Advances in the tumor microenvironment have facilitated the development of novel anticancer drugs and delivery vehicles for improved therapeutic efficacy and decreased side effects. Disulfide bonds with unique chemical and biophysical properties can be used as cleavable linkers for the delivery of chemotherapeutic drugs. Accordingly, small molecule-, peptide-, polymer- and protein-based multifunctional prodrugs bearing cleavable disulfide bonds are well accepted in clinical settings. Herein, we first briefly introduce a number of prodrugs and divide them into three categories, namely, disulfide-containing small molecule conjugates, disulfide-containing cytotoxic agent–targeted fluorescent agent conjugates, and disulfide-containing cytotoxic agent–macromolecule conjugates. Then, we discuss the complex redox environment and the underlying mechanism of free drug release from disulfide based prodrugs in in vivo settings. Based on these insights, we analyze the impact of electronics, steric hindrance and substituent position of the disulfide linker on the extracellular stability and intracellular cleavage rate of disulfide containing prodrugs. Current challenges and future opportunities for the disulfide linker are provided at the end.
Prodrugs
Prodrugs are molecules with little or no biological activity that can be metabolized into biologically active parent drugs in the body through enzymatic or chemical reactions or a combination of both. Over the past decade, prodrugs have accounted for more than 10% of the approved new chemical entities per year, making an amazing contribution to the arsenal of fighting disease. An attractive prodrug design strategy is to combine two or more different functional motifs with cleavable linkers. The rationale for using such a prodrug is to take advantage of the potential synergistic or targeted effects of multi-component prodrugs, thereby improving pharmacokinetics and reducing toxicity.
Disulfide
Disulfide bonds are the most important redox-reactive covalent bonds, formed by two cysteine residues in proteins. Disulfide bonds have already been widely found in proteins and play an important role in several important biological processes. Their key function is to accurately guide protein folding and enhance the stability of its tertiary and quaternary structures. Disulfide bonds can be used as cellular redox switches, involved in signal transmission through cascade reaction of thiol–disulfide conversion. With regard to the redox processes in vivo, the thiol pools in different biological compartments determine the redox-biological fate. It mainly includes glutathione/glutathione disulfide (GSH/GSSG), cysteine/cystine (Cys/CySS), thioredoxin-1 (Trx1), glutaredoxin (Grx) and protein disulfide isomerase (PDI). Furthermore, both the components and concentrations of thiol pools are largely different from the blood vessels to the intracellular environment. In plasma, the main thiol species is human serum albumin (HSA, 66.5 kDa) (∼422 μM). HSA’s 585 amino acids residues possess 17 disulfide bridges and only one free thiol at Cys-34, which provides more than 80% of the free thiols in plasma. However, Cys-34 is located in a crevice with limited solvent exposure, severely hindering thiol–disulfide conversion. In contrast to the low free thiol concentration in plasma, GSH, an cysteine-containing tripeptide, is on average 1–10 mM in the cytoplasm. Moreover, tumor cells with active metabolism typically exhibit an elevated production of GSH in the cytoplasm. Therefore, the different thiol pools and the large differences in redox potential from blood vessels to the intracellular environment provide prerequisites for the specific drug release of disulfide-containing prodrug systems. Inspired by its chemical properties and functional roles, disulfide bonds have been used as candidate cleavable linkers in antitumor prodrug design. Connecting chemical units of different functions with disulfide bonds can form multifunctional anticancer prodrugs and achieve tumor-specific release. These results highlight that cleavable disulfide-containing linkers could be a preferred choice in the designing of drug–drug conjugates.
Nanomedicine
Nano drug delivery systems have also been extensively pursued for the delivery of camptothecin (CPT). Nonetheless, the conventional nanocarriers, such as liposomes, micelles, dendrimers, hyperbranched polymers, inorganic nanoparticle and so on, suffer from several drawbacks, including complicated synthesis, uncontrollable structure, low drug loading capacity, high reticuloendothelial system (RES) accumulation and potential immunogenic response. These deficiencies mirror the current state of the limited number of marketed nanomedicines.
Representative disulfide-containing ADCs
| ADC | Target | Cytotoxic payload | Clinical phase | Indications | Sponsor (licensee) |
| Gemtuzumab ozogamicin (Mylotarg®) | CD33 | Calicheamicin derivative | Approved | CD33-positive AML; relapsed or refractory AML | Pfizer |
| Inotuzumab ozogamicin (Besponsa®) | CD22 | Calicheamicin derivative | Approved | Acute lymphoblastic leukaemia | Pfizer |
| Moxetumomab pasudotox (Lumoxiti®) | CD22 | Pseudomonas exotoxin A | Approved | Relapsed or refractory hairy cell leukemia | AstraZeneca |
| Mirvetuximab soravtansine (IMGN853) | FRα | DM4 | Phase III | Ovarian cancer | ImmunoGen |
| Coltuximab ravtansine (SAR3419) | CD19 | DM4 | Phase II | Diffuse large B-cell lymphoma | Sanofi |
| Lorvotuzumab mertansine (IMGN901) | CD56 | DM1 | Phase II stopped | Small cell lung cancer | ImmunoGen |
| AVE9633 | CD33 | DM4 | Phase I stopped | AML | Sanofi |
| Indatuximab ravtansine (BT-062) | CD138 | DM4 | Phase I | Multiple myeloma | Biotest |
| Anetumab ravtansine (Bay-94-9343) | Mesotherin | DM4 | Phase II | Pancreatic cancer | Bayer HealthCare |
| SAR-566658 | CA6 | DM4 | Phase I | CA6-positive advanced STsa | Sanofi |
| SAR408701 | CEACAM5 | DM4 | Phase I | Advanced STsa | Sanofi |
| SAR428926 | LAMP1 | DM4 | Phase I | Advanced STsa | Sanofi |
| HKT288 | Cadherin-6 | DM4 | Phase I | Epithelial ovarian cancer | Novartis Pharmaceuticals |
| Cantuzumab mertansine | CanAg | DM1 | Phase I stopped | CanAg-expressing advanced STsa | ImmunoGen |
| IMGN242 | CanAg | DM4 | Phase II | Gastric or gastroesophageal (GE) junction cancer | ImmunoGen |
| IMGN388 | Integrin αV | DM4 | Phase I | STsa | ImmunoGen |
| BIIB015 | Cripto | DM4 | Phase I | Relapsed/refractory STsa | Biogen |
Optimization of disulfide linkers
For disulfide-containing prodrugs to be selective and potent, the cleavable disulfide linker should be optimized to achieve two key properties: (a) high stability in circulation to avoid premature drug release. And (b) rapid self-immolation rate in tumor cells to ensure adequate parent drug release. However, only a limited number of disulfide based targeting delivery systems have undergone stability evaluation in vivo. However, due to the complex thiol pools in vivo and concentrations of these thiol pools ranging from μM to mM in different compartments, it is challenging to develop stable and potent disulfide-containing prodrugs for tumor targeting delivery.
The structure of the prodrug itself constitutes an internal factor that affects the self-immolation kinetics of disulfide bonds. While drug release from disulfide-containing prodrugs could occur in vivo via enzymatic reaction or hydrolysis in low pH environment, the plausible mechanism for disulfide cleavage and immolation is proposed.
