Guided molecular missiles for tumor-targeting chemotherapy--case studies using the second-generation taxoids as warheads

A long-standing problem in cancer chemotherapy is the lack of tumor-specific treatments. Traditional chemotherapy relies on the premise that rapidly proliferating cancer cells are more likely to be killed by a cytotoxic agent. In reality, however, cytotoxic agents have very little or no specificity, which leads to systemic toxicity, causing undesirable severe side effects. Therefore, the development of innovative and efficacious tumor-specific drug delivery protocols or systems is urgently needed. A rapidly growing tumor requires various nutrients and vitamins. Thus, tumor cells overexpress many tumor-specific receptors, which can be used as targets to deliver cytotoxic agents into tumors. This Account presents our research program on the discovery and development of novel and efficient drug delivery systems, possessing tumor-targeting ability and efficacy against various cancer types, especially multidrug-resistant tumors. In general, a tumor-targeting drug delivery system consists of a tumor recognition moiety and a cytotoxic warhead connected directly or through a suitable linker to form a conjugate. The conjugate, which can be regarded as a "guided molecular missile", should be systemically nontoxic, that is, the linker must be stable in blood circulation, but upon internalization into the cancer cell, the conjugate should be readily cleaved to regenerate the active cytotoxic warhead. These novel "guided molecular missiles" are conjugates of the highly potent second-generation taxoid anticancer agents with tumor-targeting molecules through mechanism-based cleavable linkers. These conjugates are specifically delivered to tumors and internalized into tumor cells, and the potent taxoid anticancer agents are released from the linker into the cytoplasm. We have successfully used omega-3 polyunsaturated fatty acids, in particular DHA, and monoclonal antibodies (for EGFR) as tumor-targeting molecules for the conjugates, which exhibited remarkable efficacy against human tumor xenografts in animal models. We have developed self-immolative disulfide linkers wherein the glutathione-triggered cascade drug release takes place to generate the original anticancer agent. The use of disulfide linkers is attractive beacuse it takes into account the fact that the concentration of glutathione is much higher (>1000 times) in tumor cells than in blood plasma. In order to monitor and elucidate the mechanism of tumor-targeting, internalization, and drug release, several fluorescent and fluorogenic probes using biotin as the tumor-targeting module were developed and used. Then, the progressive occurrence of the designed receptor-mediated endocytosis, drug release, and drug binding to the target protein (microtubules) has been successfully observed and confirmed by means of confocal fluorescence microscopy. These "guided molecular missiles" provide bright prospects for the development of highly efficacious new generation drugs for cancer chemotherapy.     

Macromolecular Conjugates of 4- and 8-Aminoquinoline Compounds

Some members of 4-aminoquinolines and 8-aminoquinolines have been found to provide adjuvant effects when used in combination with anti-cancer drugs. The clinical co-administration of active anti-cancer drugs with other drugs acting as potentiating agents has shown considerable merits when compared to a single-drug administration. Anti-cancer drugs are often toxic when delivered straight, but the bio-reversible drug conjugation of anticancer drugs to water-soluble macromolecular carriers has proved to enhance the therapeutic effectiveness of anticancer drugs. Following facilitated pharmacokinetics pathways, the conjugates, acting as pro-drugs, will release the active drug species in the transformed target cells and their designs are geared towards reducing pharmacological barriers of toxicity, drug resistance and poor bioavailability encountered with currently used anti-cancer drugs. This paper describes the synthesis of water-soluble macromolecular carriers containing 4- and 8-aminoquinolines that are bio-reversibly anchored with cytotoxic drugs. The conjugates and co-conjugates are isolated as water soluble solids and characterized by NMR-spectroscopy.     

Design,Synthesis and Bioevaluation of an EphA2 Receptor‐Based Targeted Delivery System

Because of its overexpression in a range of solid tumors, the EphA2 receptor is a validated target for cancer therapeutics. We recently described a new targeted delivery system based on specific EphA2‐targeting peptides conjugated with the chemotherapeutic agent paclitaxel. Here, we investigate the chemical determinants responsible for the stability and degradation of these agents in plasma. Introducing modifications in both the peptide and the linker between the peptide and paclitaxel resulted in drug conjugates that are both long‐lived in rat plasma and that markedly decrease tumor size in a prostate cancer xenograft model compared with paclitaxel alone treatment. These studies identify critical rate‐limiting degradation sites on the peptide–drug conjugates, enabling the design of agents with increased stability and efficacy. These results provide support for our central hypothesis that peptide–drug conjugates targeting EphA2 represent an innovative and potentially effective strategy to selectively deliver cytotoxic drugs to 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.     

Recent Chemical Approaches for Site-Specific Conjugation of Native Antibodies: Technologies toward Next-Generation Antibody–Drug Conjugates

Antibody–drug conjugates (ADCs), which consist of three components, antibody, linker, and payload, can function as “magic bullets”. These conjugates offer the ability to target drug delivery to specific cells, based on cell-specific recognition and the binding of an antigen by a monoclonal antibody (mAb). In particular, by delivering a cytotoxic payload to cancer cells, ADCs are expected to provide a breakthrough in oncology treatments by providing a way to increase efficacy and decrease toxicity, in comparison with traditional chemotherapeutic treatments. The development of ADC therapeutics has dramatically progressed in the past decade and two ADCs have been approved and used as anticancer drugs in the clinic. However, several critical issues regarding the performance of ADCs are still being discussed and investigated. Indeed, in the past few years, several groups have reported that, changing the number and position of the drug payloads in the ADCs, affects the pharmacokinetics, drug release rates, and biological activity. The use of conventional heterogeneous conjugation methods for ADC preparation results in the drug/antibody ratio and connecting position of the payload having stochastic distributions. Therefore, it is important to investigate how these potential problems can be circumvented through site-specific conjugation. Herein, various site-specific chemical conjugation strategies with native mAbs that are currently used for the production of ADCs, including residue-selective labeling for generating ADCs, disulfide rebridging, and affinity-peptide-mediated site-specific chemical conjugation technologies, are reviewed and described.     

Conjugation of Cisplatin Analogues and Cyclooxygenase Inhibitors to Overcome Cisplatin Resistance

Cyclooxygenase (COX) is an enzyme involved in tumorigenesis and is associated with tumor cell resistance against platinum‐based antitumor drugs. Cisplatin analogues were conjugated with COX inhibitors (indomethacin, ibuprofen) to study the synergistic effects that were previously observed in combination treatments. The conjugates ensure concerted transport of both drugs into cells, and subsequent intracellular cleavage enables a dual‐action mode. Whereas the platinum(II) complexes showed cytotoxicities similar to those of cisplatin, the platinum(IV) conjugates revealed highly increased cytotoxic activities and were able to completely overcome cisplatin‐related resistance. Although some of the complexes are potent COX inhibitors, the conjugates appear to execute their cytotoxic action via COX‐independent mechanisms. Instead, the increased lipophilicity and kinetic inertness of the conjugates seem to facilitate cellular accumulation of the platinum drugs and thus improve the efficacy of the antitumor agents. These conjugates are important tools for the elucidation of the direct influence of COX inhibitors on platinum‐based anticancer drugs in tumor cells.     

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).     

Exploiting Radiation Induction of Antigens in Cancer: Targeted Drug Delivery

Therapeutic antibodies used to treat cancer are effective in patients with advanced-stage disease. For example, antibodies that activate T-lymphocytes improve survival in many cancer subtypes. In addition, antibody–drug conjugates effectively target cytotoxic agents that are specific to cancer. This review discusses radiation-inducible antigens, which are stress-regulated proteins that are over-expressed in cancer. These inducible cell surface proteins become accessible to antibody binding during the cellular response to genotoxic stress. The lead antigens are induced in all histologic subtypes and nearly all advanced-stage cancers, but show little to no expression in normal tissues. Inducible antigens are exploited by using therapeutic antibodies that bind specifically to these stress-regulated proteins. Antibodies that bind to the inducible antigens GRP78 and TIP1 enhance the efficacy of radiotherapy in preclinical cancer models. The conjugation of cytotoxic drugs to the antibodies further improves cancer response. This review focuses on the use of radiotherapy to control the cancer-specific binding of therapeutic antibodies and antibody–drug conjugates.     

Gold(I) N‐Heterocyclic Carbene Complexes with Naphthalimide Ligands as Combined Thioredoxin Reductase Inhibitors and DNA Intercalators

Organometallic conjugates consisting of a gold(I) N‐heterocyclic carbene (NHC) moiety and a naphthalimide were prepared and investigated as cytotoxic agents that interact with both DNA and the disulfide reductase enzyme thioredoxin reductase (TrxR). The complexes were potent DNA intercalators related to their naphthalimide partial structure, inhibited TrxR as a consequence of the incorporation of the gold(I) moiety, and triggered efficient cytotoxic effects in MCF‐7 breast and HT‐29 colon adenocarcinoma cells. Strong effects on tumor cell metabolism were noted for the most cytotoxic complex, chlorido[1‐(3′‐(4′′‐ethylthio‐1′′,8′′‐naphthalimid‐N′′‐yl))‐propyl‐3‐methyl‐imidazol‐2‐ylidene]gold(I) ( 4 d ). In conclusion, the conjugation of naphthalimides with gold(I) NHC moieties provided a useful strategy for the design of bioorganometallic anticancer agents with multiple modes of action.     

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.     

Differentiation of Cancer Stem Cells by Using Synthetic Small Molecules: Toward New Therapeutic Strategies against Therapy Resistance

Despite the existing arsenal of anti-cancer drugs, 10 million people die each year worldwide due to cancers; this highlights the need to discover new therapies based on innovative modes of action against these pathologies. Current chemotherapies are based on the use of cytotoxic agents, targeted drugs, monoclonal antibodies or immunotherapies that are able to reduce or stop the proliferation of cancer cells. However, tumor eradication is often hampered by the presence of resistant cells called cancer stem-like cells or cancer stem cells (CSCs). Several strategies have been proposed to specifically target CSCs such as the use of CSC-specific antibodies, small molecules able to target CSC signaling pathways or drugs able to induce CSC differentiation rendering them sensitive to classical chemotherapy. These latter compounds are the focus of the present review, which aims to report recent advances in anticancer-differentiation strategies. This therapeutic approach was shown to be particularly promising for eradicating tumors in which CSCs are the main reason for therapeutic failure. This general view of the chemistry and mechanism of action of compounds inducing the differentiation of CSCs could be particularly useful for a broad range of researchers working in the field of anticancer therapies as the combination of compounds that induce differentiation with classical chemotherapy could represent a successful approach for future therapeutic applications.     

Tumor Targeting by Conjugation of Chlorambucil with Zwitterionic Near-Infrared Fluorophore for Cancer Phototherapy

Improving the tumor targeting of anticancer drugs to minimize systemic exposure remains challenging. The chemical conjugation of anticancer drugs with various near-infrared (NIR) fluorophores may provide an effective approach to improve NIR laser-induced cancer phototherapy. Towards this end, the selection of NIR fluorophores conjugated with hydrophobic anticancer drugs is an important consideration for targeted cancer photothermal therapy (PTT). In this study, a highly water-soluble zwitterionic NIR fluorophore (ZW800) was prepared to conjugate with a water-insoluble anticancer drug, chlorambucil (CLB), to improve tumor targeting, in vivo biodistribution, and PTT performance. The in vivo results using an HT-29 xenograft mouse model demonstrated that the CLB-ZW800 conjugate not only exhibited high tumor accumulation within 4 h after injection, but also showed rapid body clearance behavior for less systemic toxicity. Furthermore, the tumor tissue targeted by the CLB-ZW800 conjugate was exposed to 808 nm NIR laser irradiation to generate photothermal energy and promote apoptotic cell death for the effective PTT of cancer. Therefore, this study provides a feasible strategy for developing bifunctional PTT agents capable of tumor-targeted imaging and phototherapy by the conjugation of small molecule drugs with the versatile zwitterionic NIR fluorophore.     

Tumor Targeting with Methotrexate-Conjugated Zwitterionic Near-Infrared Fluorophore for Precise Photothermal Therapy

Targeted tumor imaging can effectively enable image-guided surgery and precise cancer therapy. Finding the right combination of anticancer drugs and near-infrared (NIR) fluorophores is the key to targeted photothermal cancer treatment. In this study, a tumor-targetable NIR fluorophore conjugate with rapid body clearance was developed for accurate tumor imaging and effective photothermal therapy (PTT). The methotrexate (MTX) and zwitterionic NIR fluorophore conjugate (MTX-ZW) were prepared by conjugating a folate antagonist MTX with an aminated ZW800-1 analog to increase the tumor targetability for NIR laser-based PTT of cancer. The MTX, known as a poor tumor-selective drug, showed high tumor accumulation and rapid background clearance after conjugation with the highly water-soluble zwitterionic NIR fluorophore up to 4 h post-injection. The photothermal energy was generated from the MTX-ZW conjugate to induce necrotic cell death in the targeted tumor site under 808 nm laser irradiation. Compared with the previously reported MTX conjugates, the MTX-ZW conjugate can be a great candidate for targeted tumor imaging and fluorescence-guided photothermal cancer therapy. Therefore, these results provide a strategy for the design of drug-fluorophore conjugates and elaborate therapeutic platforms for cancer phototherapy.     

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.     

Monoclonal antibody‐targeted polymeric nanoparticles for cancer therapy – future prospects

Although combination therapy for cancer utilising monoclonal antibodies in conjunction with chemotherapeutic drugs has resulted in increases in 5 year survivals, there nevertheless remains significant morbidity and mortality associated with systemic delivery of cytotoxic drugs. The advent of living radical polymerisation has resulted in complex and elegant nanoparticle structures that can be engineered to passively target a drug payload for cancer treatment. This presents a therapeutic modality whereby biodistribution and consequently systemic toxicity can be reduced, while focusing drug delivery to the tumour site. Nanoparticle delivery can be enhanced by attachment of a targeting monoclonal antibody fragment to facilitate tumour cell uptake through endocytosis, and so increase therapeutic efficacy. In this way, monoclonal antibodies can be supercharged by carrying a payload consisting of a cocktail of conventional chemotherapeutic drugs and siRNA. This review will focus on antibody‐targeted polymeric nanoparticles to cancer cells, and methods and technologies for synthesising such antibody‐targeted nanoparticles. The review is confined to polymeric‐based nanoparticles as these offer some advantages over liposomal nanoparticles and may circumvent some of the pitfalls in nanomedicine. Development of these antibody based polymeric nanoparticles and future directions for therapy are highlighted in this review. © 2014 Society of Chemical Industry     

Polymeric Co-Conjugates of Curcumin

The current cancer chemotherapy often results in high toxicity, drug resistance and some other side effects. Multidrug chemotherapy has been found to reduce the side effects associated with the currently used chemotherapy. In this research, we have demonstrated the polymer-drug co-conjugation by incorporating two bioactive agents that is, curcumin and an analogue of ferrocene (both are anticancer agents) to a polymer by a hydrazone linker. The conjugates prepared were characterized by NMR spectroscopy.     

Current Trends and Future Prospects of Molecular Targeted Therapy in Head and Neck Squamous Cell Carcinoma

In recent years, advances in drug therapy for head and neck squamous cell carcinoma (HNSCC) have progressed rapidly. In addition to cytotoxic anti-cancer agents such as platinum-based drug (cisplatin and carboplatin) and taxane-based drugs (docetaxel and paclitaxel), epidermal growth factor receptor-tyrosine kinase inhibitors (cetuximab) and immune checkpoint inhibitors such as anti-programmed cell death-1 (PD-1) antibodies (nivolumab and pembrolizumab) have come to be used. The importance of anti-cancer drug therapy is increasing year by year. Therefore, we summarize clinical trials of molecular targeted therapy and biomarkers in HNSCC from previous studies. Here we show the current trends and future prospects of molecular targeted therapy in HNSCC.     

Design and synthesis of polysapartamide co-drugs of platinum and methotrexate as anticancer agents

Literature reports several recent attempts to load a single drug onto one carrier to improve drug efficacy. An ideal anticancer drug would result from anchoring two anticancer drugs on a single carrier to exploit the advantage of possible synergistic interactions between the drugs, whilst targeting different sites in the cancer cell. This work presents the results of the synthesis and analysis of water-soluble polyaspartamide carriers, which were loaded with platinum along with methotrexate. Platinum was anchored by coordination and methotrexate by amide bonds. In all cases, drug incorporation in the molecule was assessed to be 100%. NMR was used for methotrexate conjugate analysis, while platinum incorporation was evaluated by CHN analysis. The in vitro antiproliferative activity against breast cancer displayed a very good cytotoxic activity by the co-conjugates over the free drugs and their simple conjugates.     

Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment

The chimeric anti-CD30 IgG1, cAC10, conjugated to eight equivalents of monomethyl auristatin E (MMAE) was previously shown to have potent antitumor activity against CD30-expressing tumors xenografts in mice. Moreover, the therapeutic index was increased by lowering the stoichiometry from 8 drugs/antibody down to 2 or 4. Limitations of such 'partially-loaded' conjugates are low yield (10-30%) as they are purified from mixtures with variable stoichiometry (0-8 drugs/antibody), and heterogeneity as the 2 or 4 drugs are distributed over eight possible cysteine conjugation sites. Here, the solvent-accessible cysteines that form the interchain disulfide bonds in cAC10 were replaced with serine, to reduce the eight potential conjugation sites down to 4 or 2. These Cys-->Ser antibody variants were conjugated to MMAE in near quantitative yield (89-96%) with defined stoichiometries (2 or 4 drugs/antibody) and sites of drug attachment. The engineered antibody-drug conjugates have comparable antigen-binding affinities and in vitro cytotoxic activities with corresponding purified parental antibody-drug conjugates. Additionally, the engineered and parental antibody-drug conjugates have similar in vivo properties including antitumor activity, pharmacokinetics and maximum tolerated dose. Our strategy for generating antibody-drug conjugates with defined sites and stoichiometries of drug loading is potentially broadly applicable to other antibodies as it involves engineering of constant domains.     

Controlling Toxicity of Peptide–Drug Conjugates by Different Chemical Linker Structures

The side effects of chemotherapy can be overcome by linking toxic agents to tumor‐targeting peptides with cleavable linkers. Herein, this concept is demonstrated by addressing the human Y₁ receptor (hY₁R), overexpressed in breast tumors, with analogues of the hY₁R‐preferring [F⁷,P³⁴]NPY. First, carboxytetramethylrhodamine was connected to [F⁷,P³⁴]NPY by an amide, ester, disulfide, or enzymatic linkage. Live imaging revealed hY₁R‐mediated delivery and allowed visualization of time‐dependent intracellular release. Next, the fluorophore was replaced by the toxic agent methotrexate (MTX). In addition to linkage through the amide, ester, disulfide bond, or enzymatic cleavage site, a novel disulfide/ester linker was designed and coupled to [F⁷,P³⁴]NPY by solid‐phase peptide synthesis. Internalization studies showed hY₁R subtype selective uptake, and cell viability experiments demonstrated hY₁R‐mediated toxicity that was clearly dependent on the linkage type. Fast release profiles for fluorophore‐[F⁷,P³⁴]NPY analogues correlated with high toxicities of MTX conjugates carrying the same linker types and emphasize the relevance of new structures connecting the toxophore and the carrier.