Old wine in new bottles: Advanced drug delivery systems for disulfiram-based cancer therapy

Journal Pre-proof Old wine in new bottles: Advanced drug delivery systems for disulfiram-based cancer therapy

Anne McMahon, Wu Chen, Feng Li PII:

S0168-3659(20)30009-2

DOI:

https://doi.org/10.1016/j.jconrel.2020.01.001

Reference:

COREL 10097

To appear in:

Journal of Controlled Release

Received date:

20 November 2019

Revised date:

1 January 2020

Accepted date:

2 January 2020

Please cite this article as: A. McMahon, W. Chen and F. Li, Old wine in new bottles: Advanced drug delivery systems for disulfiram-based cancer therapy, Journal of Controlled Release (2019), https://doi.org/10.1016/j.jconrel.2020.01.001

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© 2019 Published by Elsevier.

Journal Pre-proof Old Wine in New Bottles: Advanced Drug Delivery Systems for Disulfiram-based Cancer Therapy Anne McMahon, Wu Chen, and Feng Li* Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL 36849 USA

Fe ng Li, PhD

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720 S. Donahue Dr. Auburn, AL 36849, USA

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* Corresponding author:

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Te l:334- 844- 7406

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Email:FZL0023@a ub ur n.ed u ABSTRACT

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Disulfiram (DSF) is an FDA-approved drug that has been repurposed for cancer treatment. It

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showed excellent anticancer efficacy in combination with copper ions (Cu). Several active clinical trials testing the anticancer efficacy of DSF against various cancers are underway. In this

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review article, we summarized different delivery strategies for DSF-based cancer therapy. In

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many studies, DSF and Cu were delivered in two separate formulations. DSF and Cu formed copper diethyldithiocarbamate [Cu(DDC)2 ] complex which was reported as a major active anticancer ingredient for DSF/Cu combination therapy. Various delivery systems for DSF and Cu were developed to enhance their delivery into tumors. The administration of preformed Cu(DDC)2 complex was also explored to achieve better anticancer efficacy. Several studies developed formulations that were capable of delivering Cu(DDC)2 complex in a single formulation. These novel formulations will address drug delivery challenges and have great potential to improve the efficacy of DSF-based cancer therapy. DSF is an off-patent drug

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Journal Pre-proof molecule. The novel drug formulations of DSF will also serve as a good strategy for developing intellectual properties which will be critical for product development and commercialization. Keywords: Disulfiram; Copper Diethyldithiocarbamate; Cancer Therapy; Drug Delivery Systems; Conjugates; Pro-drugs 1. INTRODUCTION Increasing demand for effective anti-cancer drugs has resulted in researchers looking for FDA

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approved drugs that could be repurposed as anticancer chemotherapeutic agents. The drug

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repurpose approach can significantly reduce the risk of failure in drug development and save

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R&D costs. Disulfiram (DSF), an FDA-approved drug for treating alcoholism, has shown

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promising antineoplastic effects against a wide variety of cancers.[1-3] Several clinical trials are testing the anticancer efficacy of DSF-based therapies for treating various types of cancers

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including breast cancer, prostate cancer, glioma, and others (Table 1). Many clinical trials used

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DSF monotherapy. DSF is metabolized in the body and converted to diethyldithiocarbamate (DDC) which can chelate with copper ions and forms the Cu(DDC) 2 complex.[3] It is believed

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that Cu(DDC)2 plays a significant role in DSF-based cancer therapy. The anticancer efficacy of

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DSF is copper ion-dependent. Since many cancers have higher levels of copper ions than normal tissues, it is hypothesized that DSF could form relatively higher levels of Cu(DDC) 2 in cancer and selectively kill cancer cells. In many other clinical studies, DSF was used in combination with copper which can increase the intratumor copper concentrations and generate higher levels of Cu(DDC)2 to more effectively kill cancer cells. In most of these clinical trials, the oral administration of DSF yields low concentrations of Cu(DDC)2 in tumor regions because DSF has poor bioavailability and it is rapidly metabolized and degraded in the body. Furthermore, the administration of DSF and copper ions in two separate formulations will have difficulties in

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Journal Pre-proof synchronizing the exposure of DSF and copper ions in tumor tissues due to their different pharmacokinetic profiles. Therefore, there is a great need for advanced delivery systems for DSF-based cancer therapy. These delivery systems will have the promise to enhance intratumor drug delivery, improve anticancer potency, and reduce toxicity.[4] Here, we will briefly summarize the anticancer mechanism of DSF-based cancer therapy and discuss recent progress in developing advanced delivery systems for DSF-based cancer therapy.

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Table 1. List of clinical trials for disulfiram-based cancer therapy.

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Journal Pre-proof Tumor type

Drugs

Status

Identifier

Disulfiram/Copper Supplement

Phase II, Recruiting

NCT03323346

Germ Cell Tumor

Disulfiram

Phase II, Recruiting

NCT03950830

Glioblastoma

Disulfiram/Copper Gluconate

Early Phase I, Completed

NCT01907165

Glioblastoma

Disulfiram Metformin

Early Phase I, Recruiting

NCT03151772

Glioblastoma

Disulfiram/Copper Gluconate Temozolomide

Phase II, Recruiting

NCT03363659

Glioblastoma

Disulfiram/Copper Temozolomide Disulfiram/Copper Temozolomide Disulfiram/Copper Gluconate

Phase II, Not Yet Recruiting

NCT01777919

Glioblastoma (Recurrent)

Melanoma

NCT02715609

Phase II, Completed

NCT03034135

Phase I, Active Not Recruiting

NCT02770378

Phase II, Completed

NCT02101008

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Disulfiram/Copper Temozolomide Disulfiram Metronomic temozolomide Disulfiram and Zinc

Phase I/II, Recruiting

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Glioblastoma (Recurrent )

NCT02678975

Phase II/III, Recruiting

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Glioblastoma

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Glioblastoma

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Breast Cancer (Metastatic)

Disulfiram Arsenic trioxide

Phase I, Terminated due to lack of funding

NCT00571116

Melanoma (Stage IV )

Disulfiram

Phase I/II, Completed

NCT00256230

Phase II/III, Completed

NCT00312819

Phase II, Not Yet Recruiting

NCT03714555

Pancreatic Cancer (Metastatic, Disulfiram Recurrent) Gemcitabine Prostate Cancer (Metastatic Disulfiram/Copper Gluconate Castrate-resistant )

Phase I, Recruiting

NCT02671890

Phase I, Active Not Recruiting

NCT02963051

Prostate Cancer (Recurrent)

Completed

NCT01118741

Phase I, Completed

NCT00742911

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Melanoma (Metastatic)

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Non-small Cell Lung Cancer Disulfiram Chemotherapy Pancreatic Cancer (Metastatic )Disulfiram/Copper Gluconate

Disulfiram

Solid Cancer (Refectory, liver) Disulfiram/Copper Gluconate

2. ANTICANCER M ECHANISMS OF DSF The anticancer mechanisms of disulfiram-based therapy were discussed extensively in recent publications.[1, 5] In this review article, we will briefly summarize the anticancer mechanism (Figure 1) and mainly focus on the introduction of different delivery strategies for disulfiram-

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Journal Pre-proof based cancer therapy. DSF is metabolized into DDC in the body. DDC complexes with copper ions to produce Cu(DDC)2, which is believed to be a major anticancer ingredient for DSF-based cancer therapy. Cells treated with Cu(DDC)2 showed similar phenotypic characteristics as those treated with proteasome inhibitors, including the accumulation of poly- ubiquitinated proteins in the cytoplasm.[1-3] Because of these observations, early studies believed that DSF was a proteasome inhibitor.[6, 7] Recent studies revealed that Cu(DDC)2 did not directly inhibit

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proteasomes but rather targeted the p97-NPL4-UFDI pathway.[3] The binding of Cu(DDC)2 to

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the zinc (II) binding thiolate site present in NPL4 proteins resulted in the aggregation of

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NPL4.[8] The aggregation of NPL4 caused the deactivation of P97 segregase, which caused the

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accumulation of misfolded proteins in the endoplasmic reticulum (ER) and eventually cell death. Although the DSF monotherapy could be used for treating cancers, the combination therapy of

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copper will be more effective because of the presence of additional coppers will facilitate the

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formation of more Cu(DDC)2 complex to more effectively kill cancer cells. Studies indicated that Cu(DDC)2 killed cancer cells through the induction of paraptosis.[9] Paraptosis is a non-

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apoptotic cell death that does not need the activation of caspases. Therefore, it is a promising

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approach to kill resistant cancer cells with defective apoptotic pathways. In addition, DSF or DSF/Cu could effectively overcome drug resistance caused by multiple other mechanisms including the presence of cancer stem cells (CSCs), over-expression of drug-resistant transporters, and others. (1) Several studies demonstrated the inhibition of CSCs by DSF. [10-12] CSCs are a subpopulation of cancer cells associated with the development of drug resista nce and cause the relapse of cancers.[13] CSCs often have over-expressed alcohol dehydrogenases (ALDH). DSF could effectively inhibit ALDH and thus deplete CSCs.[14, 15] Hypoxic tumor microenvironments are also responsible, in part, for the self-renewal property of CSCs. The

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Journal Pre-proof hypoxic environment activates NFkB, a transcription factor that promotes cell proliferation, survival, invasion, chemoresistance, and migration. A recent study showed that DSF inhibited the NFkB pathway via stabilization of IkB and thus increasing the efficacy of DSF against CSCs.[16] (2) Over-expression of drug-resistant transporters leads to drug resistance in many cancers. These transporters actively transport their substrates from the cytosol to the extracellular space. Many chemotherapy drugs (e.g., paclitaxel and doxorubicin) are substrates of drug-

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resistant transporters.[17, 18] Over-expression of drug-resistant transporters reduced drug

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concentrations in tumors and resulted in poor therapeutic efficacy. In several studies, DSF

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demonstrated its ability to effectively inhibit drug-resistant transporters and overcome drug

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resistance. Its inhibition of transporters was achieved through the covalent modification of the cysteine residues of drug transporters at both the ATP site and the drug-binding site. DSF can

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inhibit multiple transporters such as P-glycoprotein and multidrug resistance protein 1.[19, 20]

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Thus, the combination of DSF and chemotherapy drugs were used in multiple studies to improve anticancer efficacy in drug-resistant cancers.[21-23] (3) DSF was also used in combination with

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poly(L-glutamic acid)-cisplatin conjugates to treat cisplatin-resistant cancers. DSF can inhibit

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NKkB activation, decrease the intracellular level of glutathione(GSH), increase the expression of pro-apoptotic Bax, and reduce the expression of anti-apoptotic Bcl-2. DSF significantly enhanced the efficacy of cisplatin against resistant lung cancers.[24] Another study also indicated that DSF NPs could overcome drug resistance in ovarian cancer through the inhibition of NKkB. [25] In summary, DSF or DSF/C u has been used alone or in combination with other drugs to effectively treat drug-resistant cancer through multiple different mechanisms.

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MDR transporters

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P97 Segregase ER Stress

NK-kB

Paraptosis Apoptosis

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Cancer Stem Cells

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Figure 1. Anticancer Mechanisms of Disulfiram.

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3. D ELIVERY SYSTEMS FOR DSF Although the oral dosage form of DSF has been used for alcohol aversion, this dosage form is

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ineffective in cancer treatment due to the poor stability of DSF in the gastric environment and

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rapid degradation of DSF in the body.[26] Therefore, a more efficient drug delivery system is needed for the clinical use of DSF as an anticancer drug. DSF formulations (e.g., DSF

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encapsulated nanocarriers or DSF-conjugates) can prevent or minimize the degradation of DSF

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during the circulation in the blood, significantly improve their circulation half- life, and overcome the rapid degradation issue. These delivery systems could also enhance the accumulation and release of DSF in tumor tissues and reduce its exposure in normal tissues. Here, we summarize various DSF delivery strategies developed in previous studies including physical encapsulation methods (Table 2, Figure 2) and conjugation methods (Figure 3). 3.1 Physical Encapsulation (A) Polymer Nanoparticles/mini-rods

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Journal Pre-proof Poly lactic-co-glycolic acid (PLGA) is an FDA-approved biodegradable polymer. The PLGA polymer has been used to prepare DSF nanoparticle (NP) formulations in multiple studies. McConville et al., prepared PLGA mini- rods using a “hot melt extrusion” method. The prepared formulation was administered through the stereotactic injection into the brain to effectively treat glioblastoma multiforme.[27] In another study, Wang et al. prepared DSF PLGA nanoparticles (NPs) via an emulsion-solvent evaporation method and used them to treat liver cancers.[10, 28]

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In this study, the NP formulation significantly improved the in vivo stability of DSF and

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prolonged its half- life in serum from 2 minutes to 7 hours. DSF NPs achieved remarkable in vivo

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anticancer efficacy. The selection of PLGA polymer, stabilizer, and sonication time all affected

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the properties of DSF PLGA NPs.[28] To further improve the drug delivery performance, PLGA NPs were modified through the attachment of hydrophilic polyethylene glycol (PEG) onto the

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surface of NPs. PEG modification is a well-established method to prevent opsonization and

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improve systemic circulation time. DSF encapsulated PEG-PLGA NPs showed prolonged systemic circulation half- life and improved delivery to tumor sites. In vivo studies showed that

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treatment with DSF PEG-PLGA NPs significantly reduced tumor size in a mice tumor

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model.[29] In another study, Song et al. developed a PEG-PLA/poly(ε-caprolactone) (PCL) hybrid NP for DSF delivery. The DSF loading capacity was increased through the optimization of PEG-PLA/PCL content ratios.[30] Furthermore, Fasehee et al. attached folate to the surface of DSF PEG-PLGA NPs.[31] This active tumor targeting strategy improved the cellular uptake of NPs in tumor cells expressing folate receptors via receptor-mediated endocytosis. [31]

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(B) Lipid NPs

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Figure 2. (A) Co-delivery of doxorubicin and disulfiram via core–shell–corona NPs (Ref. 47). Copyright 2018, Royal Society of Chemistry. (B) Nanocrystals for co-delivery of Paclitaxel and Disulfiram (Ref.21). Copyright 2019, Elsevier. (C) Disulfiram-Loaded pH-Triggered PEG-Shedding TAT Peptide-Modified Lipid NPs (Ref. 33). Copyright 2015, American Chemical Society.

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Lipid NPs have also been explored as delivery systems for DSF. Banerjee et al., designed

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vitamin E-TPGS surface- modified lipid NPs for DSF delivery with high drug encapsulation efficiency. The TPGS modified DSF lipid NPs showed improved stability and better anticancer efficacy than free DSF drug or unmodified DSF lipid NPs.[32] In another study, Zhang et al. designed pH-responsive TAT peptide-decorated lipid NPs for DSF delivery.[33] TAT peptide has been used to modify NPs to enhance their intra-tumor penetration and cellular uptake by tumor cells. However, the non-specific cellular uptake by normal cells is a concern. [34, 35] In this study, TAT peptide modified lipid NPs were further decorated with pH-responsive PEGPGA. [33] The TAT peptide was masked to prevent non-specific cellular uptake in normal

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Journal Pre-proof organs/tissues. Once these NPs were exposed to the acidic tumor microenvironment, PEG-PGA was detached and TAT peptide was exposed to facilitate cellular uptake of NPs. In another study, Liu et al. used biotin-PEG2000-distearyl phosphatidyl ethanolamine (biotin-PEG-DSPE) to modify DSF- lipid NPs to enhance tumor targeting.[36] PEG provided steric protection, while biotin on the outmost layer of NPs functioned as a tumor-targeting moiety to enhance the delivery of NPs into cancer cells overexpressing biotin receptors. In vivo studies showed that the

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biotin-PEG-DSPE modified DSF lipid NPs accumulated in tumors and effectively inhibited

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breast cancer growth in a mice tumor model.

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(C) Micelles

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Micelles have been successfully used for enhancing the delivery of hydrophobic drugs.[37-42]

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Several studies prepared micelle-based DSF delivery systems where DSF was loaded in the hydrophobic region of micelles. Tawari et al. prepared a pluronic micelle to deliver DSF for

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breast cancer treatment.[43] The poor stability of micelles is a significant concern for their in

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vivo application. To address this issue, Duan et al. prepared a cross- linked micelle formulation to

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deliver DSF.[44] Micelles cross- linked with redox-sensitive bonds remained intact during the circulation in the blood and broke apart to release encapsulated DSF under tumor redox conditions. [44] To improve DSF drug loading capacity and NP stability, Miao et al. developed mixed micelles composed of poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-PLGA), poly(e-caprolactone) (PCL), and medium-chain triglyceride (MCT).[45] PCL reduced DSF leakage from the micelles. MCT reduced the crystallinity in micelle cores and improved DSF drug loading capacity. [45] A similar strategy was used in another study where mixed micelles composed of PEG-PCL, PCL, and MCT were prepared to increase the DSF loading capacity and improve in vivo performance.[46] To optimize drug loading efficiency, authors also calculated

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Journal Pre-proof the Flory-Huggins interaction parameters to predict the miscibility between DSF and the hydrophobic core of micelles. Micelles have also been used to co-deliver DSF and doxorubicin (DOX) for combination therapy.[47, 48] In one study, poly(caprolactone)-b-poly(L-glutamic acid)-g-methoxy poly (ethylene glycol) was used for the co-delivery of hydrophobic DSF and hydrophilic DOX.[47] The polymer formed core-shell micelle NPs which loaded DSF in the hydrophobic core through

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hydrophobic interactions and DOX in the anionic shell through electrostatic interactions. The

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combination therapy showed a synergistic effect in killing breast cancer cells. These NPs also

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enhanced drug delivery into tumors and improved anticancer efficacy in vivo. In another study,

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DOX was conjugated to poly(styrene-co-maleic anhydride) (SMA) through an acid cleavable linker.[48] DSF was then encapsulated in micelle NPs formed by the polymer-DOX conjugate.

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The micelle NPs showed a fast release of DSF and a slow release of DOX in response to the

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cleavage of the acid-sensitive linker at the acidic tumor environment. DSF inhibited drugresistant transporters and sensitized resistant cancers to DOX treatment. This co-delivery system

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(D) Nanocrystals

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showed great potential for treating drug-resistant cancers.

Nanocrystals are an emerging delivery system for poorly water-soluble drugs. Nanocrystals consist of pure drug crystals without a delivery carrier or with only minimal excipients. Therefore, nanocrystals usually have higher drug loading capacities than other delivery systems. Recent studies prepared hybrid DSF-Paclitaxel (PTX) nanocrystals for co-delivery of these two drugs. DSF-PTX nanocrystals were prepared with an anti-solvent precipitation method and stabilized with β- lactoglobulin.[21, 22] The optimized formulation was rod- like NPs with a particle size of around 160 nm and high drug loading efficacies. PTX-DSF hybrid NPs entered

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Journal Pre-proof cells through caveolar endocytosis and showed significantly excellent cellular uptake. PTX-DSF NPs significantly reduced intracellular ATP and GST activities and effectively killed PTX resistant lung cancer cells. This system also showed much better efficacy than paclitaxel alone in the inhibition of tumor growth in an in vivo MDR lung tumor model. Table 2: Delivery carriers for encapsulating DSF. Materials

Cancer Type

Micelles

Ref: [28] Ref: [10] Ref: [29] Ref: [30] Folate Receptor, Ref: [31]

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Liver Cancer Breast Cancer

Ref: [33] Ref: [36]

Solid Lipid Core; TPGS

Breast Cancer

Ref: [32]

SMA-ADH-DOX poly(styrene-co-maleic anhydride) PEG5K-PCL5K, PCL5k, MCT PEG5k-PLGA2K, PCL3.4K ,MCT

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Solid Lipid Core; HS-PEG1k-TATp; PGA-g-PEG Lipid Core; Biotin-PEG2k-DSPE

Drug Resistant Breast Cancer Breast Cancer Hepatocellular carcinoma Hepatocellular carcinoma

Combination Therapy, Ref: [48] Cross-linked Micelles, Ref: [44] Ref: [46] Ref: [45]

Breast Cancer

Combination Therapy, Ref: [47]

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Lipid NPs

Non-small cell lung cancer Liver cancer stem cells Glioma Breast Cancer Breast Cancer

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Polymer NPs PLGA PLGA PEG-PLGA PEG-PLGA/PCL Folate-PEG-PLGA

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Carriers

PEG-PGlu-PCL

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Nanocrystals PTX-DSF Solution; Denatured beta-LG Drug Resistant Lung Cancer

Combination Therapy, Ref: [21,22]

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3.2 DSF Conjugates Drug conjugate is a type of prodrug in which drugs are chemically linked with small molecules,

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polymers (e.g., PEG), lipids (e.g., cholesterol), or biomacromolecules (e.g., albumin).[49-51] The drug conjugates have superior physicochemical properties and minimal pre- mature drug release or leakage. With an appropriate linker, the release of drugs can be triggered by the tumor microenvironment.[52, 53] Several studies prepared conjugate delivery systems of DDC, a reduced form metabolite of DSF (Figure 2). He et al. used lactobionic acid (LBA) modified poly[(2-(pyridin-2-yldisulfanyl) ethyl acrylate)-co-[poly(ethylene glycol)]] polymer (PDA-PEGLBA) to prepare the DDC conjugates as tumor-targeting delivery carriers.[54] DDC was conjugated to PDA-PEG-LBA through a disulfide bond. LBA served as a targeting ligand which

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Journal Pre-proof facilitated the binding and cellular uptake of drug conjugates in tumor cells via D- galactose receptor- mediated endocytosis. This conjugate showed potent anticancer activities in a peritoneal metastatic ovarian tumor model. In another study, Bakthavatsalam et al. synthesized a prodrug of DDC containing a γ-glutamyl transferase (GGT) responsive linker.[55] GGT is overexpressed in many types of cancers making it a good enzyme for tumor cell-specific activation of prodrugs.[56] This DDC prodrug conjugate successfully masked the ability of DDC to complex with

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copper ions. Once the prodrug was delivered to tumor cells, DDC was released inside the cells

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through the effects of GGT enzyme. The released DDC complexed with intracellular copper ions

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to generate the Cu(DDC)2 complex.[55] Pan et al. also synthesized an H2 O 2-response DDC

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prodrug which could release DDC and quinone methide (QM) in response to the high level of H2 O 2 in tumor tissues.[57] The released DDC formed Cu(DDC)2 complex with endogenous

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copper ions to induce cells. The QM reduced the level of intracellular GSH and amplified

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oxidative stress, and thus more effectively induced cancer cell death. The synthesis of lipid conjugates is another promising method for the delivery of anticancer drugs.[52, 53] Sheppard et

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al. synthesized a series of lipid conjugated DSF. These lipophilic DSF prodrugs have the

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potential to be used for cancer therapy, although they were used as antimicrobial agents in this study.[58] In another study, Zhou et al. reported a method to synthesize DSF polymer prodrugs. In this study DSF was used as an initiator to synthesize DS-poly(ethylene glycol) methyl ether acrylate (DS-PEGMEA) through the reversible addition- fragmentation chain transfer (RAFT) polymerization.[59] DS-PEGMEA could form NPs through self-assembly and additional free DSF were loaded into the NPs through physical encapsulation. This NP formulation could effectively induce apoptosis in melanoma cells without causing significant toxicity to normal cells.

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Figure 3. DSF conjugate prodrugs. (A) H2O2-responsive prodrug (Ref. 57); (B) g-Glutamyl Transferase-responsive prodrug (Ref. 55); (C) Lipid conjugate with disulfide bond (Ref. 58); (D) Polymer conjugate with disulfide bond (Ref. 54).

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4. D ELIVERY SYSTEMS FOR COPPER The presence of copper ions can greatly enhance the anticancer efficacy of DSF. Copper ions

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play an essential role in promoting tumor growth and many cancer cells show higher copper ion concentrations than normal cells/tissues. As a result, the administration of DSF may lead to

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higher Cu(DDC)2 complex concentrations in cancer cells than in normal cells. This difference

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may be utilized to enhance anticancer effects and reduce toxicity in normal tissues. However, in many cases, the concentration of Cu(DDC)2 in tumor cells following DSF monotherapy was insufficient to have significant anticancer efficacy. [3] Therefore, copper ions were coadministered with DSF as a combination therapy to improve the anticancer efficacy. In many clinical studies, copper was administrated orally as a form of copper gluconate or through intravenous injection of cupric chloride solution. These methods of copper administration were convenient because of the commercial availability of these copper formulations. However, direct administration of copper ions increased overall copper ion levels in the body, resulting in the loss of selectivity against cancer cells. In addition, copper ions usually have a short systemic half- life 14

Journal Pre-proof and are rapidly eliminated from the body. Delivery systems that could improve the in vivo stability and enhance the delivery of copper ions into tumors would enhance anticancer efficacy. Zhou et al. converted soluble copper ions into lipophilic copper oleate complexs and loaded the complex into membranes of PEGylated liposomes.[60] The liposome formulated copper oleate complex showed prolonged circulation time. The combination of copper oleate complex liposomes and DSF NPs showed a synergistic anticancer effect in mice bearing hepatoma

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xenografts. Wu et al. designed hollow mesoporous silica NPs for co-delivery of DSF and copper

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ions. The NPs were not toxic since DSF and copper ions were physically separated in different

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compartments of the NPs. Once the NPs reached the tumor tissues, the acidic tumor

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microenvironment triggered the release of DSF and copper ions, and the formation of Cu(DDC)2

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complexes in tumor tissues which effectively killed tumor cells.[61]

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5. CU(DDC)2 DELIVERY SYSTEMS Due to the critical role of Cu(DDC)2 in DSF-based cancer therapy, the direct administration of pre-formed Cu(DDC)2 complexes has been explored as a novel strategy to improve anticancer

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efficacy. However, the poor aqueous solubility of Cu(DDC)2 has posed an obstacle to its clinical

(Figure 4).

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use. Recently, several advanced delivery systems were developed for the delivery of Cu(DDC) 2

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Cu2+

A

B

Cu2+ Liposomes

Cu(DDC)2

Denature

DDC

Albumin

Cu(DDC)2 Liposomes

Albumin NPs

Drug Loaded Albumin NPs

C

+

D Cu 2+

Mixing Channel DDC-Na

Vortex

Cu(DDC)2 NPs

Outlet

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+ CuCl2 PLA-PEG

Cu2+

Cu(DDC)2 NPs

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DDC-

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Figure 4. Delivery systems for Cu(DDC)2. (A) Cu(DDC)2 liposomes (Ref. 62). (B) Cu(DDC)2 BSA NPs (Ref.68). (C) Cu(DDC)2 NPs prepared with SMILE technology (Ref. 9). Copyright 2018, American Chemical Society. (D) Cu(DDC)2 NPs prepared with a microfluidic device (Ref.70). Copyright 2019, Elsevier Ltd.

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5.1 Liposomes Liposomes are well-developed drug delivery systems utilized in several clinically used drug

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products. Wehbe et al. developed a method to use liposomes as nanoscale reactors to synthesize

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Cu(DDC)2 complex NPs within the aqueous core of liposomes (Figure 4A).[62-64] Briefly, copper ion- loaded liposomes were prepared first. Then, DDC was mixed with the copper ion-

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loaded liposomes. DDC crossed the liposome membranes and complexed with copper ions

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present in the aqueous core of liposomes. This liposome-based method proved to be an efficient approach to prepare Cu(DDC)2 NPs with excellent anticancer efficacy in a mice tumor model. Liposomes can be further modified with tumor-targeting moieties to improve their targeting specificity. Mareng et al. used a similar method to prepare tumor-targeting Cu(DDC)2 liposomes. They modified the surface of the liposomes with hyaluronic acids which could target CD44expressing pancreatic cancer stem cells. These liposomes showed significant inhibition of tumor growth. [65]

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Journal Pre-proof 5.2 Albumin NPs Albumin is the most abundant protein in plasma and represents 55% of blood proteins.[66] Albumin is used by the body to transport different endogenous molecules in the blood. Albumin has been frequently used to prepare biomimetic nanoscale delivery carriers because of its outstanding drug loading/conjugating abilities and inherent transporter properties. Tumor tissues often showed high concentrations of albumin and over-expression of albumin binding receptors.

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This special feature could be utilized for tumor targeting.[67] Recent studies reported the use of

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bovine serum albumin (BSA) for Cu(DDC)2 delivery (Figure 4B). Zhao et al. prepared BSA NPs through the denaturation of BSA by urea/NaBH4 . Pre-formed Cu(DDC)2 complex and

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Regorafenib (Rego) were physically encapsulated in BSA NPs.[68] In addition, the authors

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modified the surface of BSA NPs with mannoses. The developed dual- targeting NPs could target

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both secreted protein acidic and rich in cysteine (SPARC) and mannose receptors (MR). SPARC is overexpressed both in cancer cells and in pro-tumor M2 macrophages. The NP formulation

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could target both cancer cells and M2 macrophages and demonstrated enhanced anticancer

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effects through induction of apoptosis, inhibition of angiogenesis, upregulation of intracellular ROS, and re-polarization of tumor-associated macrophages (TAM). In another study, Zhou et al.

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prepared transferrin receptor binding peptide (T12) and mannose decorated dual-targeting BSA NPs to co-deliver Cu(DDC)2 and Rego for treating glioma.[69] These NPs could effectively cross the blood-brain barrier (BBB) with the assistance of the transferrin receptor (TFR) and SPARC. After crossing the BBB, NPs could also target both M2 TAM and cancer cells through MR and SPARC. This biomimetic NP formulation repolarized M2 TAM and showed enhanced anticancer efficacy in glioma bearing mice.[69]

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Journal Pre-proof 5.3 SMILE Technology Recently, our lab developed a SMILE (stabilized metal ion ligand comple x) technology as a facile method to prepare Cu(DDC)2 NPs (Figure 4C).[9] In this method, Cu(DDC)2 NPs were prepared by mixing a DDC solution and copper chloride solutions in the presence of appropriate stabilizers. The stabilizers adsorb onto the surface of in situ formed Cu(DDC)2 NPs and prevent the large aggregations. Compared with micelle formulations of Cu(DDC)2 prepared with the

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conventional film-dispersion method, the SMILE method prepared Cu(DDC)2 NPs with

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significantly higher drug concentrations. Unlike many drug- loaded NPs prepared with conventional methods where drugs were dispersed in the polymer matrix, SMILE method

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produced NPs composed of a Cu(DDC)2 complex core surrounded by stabilizers. This special

re

feature contributed to the high drug loading in NPs prepared with the SMILE technology. A

lP

variety of stabilizers have been successfully used to prepare Cu(DDC)2 NPs with the SMILE technology. The selection of stabilizers had a significant influence on the particle size, drug

na

concentration, and stability of Cu(DDC)2 NPs. An ideal stabilizer should have two essential

ur

functional components: an “anchoring” component to facilitate the adsorption of stabilizers onto the surface of Cu(DDC)2 core, and a hydrophilic component to provide sufficient steric

Jo

protection, minimize the aggregation, and improve the stability of Cu(DDC)2 NPs. The PEGPLA is an excellent stabilizer for preparing Cu(DDC)2 NPs. The PEG block provided steric projection and reduce opsonization. The PLA block interacted with Cu(DDC)2 core through multiple forces including coordination bonds and hydrophobic interactions. The PEG-PLA Cu(DDC)2 NPs demonstrated excellent stability in serum and during long term storage. In vitro studies indicated that PEG-PLA Cu(DDC)2 NPs effectively killed drug-resistant prostate cancer cells which were resistant to paclitaxel. Anticancer mechanism studies also indicated that NPs killed cancer cells through the induction of paraptosis (a non-apoptotic cell death) without

18

Journal Pre-proof activation of caspase 3/7. To further improve the SMILE technology, a 3D-printed microfluidicdevice was developed for scalable continuously production of Cu(DDC) 2 NPs (Figure 4D).[70] By using this device, we could have better control of the production process and the ability to produce NPs on large scales. Both experimental data and computational simulation results indicated that the process parameter (i.e., flow rate) had a significant impact on the mixing process and affected the properties of generated Cu(DDC)2 NPs. This method has been

of

successfully used to prepare Cu(DDC)2 NPs with BSA as the stabilizer. The BSA Cu(DDC) 2

ro

NPs demonstrate excellent anticancer efficacy against triple-negative breast cancers.

re

-p

6. FUTURE PERSPECTIVES DSF has a great promise to be used as an anticancer agent. The development of an appropriate formulation is critical for its clinical use. In this review article, we summarized different delivery

lP

strategies have been developed for DSF-based cancer therapy. Most of these systems were tested

na

with proof-of-concept in vitro studies or in vivo pre-clinical animal studies. To achieve optimal anticancer efficacy and reduce systemic toxicity, we should optimize the physicochemical

ur

properties of NPs (e.g., particle size, zeta potential, and surface functionalization) which are

Jo

critical for in vivo anticancer performance. It is expected that additional novel materials, formulations, and fabrication methods will be developed to further improve in vivo delivery performances. Furthermore, the use of novel tumor-targeting molecules including antibodies, nanobodies, aptamers, peptides, and others will be another valuable strategy to improve delivery into tumors via active tumor targeting. The biological barriers associated with tumor microenvironments are a significant obstacle preventing effective drug delivery into tumors. Strategies targeting these barriers (e.g., cancer-associated fibrosis cells, and extracellular matrixes) could be utilized to improve the delivery of NPs into tumors.

19

Journal Pre-proof Many different delivery strategies have been developed and have demonstrated great potentials for DSF-based cancer therapy in pre-clinical studies. To facilitate clinical translations, these delivery systems should be extensively evaluated with additional studies to characterize their safety and anticancer efficacy. Although new materials might offer additional benefits, the use of excipients that have already been approved by the FDA or from the generally recognized as safe (GRAS) list could minimize regulatory obstacles and shorten the R&D time. The development of

of

a scalable production process is also critical for the manufacturing and commercialization of

ro

DSF-based formulations. In addition to these technical issues, there are also additional regulatory

-p

and marketing issues that may affect the development and commercialization of DSF derived

re

drug products. Due to the lack of patent protection on DSF drug molecule per se, there is a lack of strong motivation for pharma companies to invest in clinical trials for DSF-based cancer

lP

therapies. Therefore, strategies for developing intellectual properties and utilizing proper

na

regulatory guidelines will be critical for the success of product development and commercialization.[71] In this case, the design of novel formulations will not only improve the

Jo

product development.

ur

anticancer efficacy but also contribute to the development of intellectual properties to facilitate

ACKNOWLEDGEMENT

This project was supported by Auburn University start-up fund (F. Li) and Launch Innovation Award (F, Li). REFERENCES [1] E. Ekinci, S. Rohondia, R. Khan, Q.P. Dou, Repurposing Disulfiram as An Anti-Cancer Agent: Updated Review on Literature and Patents, Recent Pat Anticancer Drug Discov, 14 (2019) 113-132. [2] N. Ding, Q. Zhu, Disulfiram combats cancer via crippling valosin-containing protein/p97 segregase adaptor NPL4, Transl Cancer Res, 7 (2018) S495-S499. [3] Z. Skrott, M. Mistrik, K.K. Andersen, S. Friis, D. Majera, J. Gursky, T. Ozdian, J. Bartkova, Z. Turi, P. Moudry, M. Kraus, M. Michalova, J. Vaclavkova, P. Dzubak, I. Vrobel, P.

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Breast Cancer (Metastatic)

Drugs

Status

Jo

Tumor type

ur

Table 1. List of clinical trials for disulfiram-based cancer therapy. Identifier

Disulfiram/Copper Supplement

Phase II, Recruiting

NCT03323346

Germ Cell Tumor

Disulfiram

Phase II, Recruiting

NCT03950830

Glioblastoma

Disulfiram/Copper Gluconate

Early Phase I, Completed

NCT01907165

25

Journal Pre-proof Glioblastoma

Disulfiram

Early Phase I, Recruiting

NCT03151772

Metformin Disulfiram/Copper Gluconate Temozolomide

Phase II, Recruiting

NCT03363659

Glioblastoma

Disulfiram/Copper Temozolomide

Phase II, Not Yet Recruiting

NCT01777919

Glioblastoma

Disulfiram/Copper Temozolomide

Glioblastoma

Disulfiram/Copper Gluconate

Glioblastoma (Recurrent )

Disulfiram/Copper

-p

ro

of

Glioblastoma

NCT02678975

Phase I/II, Recruiting

NCT02715609

Phase II, Completed

NCT03034135

Phase I, Active Not Recruiting

NCT02770378

Phase II, Completed

NCT02101008

Jo

ur

na

lP

re

Phase II/III, Recruiting

Temozolomide

Glioblastoma (Recurrent)

Disulfiram Metronomic temozolomide

Melanoma

Disulfiram and Zinc

26

Journal Pre-proof Melanoma (Metastatic)

Disulfiram

Phase I, Terminated due to lack of funding

NCT00571116

Arsenic trioxide Disulfiram

Phase I/II, Completed

NCT00256230

Non-small Cell Lung Cancer

Disulfiram

Phase II/III, Completed

NCT00312819

of

Melanoma (Stage IV )

Disulfiram/Copper Gluconate

Pancreatic Cancer (Metastatic, Recurrent)

Disulfiram

Phase II, Not Yet Recruiting

NCT03714555

NCT02671890

Phase I, Active Not Recruiting

NCT02963051

lP

Phase I, Recruiting

ur

Disulfiram/Copper Gluconate

Jo

Prostate Cancer (Metastatic Castrateresistant )

na

Gemcitabine

re

-p

Pancreatic Cancer (Metastatic )

ro

Chemotherapy

Prostate Cancer (Recurrent)

Disulfiram

Completed

NCT01118741

Solid Cancer (Refectory, liver)

Disulfiram/Copper Gluconate

Phase I, Completed

NCT00742911

27

Journal Pre-proof

Table 2: Delivery carriers for encapsulating DSF. Carriers

Materials

Cancer Type

R R

Solid Lipid Core; TPGS

Breast Cancer

R

SMA-ADH-DOX poly(styrene-co-maleic anhydride) PEG5K-PCL5K, PCL5k, MCT PEG5k-PLGA2K, PCL3.4K ,MCT

Drug Resistant Breast Cancer Breast Cancer Hepatocellular carcinoma Hepatocellular carcinoma

C C R R

PEG-PGlu-PCL

Breast Cancer

C

-p

ro

Liver Cancer Breast Cancer

na

lP

Micelles

R R R R F

Solid Lipid Core; HS-PEG1k-TATp; PGA-g-PEG Lipid Core; Biotin-PEG2k-DSPE

re

Lipid NPs

Non-small cell lung cancer Liver cancer stem cells Glioma Breast Cancer Breast Cancer

of

Polymer NPs PLGA PLGA PEG-PLGA PEG-PLGA/PCL Folate-PEG-PLGA

ur

Nanocrystals PTX-DSF Solution; Denatured beta-LG Drug Resistant Lung Cancer

Jo

Figure 1. Anticancer Mechanisms of Disulfiram. Figure 2. (A) Co-delivery of doxorubicin and disulfiram via core–shell–corona NPs (Ref. 47). Copyright 2018, Royal Society of Chemistry. (B) Nanocrystals for co-delivery of Paclitaxel and Disulfiram (Ref.21). Copyright 2019, Elsevier. (C) Disulfiram-Loaded pH-Triggered PEG-Shedding TAT Peptide-Modified Lipid NPs (Ref. 33). Copyright 2015, American Chemical Society. Figure 3. DSF conjugate prodrugs. (A) H2O2-responsive prodrug (Ref. 57); (B) g-Glutamyl Transferase-responsive

28

C

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prodrug (Ref. 55); (C) Lipid conjugate with disulfide bond (Ref. 58); (D) Polymer conjugate with disulfide bond (Ref. 54). Figure 4. Delivery systems for Cu(DDC)2. (A) Cu(DDC)2 liposomes (Ref. 62). (B) Cu(DDC)2 BSA NPs (Ref.68). (C) Cu(DDC)2 NPs prepared with SMILE technology (Ref. 9). Copyright 2018, American Chemical Society. (D) Cu(DDC)2 NPs prepared with a microfluidic device (Ref.70). Copyright 2019, Elsevier Ltd.

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HIGHLIGHTS Anticancer mechanisms of disulfiram.

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Development of advanced delivery systems for disulfiram-based cancer therapy.

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