Rational Design of Multifunctional Polymer Therapeutics for Cancer Theranostics

Although anti-angiogenic agents offer great therapeutic potential, preclinical and clinical studies suggest that these agents, used as monotherapies, have a delayed onset of activity and may have only limited effects on advanced malignancies. Multimodality targeted polymer therapeutics that include anti-angiogenic agents and chemotherapeutics offer the potential for improved efficacy and diminished toxicity in the treatment of cancer and other angiogenesis-dependent diseases. We have recently designed and characterized novel combined anti-angiogenic and antitumor polymer–drug conjugates that target both the tumor and its microenvironment. These conjugates include combined anti-angiogenic and chemotherapeutic drugs, such as TNP-470 and paclitaxel, respectively. Several conjugates also incorporate bisphosphonates as targeting moieties for bone metastases and osteosarcomas or RGD peptidomimetics that target integrins overexpressed on tumor endothelial cells and several tumor cells. Using molecular imaging techniques, we have successfully established dormant and fast-growing tumor mouse models to intravitally non-invasively follow-up tumor progression and response to novel polymer therapeutics. Our results point at our polymer therapeutics as novel bi-specific conjugates targeting both the tumor epithelial and endothelial compartments, warranting their use on a wide spectrum of primary as well as metastatic tumors. The use of these novel architectures will potentially shed light on the molecular mechanisms underlying tumor dormancy and hopefully transform cancer into a chronically-manageable disease.     

Synergistic Interaction of CPP2 Coupled with Thiazole Derivates Combined with Clotrimazole and Antineoplastic Drugs in Prostate and Colon Cancer Cell Lines

Cell-penetrating peptides (CPPs) are small peptide sequences used mainly as cellular delivery agents that are able to efficiently deliver cargo into cells. Some CPPs also demonstrate intrinsic anticancer properties. Previously, our group developed a new family of CPP2-thiazole conjugates that have been shown to effectively reduce the proliferation of different cancer cells. This work aimed to combine these CPP2-thiazole conjugates with paclitaxel (PTX) and 5-fluorouracil (5-FU) in PC-3 prostate and HT-29 colon cancer cells, respectively, to evaluate the cytotoxic effects of these combinations. We also combined these CPP2-thiazole conjugates with clotrimazole (CLZ), an antifungal agent that has been shown to decrease cancer cell proliferation. Cell viability was evaluated using MTT and SRB assays. Drug interaction was quantified using the Chou–Talalay method. We determined that CPP2 did not have significant activity in these cells and demonstrate that N-terminal modification of this peptide enhanced its anticancer activity in both cell lines. Our results also showed an uneven response between cell lines to the proposed combinations. PC-3 cells were more responsive to the combination of CPP2-thiazole conjugates with CLZ than PTX and were more sensitive to these combinations than HT-29 cells. In addition, the interaction of drugs resulted in more synergism in PC-3 cells. These results suggest that N-terminal modification of CPP2 results in the enhanced anticancer activity of the peptide and demonstrates the potential of CPPs as adjuvants in cancer therapy. These results also validate that CLZ has significant anticancer activity both alone and in combination and support the strategy of drug repurposing coupled to drug combination for prostate cancer therapy.     

Targeting EphA2‐Sam and Its Interactome: Design and Evaluation of Helical Peptides Enriched in Charged Residues

The EphA2 receptor controls diverse physiological and pathological conditions and its levels are often upregulated in cancer. Targeting receptor overexpression, through modulation of endocytosis and consequent degradation, appears to be an appealing strategy for attacking tumor malignancy. In this scenario, the Sam domain of EphA2 plays a pivotal role because it is the site where protein regulators of endocytosis and stability are recruited by means of heterotypic Sam–Sam interactions. Because EphA2‐Sam heterotypic complexes are largely based on electrostatic contacts, we have investigated the possibility of attacking these interactions with helical peptides enriched in charged residues. Several peptide sequences with high predicted helical propensities were designed, and detailed conformational analyses were conducted by diverse techniques including NMR, CD, and molecular dynamics (MD) simulations. Interaction studies were also performed by NMR, surface plasmon resonance (SPR), and microscale thermophoresis (MST) and led to the identification of two peptides capable of binding to the first Sam domain of Odin. These molecules represent early candidates for the generation of efficient Sam domain binders and antagonists of Sam–Sam interactions involving EphA2.     

RAFT-derived polymer-drug conjugates: poly(hydroxypropyl methacrylamide) (HPMA)-7-ethyl-10-hydroxycamptothecin (SN-38) conjugates

A series of well‐defined polymer–drug conjugates were prepared in order to modify the physical properties of a known cytotoxic drug, 7‐ethyl‐10‐hydroxycamptothecin (SN‐38), the active metabolite of irinotecan (CPT‐11). Reversible addition–fragmentation chain transfer (RAFT) polymerisation was used to covalently and site‐specifically append a defined N‐(2‐hydroxypropyl)methacrylamide (HPMA) polymer to SN‐38 using a graft‐from process. These poly‐HPMA–SN‐38 conjugates displayed excellent aqueous solubility and stability, whilst retaining the cytotoxic activity of the parent SN‐38. In vitro co‐culture assays containing both cancer and noncancer cell lines demonstrated the specificity of RAFT‐derived poly‐HPMA–SN‐38 conjugates for cancerous cells. The concept of post‐optimisation modification of small‐molecule drugs through a graft‐from polymer conjugation method is introduced.     

Carbohydrate-based Biomedical Copolymers for Targeted Delivery of Anticancer Drugs

A variety of strategies and carrier molecules have been used to direct therapeutic agents to tumor sites. The incorporation of a specific targeting moiety to drug carrier may result in active drug uptake by malignant cells. Carbohydrates are important mediators of cell–cell recognition events and have been implicated in related processes such as cell signaling regulation, cellular differentiation, and immune response. The biocompatibility of carbohydrates and their ability to be specifically recognized by cell-surface receptors indicate their potential utility as ligands in targeted drug delivery for therapeutic applications. Yet, carbohydrates are not ideal targeting ligands because they are difficult to synthesize, bind weakly to carbohydrate receptors, and are prone to suffer from enzyme degradation due to labile glycosidic linkages. This review describes the design and development of HPMA-based biomedical copolymers to facilitate the selective delivery of drugs to tumor tissues via carbohydrate–endogenous lectin interactions. Various carbohydrate-decorated HPMA copolymer–drug conjugates are presented and the application of the copolymers for drug delivery is discussed. Current efforts to increase the affinity of carbohydrate ligands for their target receptors through multivalent display are also discussed. These novel HPMA copolymer carbohydrate conjugates hold promise as clinically relevant drug delivery systems for cancer therapy.     

Introducing Glycolinkers for the Functionalization of Cytotoxic Drugs and Applications in Antibody–Drug Conjugation Chemistry

Antibody–drug conjugates (ADCs) are promising alternatives to naked antibodies for selective drug‐delivery applications and treatment of diseases such as cancer. Construction of ADCs relies upon site‐selective, efficient and mild conjugation technologies. The choice of a chemical linker is especially important, as it affects the overall properties of the ADC. We envisioned that hydrophilic bifunctional chemical linkers based on carbohydrates would be a useful class of derivatization agents for the construction of linker–drug conjugates and ADCs. Herein we describe the synthesis of carbohydrate‐based derivatization agents, glycolinker–drug conjugates featuring the tubulin inhibitor monomethyl auristatin E and an ADC based on an anti‐EGFR antibody. In addition, an initial in vitro cytotoxicity evaluation of the individual components and the ADC is provided against EGFR‐positive cancer cells.     

Design,Synthesis, and in vitro Evaluation of Multivalent Drug Linkers for High‐Drug‐Load Antibody–Drug Conjugates

A series of novel multivalent drug linkers (MDLs) containing cytotoxic agents were synthesized and conjugated to antibodies to yield highly potent antibody–drug conjugates (ADCs) with drug/antibody ratios (DARs) higher than those typically reported in the literature (10 vs. ≈4). These MDLs contain two copies of a cytotoxic agent attached to biocompatible scaffolds composed of a branched peptide core and discrete polyethylene glycol (PEG) chains to enhance solubility and decrease aggregation. These drug linkers produced well‐defined ADCs, whose DARs could be accurately determined by LC–MS. Using this approach, ADCs with significantly lower aggregation and higher DAR than those of conventional drug linker design were obtained with highly hydrophobic cytotoxic agents such as monomethyldolastatin 10 (MMAD). The in vitro potencies of the MDL‐derived conjugates matched that of ADCs of similar DAR with conventional linkers, and the potency increased proportionally with drug loading. This approach may provide a means to prepare highly potent ADCs from a broader range of drugs, including those with lower cytotoxicity or poor solubility, which otherwise limits their use for antibody–drug conjugates. This may also provide a means to further improve the potency achievable with cytotoxins currently used in ADCs.     

A Combination Strategy to Inhibit Pim‐1: Synergism between Noncompetitive and ATP‐Competitive Inhibitors

Pim‐1 is a serine/threonine kinase critically involved in the initiation and progression of various types of cancer, especially leukemia, lymphomas and solid tumors such as prostate, pancreas and colon, and is considered a potential drug target against these malignancies. In an effort to discover new potent Pim‐1 inhibitors, a previously identified ATP‐competitive indolyl‐pyrrolone scaffold was expanded to derive structure–activity relationship data. A virtual screening campaign was also performed, which led to the discovery of additional ATP‐competitive inhibitors as well as a series of 2‐aminothiazole derivatives, which are noncompetitive with respect to both ATP and peptide substrate. This mechanism of action, which resembles allosteric inhibition, has not previously been characterized for Pim‐1. Notably, further evaluation of the 2‐aminothiazoles indicated a synergistic inhibitory effect in enzymatic assays when tested in combination with ATP‐competitive inhibitors. A synergistic effect in the inhibition of cell proliferation by ATP‐competitive and ATP‐noncompetitive compounds was also observed in prostate cancer cell lines (PC3), where all Pim‐1 inhibitors tested in showed synergism with the known anticancer agent, paclitaxel. These results further establish Pim‐1 as a target in cancer therapy, and highlight the potential of these agents for use as adjuvant agents in the treatment of cancer diseases in which Pim‐1 is associated with chemotherapeutic resistance.     

Design and In vitro Evaluation of Branched Peptide Conjugates: Turning Nonspecific Cytotoxic Drugs into Tumor‐Selective Agents

The use of peptide receptors as targets for tumor‐selective therapies was envisaged years ago with the findings that receptors for different endogenous regulatory peptides are overexpressed in several primary and metastatic human tumors, and can be used as tumor antigens. Branched peptides can retain or even increase, through multivalent binding, the biological activity of a peptide and are very resistant to proteolysis, thus having a markedly higher in vivo activity compared with the corresponding monomeric peptides. Oligo‐branched peptides, containing the human regulatory peptide neurotensin (NT) sequence, have been used as tumor‐specific targeting agents. These peptides are able to selectively and specifically deliver effector units, for cell imaging or killing, to tumor cells that overexpress NT receptors. Results obtained with branched NT conjugated to different functional units for tumor imaging and therapy indicate that branched peptides are promising novel multifunctional targeting molecules. This study is focused on the role of the releasing pattern of drug‐conjugated branched NT peptides. We present results obtained with oligo‐branched neurotensin peptides conjugated to 6‐mercaptopurin (6‐MP), combretastain A‐4 (CA4) and monastrol (MON). Drugs were conjugated to oligo‐branched neurotensin through different linkers, and the mode‐of‐release, together with cytotoxicity, was studied in different human cancer cell lines. The results show that branched peptides are very promising pharmacodelivery options. Among our drug‐armed branched peptides, NT4–CA4 was identified as a candidate for further development and evaluation in preclinical pharmacokinetic and pharmacodynamic studies. This peptide–drug exhibits significant activity against pancreas and prostate human cancer cells. Consequently, this derivative is of considerable interest due to the high mortality rates of pancreas neuroendocrine tumors and the high incidence of prostate cancer.     

Hunting for Novel Routes in Anticancer Drug Discovery: Peptides against Sam-Sam Interactions

Among the diverse protein binding modules, Sam (Sterile alpha motif) domains attract attention due to their versatility. They are present in different organisms and play many functions in physiological and pathological processes by binding multiple partners. The EphA2 receptor contains a Sam domain at the C-terminus (EphA2-Sam) that is able to engage protein regulators of receptor stability (including the lipid phosphatase Ship2 and the adaptor Odin). Ship2 and Odin are recruited by EphA2-Sam through heterotypic Sam-Sam interactions. Ship2 decreases EphA2 endocytosis and consequent degradation, producing chiefly pro-oncogenic outcomes in a cellular milieu. Odin, through its Sam domains, contributes to receptor stability by possibly interfering with ubiquitination. As EphA2 is upregulated in many types of tumors, peptide inhibitors of Sam-Sam interactions by hindering receptor stability could function as anticancer therapeutics. This review describes EphA2-Sam and its interactome from a structural and functional perspective. The diverse design strategies that have thus far been employed to obtain peptides targeting EphA2-mediated Sam-Sam interactions are summarized as well. The generated peptides represent good initial lead compounds, but surely many efforts need to be devoted in the close future to improve interaction affinities towards Sam domains and consequently validate their anticancer properties.     

In vitro degradability and stability of hydrophobically modified pH‐sensitive micelles using MPEG‐grafted poly(β‐amino ester) for efficient encapsulation of paclitaxel

Methoxypoly(ethylene glycol)‐grafted poly(β‐amino ester) was synthesized for the fabrication of pH‐sensitive micelles, and these micelles were modified with deoxycholic acid to facilitate the hydrophobic interaction between the micellar core and paclitaxel. The micelle properties were studied by dynamic light scattering and fluorescence spectrometry. An in vitro degradation study showed that the synthesized polymers degraded hydrolytically within 24 h under physiological conditions. The stability of paclitaxel‐loaded pH‐sensitive micelles was evaluated in vitro. The introduced deoxycholic acid more stabilized the micelles at pH 7.4 compared to the micelles without modification. But the pH‐sensitive region of the micelles was lowered from pH 6.8 to pH 5.8. These results indicate that pH‐sensitive micelles with improved stability have great potential as hydrophobic drug carriers for tumor targeting. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Molecular‐Target‐Based Anticancer Photosensitizer: Synthesis and in vitro Photodynamic Activity of Erlotinib–Zinc(II) Phthalocyanine Conjugates

Targeted photodynamic therapy is a new promising therapeutic strategy to overcome growing problems in contemporary medicine, such as drug toxicity and drug resistance. A series of erlotinib–zinc(II) phthalocyanine conjugates were designed and synthesized. Compared with unsubstituted zinc(II) phthalocyanine, these conjugates can successfully target EGFR‐overexpressing cancer cells owing to the presence of the small molecular‐target‐based anticancer agent erlotinib. All conjugates were found to be essentially non‐cytotoxic in the absence of light (up to 50 μM ), but upon illumination, they show significantly high photo‐cytotoxicity toward HepG2 cells, with IC₅₀ values as low as 9.61–91.77 nM under a rather low light dose (λ=670 nm, 1.5 J cm^?2). Structure–activity relationships for these conjugates were assessed by determining their photophysical/photochemical properties, cellular uptake, and in vitro photodynamic activities. The results show that these conjugates are highly promising antitumor agents for molecular‐target‐based photodynamic therapy.     

Peptide Fragments of Odin‐Sam1: Conformational Analysis and Interaction Studies with EphA2‐Sam

Odin is a protein belonging to the ANKS family, and has two tandem Sam domains. The first, Odin‐Sam1, binds to the Sam domain of the EphA2 receptor (EphA2‐Sam); this interaction could be crucial for the regulation of receptor endocytosis and might have an impact on cancer. Odin‐Sam1 associates with EphA2‐Sam by adopting a “mid‐loop/end‐helix” model. In this study three peptide sequences, encompassing the mid‐loop interacting portion of Odin‐Sam1 and its C‐terminal α5 helix, were designed. Their conformational properties were analyzed by CD and NMR. In addition, their abilities to interact with EphA2‐Sam were investigated by SPR studies. The peptides adopt a predominantly disordered state in aqueous buffer, but a higher helical content is evident in the presence of the cosolvent trifluoroethanol. Dissociation constants towards EphA2‐Sam were in the high micromolar range. The structural findings suggest further routes for the design of potential anti‐cancer therapeutics as inhibitors of EphA2‐Sam heterotypic interactions.     

Development and Biochemical Characterization of Self-Immolative Linker Containing GnRH-III-Drug Conjugates

The human gonadotropin releasing hormone (GnRH-I) and its sea lamprey analogue GnRH-III specifically bind to GnRH receptors on cancer cells and can be used as targeting moieties for targeted tumor therapy. Considering that the selective release of drugs in cancer cells is of high relevance, we were encouraged to develop cleavable, self-immolative GnRH-III-drug conjugates which consist of a p-aminobenzyloxycarbonlyl (PABC) spacer between a cathepsin B-cleavable dipeptide (Val-Ala, Val-Cit) and the classical anticancer drugs daunorubicin (Dau) and paclitaxel (PTX). Alongside these compounds, non-cleavable GnRH-III-drug conjugates were also synthesized, and all compounds were analyzed for their antiproliferative activity. The cleavable GnRH-III bioconjugates revealed a growth inhibitory effect on GnRH receptor-expressing A2780 ovarian cancer cells, while their activity was reduced on Panc-1 pancreatic cancer cells exhibiting a lower GnRH receptor level. Moreover, the antiproliferative activity of the non-cleavable counterparts was strongly reduced. Additionally, the efficient cleavage of the Val-Ala linker and the subsequent release of the drugs could be verified by lysosomal degradation studies, while radioligand binding studies ensured that the GnRH-III-drug conjugates bound to the GnRH receptor with high affinity. Our results underline the high value of GnRH-III-based homing devices and the application of cathepsin B-cleavable linker systems for the development of small molecule drug conjugates (SMDCs).     

Glucose Conjugation of Anti‐HIV‐1 Oligonucleotides Containing Unmethylated CpG Motifs Reduces Their Immunostimulatory Activity

Antisense oligodeoxynucleotides (ODNs) are short synthetic DNA polymers complementary to a target RNA sequence. They are commonly designed to halt a biological event, such as translation or splicing. ODNs are potentially useful therapeutic agents for the treatment of different human diseases. Carbohydrate–ODN conjugates have been reported to improve the cell‐specific delivery of ODNs through receptor mediated endocytosis. We tested the anti‐HIV activity and biochemical properties of the 5′‐end glucose‐conjugated GEM 91 ODN targeting the initiation codon of the gag gene of HIV‐1 RNA in cell‐based assays. The conjugation of a glucose residue significantly reduces the immunostimulatory effect without diminishing its potent anti‐HIV‐1 activity. No significant effects were observed in either ODN stability in serum, in vitro degradation of antisense DNA–RNA hybrids by RNase H, cell toxicity, cellular uptake and ability to interfere with genomic HIV‐1 dimerisation.     

Improved stability and tumor targeting of 5‐fluorouracil by conjugation with hyaluronan

To circumvent the rapid clearance in vivo and consequent low tumor targeting of 5‐fluorouracil (5‐Fu), hyaluronan‐5‐fluorouracil conjugate (HA‐5‐Fu) was firstly synthesized and characterized. The stability of HA‐5‐Fu in vitro was evaluated by incubation with phosphate buffered saline, hyaluronidase solution, and mice plasma, respectively. The tumor targeting was tested by in vitro cytotoxicity evaluation and in vivo pharmacokinetics study in plasma and tumor. HA‐5‐Fu with drug loading of 87.674 mg/g was successfully obtained and confirmed. HA‐5‐Fu showed high stability in acidic environment and moderate stability under enzymatic cleavage. The enhanced cytotoxicity of HA‐5‐Fu over 5‐Fu depended on drug concentration, incubation time, and cell lines type. The t_1/2 of HA‐5‐Fu in plasma after injection of prodrug was extended up to 10 times compared with that of 5‐Fu. Notably, AUC_0–t in tumors of HA‐5‐Fu was 3.6 times higher than 5‐Fu, demonstrating its excellent tumor targeting and quite promising prospect in anti‐cancer therapy. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 927‐932, 2013     

Target Hopping as a Useful Tool for the Identification of Novel EphA2 Protein–Protein Antagonists

Lithocholic acid (LCA), a physiological ligand for the nuclear receptor FXR and the G‐protein‐coupled receptor TGR5, has been recently described as an antagonist of the EphA2 receptor, a key member of the ephrin signalling system involved in tumour growth. Given the ability of LCA to recognize FXR, TGR5, and EphA2 receptors, we hypothesized that the structural requirements for a small molecule to bind each of these receptors might be similar. We therefore selected a set of commercially available FXR or TGR5 ligands and tested them for their ability to inhibit EphA2 by targeting the EphA2‐ephrin‐A1 interface. Among the selected compounds, the stilbene carboxylic acid GW4064 was identified as an effective antagonist of EphA2, being able to block EphA2 activation in prostate carcinoma cells, in the micromolar range. This finding proposes the “target hopping” approach as a new effective strategy to discover new protein–protein interaction inhibitors.     

Synthesis,Characterization, and Initial Biological Evaluation of [99mTc]Tc‐Tricarbonyl‐labeled DPA‐α‐MSH Peptide Derivatives for Potential Melanoma Imaging

α‐Melanocyte stimulating hormone (α‐MSH) derivatives target the melanocortin‐1 receptor (MC1R) specifically and selectively. In this study, the α‐MSH‐derived peptide NAP‐NS1 (Nle‐Asp‐His‐d ‐Phe‐Arg‐Trp‐Gly‐NH₂) with and without linkers was conjugated with 5‐(bis(pyridin‐2‐ylmethyl)amino)pentanoic acid (DPA‐COOH) and labeled with [^99mTc]Tc‐tricarbonyl by two methods. With the one‐pot method the labeling was faster than with the two‐pot method, while obtaining similarly high yields. Negligible trans‐chelation and high stability in physiological solutions was determined for the [^99mTc]Tc‐tricarbonyl–peptide conjugates. Coupling an ethylene glycol (EG)‐based linker increased the hydrophilicity. The peptide derivatives displayed high binding affinity in murine B16F10 melanoma cells as well as in human MeWo and TXM13 melanoma cell homogenates. Preliminary in vivo studies with one of the [^99mTc]Tc‐tricarbonyl–peptide conjugates showed good stability in blood and both renal and hepatobiliary excretion. Biodistribution was performed on healthy rats to gain initial insight into the potential relevance of the ^99mTc‐labeled peptides for in vivo imaging.     

Molecular assembly of multifunctional ⁹⁹(m)Tc radiopharmaceuticals using "clickable" amino acid derivatives

Synthetic strategies that enable the efficient and selective combination of different biologically active entities hold great promise for the development of multifunctional hybrid conjugates useful for biochemical and medical applications. Starting from side‐chain‐functionalized N(α)‐propargyl lysine derivatives, conjugates containing a ^99mTc‐based imaging probe for SPECT and two different moieties (e.g., tumor‐targeting vectors, pharmacological modifiers, affinity tags, or second imaging probes) can be assembled using the Cu^I‐catalyzed alkyne–azide cycloaddition in efficient one‐pot protocols. This strategy was successfully applied to the preparation of a ^99mTc‐labeled conjugate comprising a tumor‐targeting peptide sequence (bombesin(7–14)) and a low‐molecular‐weight albumin binder, a pharmacological modifier that prolongs the blood circulation time of the conjugate. Evaluation of the conjugate in vitro and in vivo provided promising results for its use as an imaging agent for the visualization of tumors positive for the gastrin‐releasing peptide receptor. The methodology presented herein provides an attractive synthetic tool for the preparation of multifunctional ^99mTc‐based radiopharmaceuticals with significant potential for a multitude of applications.     

Recent Progress on Cyclic Peptides’ Assembly and Biomedical Applications

Cyclic peptides are important building blocks for forming functional structures and have been applied in various fields. Considering the significant structural and functional roles of cyclic peptides in materials science and the attributed biophysical advantages, we provide an overview of cyclic peptide types that can self-assemble to form nanotubes, recent progress in stimuli-triggered cyclic peptide assembly, and methods to construct peptide and polymer conjugates based on cyclic peptides with alternative chirality. Specifically, we highlight the roles that stimuli-triggered cyclic peptides and their conjugates play in biomedical applications. Recent progress in other cyclic peptides acting as gelators in drug delivery and biomedicine are also summarized. These cyclic peptides with self-assembly properties are expected to act as adaptive systems for drug delivery and selective disease targeting.