing units, together with 64Cu-DOTA moieties, were studied in vivo and ex vivo by the Liu group using PET imaging and Raman spectroscopy [158]. In particular, mice bearing subcutaneous v3-positive U87MG tumors were in-

Fig. (42). Schematic drawing of RGD-based multimodal optical/MRI PAMAM dendrimers (160) [155].



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Fig. (43). Schematic drawing of RGD-based multimodal optical/MRI manganese nanoparticles (161) [156].

ID, a significant increase from ca 3-4% ID for the RGD-free counterpart. The high tumor accumulation was the result of the long blood circulation time, the specific high tumor binding affinity of the conjugate, and the multivalency effect exerted by multiple copies of RGD units in the nanotube. Also, exploiting the intrinsic optical properties of the nanotube core, Raman spectroscopy was employed to directly detect SWNTs in the various tissues of a mouse carrying U87MG tumor. By this optical technique, the amounts of (162) in each of the tissue samples were quantified; and the values proved in reasonable agreement with the PET data based on the nanotube radioactivity. Integrated, multimodal nanotherapeutic systems which can diagnose and deliver targeted therapeutics, while monitoring the response to therapy, are precious tools to address the challenges of contemporary molecular medicine. In a brilliant, very recent study, Park and Cheon [159] developed “all-in-one” magnetic nanoparticle probes for the simultaneous molecular imaging and siRNA delivery to specific cancer cells. A schematic display of multimodal particles (163) is shown in Fig. (44). Thus, manganese-doped magnetismengineered iron oxide (MnMEIO) particles coated with bovine serum albumin were first derivatized into SPDPcarrying derivatives (SPDP = n-succinimidyl-3-(2pyridyldithio)propionate), and then treated with thiolated PEG3400 terminating with a c(RGDfK) ligand. The resulting nanoparticles were finally treated with Cy5 dye-labelled thiolated siRNA to produce the multimodal construct (163). Of note, both the RGD-recognizing unit and siRNA therapeutic gene are attached to the surface of the nanoparticle through disulfide bonds, which could be cleaved readily in an intracellular reductive environment. Overall, (163) incorporates a PEGylated RGD for nanoparticle stabilization and cancer cell targeting, as well as a fluorescent cypatemodified siRNA for the inhibition of gene expression and fluorescence imaging. In addition, the inner metal core of the particle serves as MRI beacon due to its intrinsic paramagnetic MRI active nature. Firstly, the specific cellular targeting ability of (163) was assayed for v3-positive breast can-

cer cells MDA-MB-435 and v3-negative lung-carcinoma cells A549. Each cell line was treated with (163), and then simultaneously imaged by MRI and fluorescence confocal microscopy. The results of this study clearly showed that RGD-based nanoparticles were able to bind to v3-positive cells, solely. Next, the subcellular distribution of (163) was visualized by fluorescence imaging, which demonstrated its exclusive internalization into v3-positive MDA-MB-435 cells. Furthermore, CMI revealed that a considerable amount of siRNA escaped from the endosome into the cytoplasm of such cells. To probe the inhibition of the expression of green fluorescent protein (GFP), siRNA-carrying nanoparticles (163) were transfected into GFP-expressing MDA-MB-435 and A549 cells, and the effects monitored by fluorescence imaging; this demonstrated the effective RGD-mediated target gene-silencing effects of the nanoparticles. Finally, a TEM study on (163)-administered MDA-MB-435 cells provided detailed information on the spatial distribution into the cells, underlying that the nanoparticles were distributed in the endosome and cytoplasm, with no penetration into the nucleus. Additional “theranostic” particles, that is c(RGDfK) carrying fluorescently labelled liposomal nanoparticles LNs and loaded with potent anticancer drug doxorubicin, were manufactured by Cressman et al. [160], and their behaviour within v3-expressing cells assayed. In particular, the cellular uptake of doxo-encapsulated LNs embodying the RGD/methoxycoumarin pair was monitored by fluorescence microscopy using three cell lines with decreasing levels of v3 expression, namely HUVEC, M21, and M21L cells. As expected, RGD-LNs delivered more drug into the cytosol of HUVECs than into M21 and M21L cells, supporting the notion that the multivalent RGD presentation is neatly associated with superior v3-mediated cellular internalization of the targeted supramolecular lipid structures. All in all, these last studies highlight three major points: first, the multivalency of the nanoparticles is beneficial for accommodating a variety of active units in view of a multi-


Auzzas et al.

Fig. (44). Schematic drawings of RGD-based SWNT and MnMEIO nanoparticles (162) and (163) [158,159].

modal platform; second, the RGD-targeting moieties associated with PEGylated polymer chains are useful for the specific targeting of cancer cells thereby reducing unwanted organ uptake; and third, the simultaneous diagnosis and therapeutic treatment (theragnosis) may minimize the invasiveness and side effects in living subjects. 5. CONCLUDING REMARKS From a far, one could envision the realm of integrins which had already seen us actively involved in research - as a well compiled “carte des vins” which, yet, only recommends a single wine; nonetheless, this choice of “bottle” has proved appealing and “mouth-watering” to a wide number of chemists, biologists, medical chemists and doctors. In truth, after having confronted this topic, we were able to fathom how varied and vast the scientific literature dealing with integrins and their antagonists was. Even after narrowing the field to the v3 receptor sub-type, and considering just those peptide or semi-peptide antagonists bearing a cyclic RGD consensus sequence, the number of studies concerning design, synthesis and applications remained astonishing. In this review we have tried to give an extensive analysis of the judicious approach that led to the discovery of important v3 integrin inhibitors, and we have emphasised the latest

results where several of these prototypes have found applications in pharmaceutical and biomedical fields. The evolution of research studies, in time, has highlighted a clear shift of interest within this sector. Thus, whereas the search for and selection of potent v3 antagonist leads can be considered an established science, with examples bearing good binding properties and selectivity already identified and even commercialized, the focal point of study has shifted towards the use of a selected number of structural motifs (e.g. c(RGDfK), c(RGDyK) and their multimeric versions) as intelligent and selective agents in drug delivery, as imaging beacons or, even, as both towards tumour masses. As a result, the meticulous chemical and biological aspects of these studies have embraced and merged with the pharmacological, medical, engineering and technopharmaceutical disciplines. In prospective, we maintain that a prolific approach to integrin research should, on one hand, follow through to studying the vocational applications of these molecules, in particular the construction and application of multifunctional and multivalent systems in their nano-dimensional formats; and on the other hand, of no lesser importance, it should open out towards the selection of new structural motifs which surpass existing ones in efficiency and selectivity. Thus, the alliance



between academic research and industrial development, which has already given positive and tangible results, may continue to thrive. These frontiers will hail the near future of research in this exciting, fruitful domain. ACKNOWLEDGEMENTS The authors thank Dr Lucia Marzocchi for critical manuscript review. LA thanks Consiglio Nazionale delle Ricerche (CNR), Italy, for a leave of absence (Short-Term Mobility Grant 2009). A postdoctoral grant from University of Milan, Italy, and a fellowship from Regione Autonoma della Sardegna (Master and Back 2008), Italy, are kindly acknowledged by PB and PC, respectively. Supported, in part, by Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR, PRIN 2006-2008). REFERENCES [1] [2]

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[4]

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Received: November 30, 2009

Revised: February 05, 2010

Accepted: February 06, 2010


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