Hybrid Prodrugs Research Articles

Hybrid nanoassembly indicating a synthetic lethality relationship induces mitotic catastrophe-mediated tumor elimination

Although DNA damage-based chemotherapy has been well investigated for clinical oncology, their effectiveness is greatly hindered by the DNA damage defenses including DNA damage repair and cell cycle arrest. Herein, we propose hybrid prodrug nanoassemblies using core-matched strategy and self-assembly technology to facilitate a synthetic lethal relationship between DNA damage and DNA damage defense. Hydroxycamptothecin (HCPT), a topoisomerase I inhibitor, is chosen as the DNA toxic chemotherapeutic. Norcantharidin (NCTD), a PP2A inhibitor, serves to abrogate homologous recombination repair and G2/M cell cycle arrest initiated by HCPT. Hybrid prodrug nanoassemblies efficiently co-deliver the two drugs with notably different physicochemical properties and lay solid foundation for their synthetic lethal synergy. The developed synthetic lethal synergy potently damages DNA and successfully attenuates DNA damage defense. More intriguingly, such synthetic lethality synergy induces tumor apoptosis by the cell death mechanism of mitotic catastrophe. In both breast tumor and colon tumor mouse models, hybrid prodrug nanoassemblies remarkably suppresses tumor proliferation and improves the safety of chemotherapy. Our results provide a promising strategy to potentiate DNA damage-based translational medicine.

Dual-Stimuli-Activatable Hybrid Prodrug for the Self-Immolative Delivery of an Anticancer Agent and Hydrogen Sulfide with Turn-On Fluorescence.

Stimuli-responsive fluorogenic prodrugs are advantageous for the targeted drug delivery enabling real-time non-invasive monitoring with turn-on fluorescence. We report herein the dual-stimuli (ROS and CA)-responsive thiocarbamate-based prodrug (AM-TCB) for the turn-on fluorogenic delivery of the naphthalimide-based anticancer agent amonafide along with the gasotransmitter hydrogen sulfide (H2 S). A carbamate-based prodrug AM-CB was also designed, capable of releasing the anticancer agent amonafide without any H2 S. The prodrugs were synthesized using multi-step organic synthesis. UV-Vis and fluorescence spectroscopic studies revealed selective reactivity of the boronate ester group of prodrugs towards ROS (primarily H2 O2 ) with the release of amonafide and COS/CO2 via self-immolative processes. Hydrolysis of the generated COS by carbonic anhydrase (CA) produces H2 S. While the prodrug AM-TCB retained the anticancer activity of free amonafide in cancer cells (MDA-MB-231 and HeLa), unlike amonafide, it enhanced the cellular viability of the non-malignant cells (HEK-293). Fluorescence imaging in HeLa cells revealed the simultaneous delivery of the anticancer agent and H2 S from AM-TCB with turn-on fluorescence. Western blot studies further revealed the cytoprotective effects of the released H2 S from AM-TCB. The present adjuvant strategy therefore would be helpful in future for ameliorating the anticancer drug-induced side-effects.

Acid-sensitive stable polymeric micelle-based oxidative stress nanoamplifier as immunostimulating anticancer nanomedicine.

Oxidative stress amplifying compounds could elicit selective killing of cancer cells with minimal toxicity to normal cells and also induce immunogenic cell death (ICD). However, compared to conventional anticancer drugs, oxidative stress amplifying compounds have inferior therapeutic efficacy. It can be postulated that the anticancer therapeutic efficacy and immunostimulating activity of oxidative stress amplifying hybrid prodrug (OSamp) could be fully maximized by employing ultrastable polymeric micelles as drug carriers. In this work, we developed tumour-targeted oxidative stress nanoamplifiers, composed of OSamp, amphiphilic poly(ethylene glycol) methyl ether-block-poly(cyclohexyloxy ethyl glycidyl ether)s (mPEG-PCHGE) and a lipopeptide containing Arg-Gly-Asp (RGD). Tumour targeted OSamp-loaded mPEG-PCHGE (T-POS) micelles exhibited excellent colloidal stability and significant cytotoxicity to cancer cells with the expression of DAMPs (damage-associated molecular patterns). In the syngeneic mouse tumour model, T-POS micelles induced significant apoptotic cell death to inhibit tumour growth without noticeable body weight changes. T-POS micelles also induced ICD and activated adaptive immune responses by increasing the populations of cytotoxic CD4+ and CD8+ T cells. Therefore, these results suggest that T-POS micelles hold great translational potential as immunostimulating anticancer nanomedicine.

Activating cGAS-STING pathway with ROS-responsive nanoparticles delivering a hybrid prodrug for enhanced chemo-immunotherapy

cGAS-STING pathway, as an essential intracellular immune response pathway, has attracted much attention in tumor immunotherapy. However, low metabolic stability of conventional STING agonists limits their clinical application. Recent study shows that chemotherapeutic drugs cisplatin and camptothecin (CPT) can activate cGAS-STING pathway and induce immune response by DNA damage. Nevertheless, the ability of chemotherapeutic drugs to activate STING is so weak that new strategies are required to improve drug delivery efficiency for enhanced DNA damage, and then efficiently activate cGAS-STING pathway. Herein, we have developed a hybrid platinum prodrug (CPT-Pt (IV)) which can be triggered to release cisplatin and CPT in tumor cells. CPT-Pt (IV) with high hydrophobicity is further self-assembled with a ROS sensitive polymer (P1) and mPEG2k-DSPE into ROS responsive nanoparticles (NPs). NPs could accumulate in the tumor site to release cisplatin and CPT, resulting in DNA double damage and finally activating cGAS-STING pathway, inducing DC cells maturation and increasing tumor infiltration of CD8+ T cells on colorectal cancer mouse model. This study showed that common DNA targeted drugs can activate the cGAS-STING pathway in situ via nano delivery system, and enhance the effect of chemotherapy and immunotherapy, which provide a new strategy for clinical antitumor therapy.

Mitigation of doxorubicin-induced cardiotoxicity with an H2O2-Activated, H2S-Donating hybrid prodrug

Doxorubicin (DOX) is one of the most effective anticancer agents in clinical oncology. Its continued use, however, is severely limited by its dose-dependent cardiotoxicity which stems, in part, from its overproduction of reactive oxygen species (ROS) and often manifests itself as full-blown cardiomyopathy in patients, years after the cessation of treatment. Therefore, identifying DOX analogs, or prodrugs, with a diminished cardiotoxic profile is highly desirable. Herein, we describe a novel, H2O2-responsive DOX hybrid codrug (mutual prodrug) that has been rationally designed to concurrently liberate hydrogen sulfide (H2S), a purported cardioprotectant with anticancer activity, in an effort to maintain the antitumor effects of DOX while simultaneously reducing its cardiotoxic side effects. Experiments with cardiomyoblast cells in culture demonstrated a rapid accumulation of prodrug into the cells, but diminished apoptotic effects compared with DOX, dependent upon its release of H2S. Cells treated with the prodrug exhibited significantly higher Nrf2 activation relative to DOX-treated cells. Preliminary indications, using a mouse triple-negative breast cancer cell line sensitive to DOX treatment, are that the prodrug maintains considerable toxicity against the tumor-inducing cell line, suggesting significant promise for this prodrug as a cardioprotective chemotherapeutic to replace DOX.

Current Drifts in Design and Development of Prodrugs

The design and development of prodrug is based on the objectivity to improve the pharmacokinetic and pharmacodynamic profiles including toxicity of the potent chemical entity. Currently, the prodrug approach is extended with conjugating the parent drug with a self immolating molecule or linker which itself assembles into nanoparticles and prolonged the blood circulation and having passive targeting ability. It also includes delivery of the drug at target site having specific pH condition, delivery of drug to brain by endogenous carrier molecule, NQO1 activable prodrug, prodrug with sustained release behavior, receptor targeted, dual action prodrug, activation via specific enzyme like human carboxylesterase, and phospholipase A2. Some other approaches to prodrug development are that the conversion of active pharmaceutical ingredient (API) into ionic liquids (ILs). In this the biocompatible ionic liquids include choline and amino acid esters are used. One of the anticancer drug methotrexate is formulated into ionic liquid form and used as potential anticancer prodrug. In addition to improvement in combination chemotherapy, there is photoresponsive hybrid prodrug that consists of a photoremovable protecting group. The release of doxorubicin and combrestatin A4 is mainly triggered by 405 nm light and 365 nm light respectively and displayed a quasi-sequential release behavior.

H2O2-activatable hybrid prodrug nanoassemblies as a pure nanodrug for hepatic ischemia/reperfusion injury

Self-assembling prodrugs are able to form stable nanoparticles without additional excipients and therefore have gained increasing interest in the field of drug delivery. As a natural derivative of vitamin A, all-trans retinoic acid (atRA) exerts antioxidant, anti-inflammatory, and immunostimulatory effects. However, the clinical translation of atRA has been hampered by its insufficient therapeutic efficacy. In this work, to fully maximize the therapeutic potential of atRA, we developed delicately designed self-assembling RABA (atRA-based hybrid prodrug) as a hybrid prodrug of atRA and hydroxybenzyl alcohol (HBA). RABA could form nanoassemblies and decompose to release atRA and HBA simultaneously in response to hydrogen peroxide (H2O2). In a mouse model of hepatic ischemia/reperfusion (IR) injury, RABA nanoassemblies accumulated in liver preferentially and exerted highly potent antioxidant, anti-inflammatory, and antiapoptotic effects, leading to effective protection of liver from IR injury. RABA nanoassemblies exhibited significantly higher therapeutic efficacy than the combination of equivalent atRA and HBA. Given its H2O2-responsiveness, self-assembling and self-immolating behaviors, and cooperative therapeutic actions, RABA nanoassemblies have great potential as a pure nanodrug for hepatic IR injury. This study also provides a new valuable addition in the development of prodrug self-assemblies that will emerge as next generation of drugs.

Enzyme-responsive hybrid prodrug of nitric oxide and hydrogen sulfide for heart failure therapy.

A hybrid prodrug was synthesized to realize the combined delivery of nitric oxide and hydrogen sulfide. The NO-H2S donor can release nitric oxide and hydrogen sulfide step by step in response to the endogenous enzymes β-galactosidase and carbonic anhydrase, providing potent therapeutic efficacy for heart failure post- myocardial infarction.

A platinum-ruthenium hybrid prodrug with multi-enzymatic activities for chemo-catalytic therapy of hypoxic tumors.

Regulation of tumor hypoxia and redox homeostasis is a promising strategy for cancer therapy. Nanocatalytic medicine has played more and more important roles in this field because it can cleverly convert the efficiency and selectivity of catalysis into high therapeutic efficiency. Herein, we developed a platinum(iv)-ruthenium hybrid prodrug, named as Pt-Ru, for efficient chemo-catalytic synergistic therapy of hypoxic tumors. The ruthenium hybridization endowed the Pt(iv) prodrug with multi-enzyme catalytic activity, that is, mimicking catalase (CAT) to generate O2 in situ, mimicking peroxidase (POD) to produce reactive oxygen species, and mimicking glutathione peroxidase (GPx) to deplete GSH, thus effectively overcoming tumor hypoxia and cisplatin resistance. As a result, Pt-Ru treatment led to a superior anticancer efficacy to cisplatin both in vitro and in vivo. This work suggested redox homeostasis regulation as a tantalizing angle for developing the next generation of platinum drugs.

Fluorescent traceable nanoparticles by co-self-assembly of carbon dot-drug conjugate and biodegradable hyperbranched polymer for diagnosis and therapy

Carbon dots (CDs) have attracted more interest in tumor theranostics, but they suffer from the rapid renal clearance due to small size and high hydrophilicity. To solve such problems, hydrophobic pH-triggered carbon dot-drug conjugate (CDs-Hy-DOX) with high doxorubicin (DOX) content of 48.23% were designed by covalent conjugation of DOX onto the CDs via acid-labile linkage with hydrazine (Hy) as bridge. Then the fluorescent traceable hybrid prodrug nanoparticles were fabricated via co-self-assembly with the CDs-Hy-DOX as pH-sensitive prodrug and a pH/reduction dual-triggered degradable hyperbranched polymer PEG-PO-Cy as polyethylene glycol (PEG)-based surfactant, as well as gatekeeper for pH/reduction dual-triggered DOX release. The hybrid prodrug nanoparticles with hydrodynamic diameter of 220 nm and DOX content of 22.99% were obtained with the optimized co-self-assembling condition. They could release 68.98% of DOX in the simulated tumor microenvironment within 3 days in a sustained release mode, with a premature drug leakage of 7.58%. After the acid-triggered DOX release from the CDs-Hy-DOX, which was accelerated by the pH/reduction dual-triggered degradation of the hyperbranched polymer, the strong fluorescence of CDs-Hy was recovered, demonstrating the promising potential in future tumor nanotheranostics.

Fluorescent turn-on carbon dot-cored pseudo unimolecular prodrug micelles for tumor-specific dual-triggered drug delivery

Polymer prodrugs have attracted intense attention in tumor chemotherapy in the form of nanoparticles, nanogels or micelles. However, it is still a challenge to control their particle size and stability. Here, carbon dot-cored star-like copolymer hybrid prodrug (CD-PMHyDOX-ssPEG) has been designed for the tumor-specific fluorescent turn-on and pH/reduction dual-triggered drug delivery. The fluorescence of CDs was almost completely quenched after the surface modification and drug conjugation via both static and dynamic quenching effects. It could easily self-assemble into pseudo unimolecular prodrug micelles in aqueous system with CDs as fluorescent off-on core, acid-labile polymer prodrug block as shell and reduction-triggered detached PEG block as corona, possessing excellent tumor-specific pH and reduction dual-triggered drug release performance. Furthermore, the fluorescence of CDs could be partially recovered after the drug release because of the relief of the dynamic quenching during the drug release.

Facile fabrication of fluorescent traceable hybrid prodrug nanosponges for tumor intracellular pH-triggered DOX release

Carbon dots (CDs) have been widely investigated in the theranostic agents owing to their excellent stability, cytocompatibility and fluorescent property. However, the CDs-based theranostic agents could be cleared rapidly via the kidney because of their small size. In the present work, CDs-based hybrid nanosponges were designed with the desired diameter as the precursor for the fluorescent traceable prodrug. After conjugation of DOX via acid-labile bonds, the prodrug nanotheranostics was obtained with a DOX content of 19.63 % and a mean hydrodynamic diameter around 120 nm, possessing excellent pH-triggered drug release performance. Furthermore, the fluorescence of the CDs was recovered during the drug release in the simulated tumor intracellular microenvironment, making the proposed fluorescent traceable hybrid prodrug nanosponges promising nanotheranostics in future tumor treatment.

Synthesis and characterization of a photoresponsive doxorubicin/combretastatin A4 hybrid prodrug

To explore the application of photoremovable protecting groups (PPGs) in the field of combination chemotherapy, we designed and synthesized a photoresponsive hybrid prodrug 4 that bearing both doxorubicin (DOX) and combretastatin A4 (CA4). Light triggered drug release investigation found that DOX release was mainly accomplished by 405 nm light while CA4 release was mainly triggered by 365 nm light, i.e., prodrug 4 exhibited a quasi-sequential release behavior when a sequential light irradiation strategy was applied. Cell viability evaluation confirmed the increased cytotoxicity of prodrug 4 compared with individual drugs towards MDA-MB-231cells, indicating that a synergistic effect was achieved.

Hybrid Prodrug Nanoparticles with Tumor Penetration and Programmed Drug Activation for Enhanced Chemoresistant Cancer Therapy.

Despite nanomedicine having shown great potential for reversing cancer cell resistance, the suboptimal transport across multiple biological obstacles seriously impedes its reaching targets at an efficacious level, which remains a challenging hurdle for clinical success in resistant cancer therapy. Here, a lipid-based hybrid nanoparticle was designed to efficiently deliver the therapeutics to resistant cells and treat resistant cancer in vivo. The hybrid nanoparticles (D-NPs/tetrandrine (TET)) are composed of a pH-responsive prodrug 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-doxorubicin (DOX), an efflux inhibitor TET, and a surfactant DSPE-[methoxy (poly(ethylene glycol))-2000] (DSPE-mPEG2000), which hierarchically combatted the sequential physiological and pathological barriers of drug resistance and exhibited prolonged blood circulation, high tumor accumulation, and deep tumor parenchyma penetration. In the meantime, the programmed stepwise activation of encapsulated TET and DOX suppressed the function of resistance-related P-glycoprotein in a timely manner and facilitated the DOX sustained accommodation in tumor cells. Through systematic studies, the results show that such a nanosystem dramatically enhances drug potency and significantly overcomes the DOX resistance of breast cancer with negligible systemic toxicities. These findings provide new strategies to systemically combat chemoresistant cancers.

Synergistic antitumor activity of a self-assembling camptothecin and capecitabine hybrid prodrug for improved efficacy

The direct use of anticancer drugs to create their own nanostructures is an emerging concept in the field of drug delivery to obtain nanomedicines of high drug loading and high reproducibility, and the combination use of two or more drugs has been a proven clinical strategy to enhance therapeutic outcomes. We report here the synthesis, assembly and cytotoxicity evaluation of self-assembling hybrid prodrugs containing both camptothecin (CPT) and a capecitabine (Cap) analogue. CPT and Cap molecules were conjugated onto a short β-sheet-forming peptide (Sup35) to yield three different self-assembling prodrugs (dCPT-Sup35, CPT-Cap-Sup35 and dCap-Sup35). We found that the chemical structure of conjugated drugs could strongly influence their assembled morphology as well as their structural stability in aqueous solution. With a decrease in number of CPT units, the resulting nanostructures exhibited a morphological transformation from nanofibers (dCPT-Sup35) to filaments (CPT-Cap-Sup35) then to spherical particles (dCap-Sup35). Notably, the hybrid CPT-Cap prodrug showed a synergistic effect and significantly enhanced potency against three esophageal adenocarcinoma cell lines compared with the two homo-prodrugs (dCPT-Sup35 and dCap-Sup35) as well as free parent drugs (CPT, 5-Fu and CPT/5-FU mixture (1:1)). We believe this work represents a conceptual advancement in integrating two structurally distinct drugs of different action mechanisms into a single self-assembling hybrid prodrug to construct self-deliverable nanomedicines for more effective combination chemotherapy.

Gemcitabine-camptothecin conjugates: a hybrid prodrug for controlled drug release and synergistic therapeutics.

Drug self-delivery systems represent an important approach to enhance the therapeutic efficacy for cancer therapy. We report the design, synthesis and characterization of a new amphiphilic small molecule prodrug based on two types of anticancer drugs, the hydrophilic gemcitabine and hydrophobic camptothecin, linked by a disulfide bond and abbreviated as GT-CPT. The obtained amphiphilic prodrug conjugates self-assembled into nanoparticles in water and showed strong micellar stability and excellent blood compatibility in vivo. The GT-CPT prodrug conjugates could realize precise drug loading as high as ∼75 wt% demonstrating a carrier-free model for efficient drug delivery. Furthermore, the reduction-responsive disulfide bond enabled controlled drug release in the presence of tumour-specific microenvironment. It was found that each of these hybrid drug components (CPT and GT) not only showed enhanced cytotoxicity individually but also exhibited a prominent synergistic effect on HeLa and MCF-7 cancer cells. This study demonstrated the promising potential of this stimuli-responsive hybrid prodrug conjugate for highly efficient co-delivery of multiple anticancer chemotherapeutics, which could inspire further applications using such hybrid prodrug conjugates for combination cancer chemotherapy.

Cancer Cell-Directed Drug Delivery and Chemopotentiating Effects by GSTP1-Activated Nitric Oxide (NO)-Releasing Prodrug

Overexpressed glutathione S-transferase P1 (GSTP1) has been associated with chemotherapy resistance. GSTP1 activated arylated diazeniumdiolates causes significant DNA damage by nitric oxide (NO) release. Poly(ADP-ribose) polymerases (PARP) inhibitors induces DNA damage. A hybrid prodrug was designed by combining the structural features of established PARP-1 inhibitors and arylated diazeniumdiolates to exacerbate DNA damage. The prodrug is activated by GSTP1 catalyzed reaction with glutathione and releases NO and a moiety that inhibits PARP-1. It has also proven to be effective as anticancer agent in both in vitro and in vivo models. The chemopotentiating effects of the prodrug were also observed in combination with microtubule targeting agents or proteasome inhibitors, accompanied by increase in DNA damage, and induction of apoptosis. Interestingly, S-nitrosation of Cys101 of GSTP1 by the prodrug upon activation by GSTP1 was observed. This modification significantly decreased the catalytic activity of GSTP1, suggesting that the prodrug executes synergistic effects through multiple cellular effects, including direct inhibitory effect on GSTP1. These findings demonstrate that S-nitrosation of GSTP1 may affect proper protein folding and induce unfolded protein and endoplasmic reticulum stress. Our strategy not only activates prodrug by GSTP1 catalytic activity but also inhibits GSTP1 upon activation and delivers the compound at the cancer site. Thus, a three-pronged attack on cancer cells may be afforded by such a novel strategy. Additionally, GSTP1 inhibition by designed prodrug may provide a translational opportunity to target GSTP1 for its role in MAP kinase cascades, which participates in cellular survival and death signaling. As future studies are warranted to confirm our hypothesis, we plan to continue our structure based design to improve the hybrid prodrug for higher GSTP1 selectivity and higher bioavailability.

Esterase-Sensitive Prodrugs with Tunable Release Rates and Direct Generation of Hydrogen Sulfide.

Prodrugs that release hydrogen sulfide upon esterase-mediated cleavage of an ester group followed by lactonization are described herein. By modifying the ester group and thus its susceptibility to esterase, and structural features critical to the lactonization rate, H2 S release rates can be tuned. Such prodrugs directly release hydrogen sulfide without the involvement of perthiol species, which are commonly encountered with existing H2 S donors. Additionally, such prodrugs can easily be conjugated to another non-steroidal anti-inflammatory agent, leading to easy synthesis of hybrid prodrugs. As a biological validation of the H2 S prodrugs, the anti-inflammatory effects of one such prodrug were examined by studying its ability to inhibit LPS-induced TNF-α production in RAW 264.7 cells. This type of H2 S prodrugs shows great potential as both research tools and therapeutic agents.

Esterase‐Sensitive Prodrugs with Tunable Release Rates and Direct Generation of Hydrogen Sulfide

AbstractProdrugs that release hydrogen sulfide upon esterase‐mediated cleavage of an ester group followed by lactonization are described herein. By modifying the ester group and thus its susceptibility to esterase, and structural features critical to the lactonization rate, H2S release rates can be tuned. Such prodrugs directly release hydrogen sulfide without the involvement of perthiol species, which are commonly encountered with existing H2S donors. Additionally, such prodrugs can easily be conjugated to another non‐steroidal anti‐inflammatory agent, leading to easy synthesis of hybrid prodrugs. As a biological validation of the H2S prodrugs, the anti‐inflammatory effects of one such prodrug were examined by studying its ability to inhibit LPS‐induced TNF‐α production in RAW 264.7 cells. This type of H2S prodrugs shows great potential as both research tools and therapeutic agents.

Prostate tumor specific peptide–peptoid hybrid prodrugs

Inspired by naturally occurring host defense peptides, cationic amphipathic peptoids provide a promising scaffold for anti-cancer therapeutics. Herein, we report a library of peptide-peptoid hybrid prodrugs that can be selectively activated by prostate cancer cells. We have identified several compounds demonstrating potent anti-cancer activity with good to moderate selectivity. We believe that these prodrugs can provide a useful design principle for next generation peptide-peptoid hybrid prodrugs.