Critical Design Strategies Supporting Optimized Drug Release from Polymer-Drug Conjugates.

The importance of an adequate linking moiety design that allows controlled drug(s) release at the desired site of action is extensively studied for polymer-drug conjugates (PDCs). Redox-responsive self-immolative linkers bearing disulfide moieties (SS-SIL) represent a powerful strategy for intracellular drug delivery; however, the influence of drug structural features and linker-associated spacers on release kinetics remains relatively unexplored. The influence of drug/spacer chemical structure and the chemical group available for conjugation on drug release and the biological effect of resultant PDCs is evaluated. A "design of experiments" tool is implemented to develop a liquid chromatography-mass spectrometry method to perform the comprehensive characterization required for this systematic study. The obtained fit-for-purpose analytical protocol enables the quantification of low drug concentrations in drug release studies and the elucidation of metabolite presence. and provides the first data that clarifies how drug structural features influence the drug release from SS-SIL and demonstrates the non-universal nature of the SS-SIL. The importance of rigorous linker characterization in understanding structure-function correlations between linkers, drug chemical functionalities, and in vitro release kinetics from a rationally-designed polymer-drug nanoconjugate, a critical strategic crafting methodology that should remain under consideration when using a reductive environment as an endogenous drug release trigger.

Self-Immolative RAFT-Polymer End Group Modification.

Reversible modifications of reversible addition-fragmentation chain transfer (RAFT)-polymerization derived end groups are usually limited to reductive degradable disulfide conjugates. However, self-immolative linkers can promote ligation and traceless release of primary and secondary amines as well as alcohols via carbonates or carbamates in ß-position to disulfides. In this study, these two strategies are combined and the concept of self-immolative RAFT-polymer end group modifications is introduced: As model compounds, benzylamine, dibenzylamine, and benzyl alcohol are first attached as carbamates or carbonates to a symmetrical disulfide, and in a straightforward one-pot reaction these groups are reversibly attached to aminolyzed trithiocarbonate end groups of RAFT-polymerized poly(N,N-dimethylacrylamide). Quantitative end group modification is confirmed by 1 H NMR spectroscopy, size exclusion chromatography, and mass spectrometry, while reversible release of attached compounds under physiological reductive conditions is successfully monitored by diffusion ordered NMR spectroscopy and thin layer chromatography. Additionally, this concept is further expanded to protein-reactive, self-immolative carbonate species that enable reversible bioconjugation of lysozyme and α-macrophage mannose receptor (MMR) nanobodies as model proteins. Altogether, self-immolative RAFT end group modifications can form the new basis for reversible introduction of various functionalities to polymer chain ends including protein bioconjugates and, thus, opening novel opportunities for stimuli-responsive polymer hybrids.

Phosphate-Based Self-Immolative Linkers for the Delivery of Amine-Containing Drugs.

Amine-containing drugs often show poor pharmacological properties, but these disadvantages can be overcome by using a prodrug approach involving self-immolative linkers. Accordingly, we designed l-lactate linkers as ideal candidates for amine delivery. Furthermore, we designed linkers bearing two different cargos (aniline and phenol) for preferential amine cargo release within 15 min. Since the linkers carrying secondary amine cargo showed high stability at physiological pH, we used our strategy to prepare phosphate-based prodrugs of the antibiotic Ciprofloxacin. Therefore, our study will facilitate the rational design of new and more effective drug delivery systems for amine-containing drugs.

Engineered Ferritin Nanoparticles for the Bioluminescence Tracking of Nanodrug Delivery in Cancer.

The identification of a highly sensitive method to check the delivery of administered nanodrugs into the tumor cells is a crucial step of preclinical studies aimed to develop new nanoformulated cures, since it allows the real therapeutic potential of these devices to be forecast. In the present work, the ability of an H-ferritin (HFn) nanocage, already investigated as a powerful tool for cancer therapy thanks to its ability to actively interact with the transferrin receptor 1, to act as an efficient probe for the monitoring of nanodrug delivery to tumors is demonstrated. The final formulation is a bioluminescent nanoparticle, where the luciferin probe is conjugated on nanoparticle surface by means of a disulfide containing linker (Luc-linker@HFn) which is subjected to glutathione-induced cyclization in tumor cell cytoplasm. The prolonged imaging of luciferase+ tumor models, demonstrated by an in vitro and an in vivo approach, associated with the prolonged release of luciferin into cancer cells by disulfide bridge reduction, clearly indicates the high efficiency of Luc-linker@HFn for drug delivery to the tumor tissues.

Disulfide-Based Self-Immolative Linkers and Functional Bioconjugates for Biological Applications.

It is of vital importance to reversibly mask and selectively activate bioactive agents for advanced therapeutic and diagnostic purposes, aiming to efficiently suppress background interferences and attenuate systemic toxicity. This strategy has been involved in diverse applications spanning from chemical/biological sensors and diagnostics to drug delivery nanocarriers. Among these, redox-responsive disulfide linkages have been extensively utilized by taking advantage of extracellular and intracellular glutathione (GSH) gradients. However, direct conjugation of cleavable triggers to bioactive agents through disulfide bonds suffers from bulky steric hindrance and limited choice of trigger-drug combinations. Fortunately, the emergence of disulfide self-immolative linkers (DSILs) provides a general and robust strategy to not only mask various bioactive agents through the formation of dynamic disulfide linkages but also make it possible to be selectively activated upon disulfide cleavage in the reductive cytoplasmic milieu. In this review, recent developments in DSILs are focused with special attention on emerging chemical design strategies and functional applications in the biomedical field.

A Mechanism-Based Approach to Screening Metagenomic Libraries for Discovery of Unconventional Glycosidases.

Functional metagenomics has opened new opportunities for enzyme discovery. To exploit the full potential of this new tool, the design of selective screens is essential, especially when searching for rare enzymes. To identify novel glycosidases that employ cleavage strategies other than the conventional Koshland mechanisms, a suitable screen was needed. Focusing on the unsaturated glucuronidases (UGLs), it was found that use of simple aryl glycoside substrates did not allow sufficient discrimination against ß-glucuronidases, which are widespread in bacteria. While conventional glycosidases cannot generally hydrolyze thioglycosides efficiently, UGLs follow a distinct mechanism that allows them to do so. Thus, fluorogenic thioglycoside substrates featuring thiol-based self-immolative linkers were synthesized and assessed as selective substrates. The generality of the approach was validated with another family of unconventional glycosidases, the GH4 enzymes. Finally, the utility of these substrates was tested by screening a small metagenomic library.

Self-immolative bioluminogenic quinone luciferins for NAD(P)H assays and reducing capacity-based cell viability assays.

Highly sensitive self-cleavable trimethyl lock quinone-luciferin substrates for diaphorase were designed and synthesized to measure NAD(P)H in biological samples and monitor viable cells via NAD(P)H-dependent cellular oxidoreductase enzymes and their NAD(P)H cofactors.

Reversible Polymer-Protein Functionalization by Stepwise Introduction of Amine-Reactive, Reductive-Responsive Self-Immolative End Groups onto RAFT-Derived Polymers.

Many promising therapeutic protein or peptide drug candidates are rapidly excreted from an organism due to their small size or their inherent immunogenicity. One way to counteract these effects is PEGylation, in which the biopolymer is shielded by synthetic polymers exploiting their stealth properties. However, these modifications are often accompanied by a reduction in the biological function of the protein. By using responsive moieties that bridge the polymer to the protein, a reversible character is provided to this type of conjugation. In this regard, the reductive-responsive nature of disulfides can be exploited via self-immolative structures for reversible linkage to aminic lysine residues and the N-terminus on the protein surface. They enable a traceless release of the intact protein without any further modification and thus preserve the protein's bioactivity. In this study, we demonstrate how this chemistry can be made broadly accessible to RAFT-derived water-soluble polymers like poly(N,N-dimethylacrylamide) (pDMA) as a relevant PEG alternative. A terminal reactive imidazole carbamate with an adjacent self-immolative motif was generated in a gradual manner onto the trithiocarbonate chain transfer moiety of the polymer by first substituting it with a disulfide-bridged alcohol and subsequently converting it into an amine reactive imidazole carbamate. Successful synthesis and complete characterization were demonstrated by NMR, size exclusion chromatography, and mass spectrometry. Finally, two model proteins, lysozyme and a therapeutically relevant nanobody, were functionalized with the generated polymer, which was found to be fully reversible under reductive conditions in the presence of free thiols. This strategy has the potential to extend the generation of reversible reductive-responsive polymer-protein hybrids to the broad field of available functional RAFT-derived polymers.

Design and synthesis of water-soluble prodrugs of rifabutin for intraveneous administration.

Acinetobacter baumannii is a gram-negative bacterium causing severe hospital-acquired infections such as bloodstream infections or pneumonia. Moreover, multidrug resistant A. baumannii becomes prevalent in many hospitals. Consequently, the World Health Organization made this bacterium a critical priority for the research and development of new antibiotics. Rifabutin, a semisynthetic product from the rifamycin class, was recently found to be very active in nutrient-limited eukaryotic cell culture medium against various A. baumannii strains, including extremely drug-resistant strains, with minimal inhibitory concentrations as low as 0.008 µg/mL. Moreover, this in vitro potency translates into in vivo efficacy. Thus, rifabutin appears to be an attractive novel antibiotic against A. baumannii. In this work, our objective was to design and synthetize rifabutin prodrugs with increased aqueous solubility to allow intraveneous use. Synthetic methodologies were developed to selectively functionalize the hydroxyl group in position 21 and to afford 17 prodrugs. We measured the water solubility of the prodrugs, the stability in human and mouse plasma and their antimicrobial activity against A. baumannii after incubation in human serum. Finally, a pharmacokinetic release study of rifabutin was performed in CD1 mice with three selected prodrugs as a proof of concept.

Post-synthetic 'Click' Synthesis of RAFT Polymers with Pendant Self-immolative Triazoles.

Self-immolative linkers offer efficient mechanisms for deprotecting 'caged' functional groups in response to specific stimuli. Herein we describe a convenient 'click' chemistry method for introducing pendant self-immolative linkers to a polymer backbone through post-polymerization modification. The introduced triazole rings serve both to anchor the stimuli-cleavable trigger groups to the polymer backbone, while also forming a functional part of the self-immolation cascade. We investigate the polymerization kinetics, post-synthetic modification, and self-immolation mechanism of a model polymer system, and discuss avenues for future studies on poly-pendant self-immolative triazoles as a modular, stimuli-responsive macromolecule platform.

Self-immolative Linkers in Prodrugs and Antibody Drug Conjugates in Cancer Treatment.

BACKGROUND: The design of anti-cancer therapies with high anti-tumour efficacy and reduced toxicity continues to be challenging. Anti-cancer prodrug and antibody-drug-conjugate (ADC) strategies that can specifically and efficiently deliver cytotoxic compounds to cancer cells have been used to overcome some of the challenges. The key to the success of many of these strategies is a self-immolative linker, which after activation can release the drug payload. Various types of triggerable self-immolative linkers are used in prodrugs and ADCs to improve their efficacy and safety. OBJECTIVE: Numerous patents have reported the significance of self-immolative linkers in prodrugs and ADCs in cancer treatment. Based on the recent patent literature, we summarise methods for designing the site-specific activation of non-toxic prodrugs and ADCs in order to improve selectivity for killing cancer cells. METHODS: In this review, an integrated view of the potential use of prodrugs and ADCs in cancer treatment are provided. This review presents recent patents and related publications over the past ten years uptill 2020. RESULTS: The recent patent literature has been summarised for a wide variety of self-immolative PABC linkers, which are cleaved by factors including responding to the difference between the extracellular and intracellular environments (pH, ROS, glutathione) through over-expressed enzymes (cathepsin, plasmin, ß-glucuronidase) or bioorthogonal activation. The mechanism for self-immolation involves the linker undergoing a 1,4- or 1,6-elimination (via electron cascade) or intramolecular cyclisation to release cytotoxic drug at the targeted site. CONCLUSION: This review provides the commonly used strategies from recent patent literature in the development of prodrugs based on targeted cancer therapy and antibody-drug conjugates, which show promise in therapeutic applications.

Synthetic Artificial Apoptosis-Inducing Receptor for On-Demand Deactivation of Engineered Cells.

The design of a fully synthetic, chemical "apoptosis-inducing receptor" (AIR) molecule is reported that is anchored into the lipid bilayer of cells, is activated by the incoming biological input, and responds with the release of a secondary messenger-a highly potent toxin for cell killing. The AIR molecule has four elements, namely, an exofacial trigger group, a bilayer anchor, a toxin as a secondary messenger, and a self-immolative scaffold as a mechanism for signal transduction. Receptor installation into cells is established via a robust protocol with minimal cell handling. The synthetic receptor remains dormant in the engineered cells, but is effectively triggered externally by the addition of an activating biomolecule (enzyme) or in a mixed cell population through interaction with the surrounding cells. In 3D cell culture (spheroids), receptor activation is accessible for at least 5 days, which compares favorably with other state of the art receptor designs.

Perspectives About Self-Immolative Drug Delivery Systems.

Self-immolative drug delivery system is one of the delivery systems, which have drawn attention, in recent research, highlighting the improvement they generate in drug selectivity and efficacy. Self-immolative linkers, or spacers, are covalent groups, which have the role of cleavaging two bonds between a protector group and a drug, in the case of drug delivery systems, after a stimuli.The cascade of reactions allows to control the release of the drug. The choice of the adequate self-immolative linker is essential and depend on many variables and goals as well. Many approaches can be explored when designing a system adequate for achieving these goals, especially prodrugs. Some of the most used stimuli-responses for self-immolative drugs - enzyme triggers, chemical triggers, as pH, redox system, 1,4-, 1,6-, 1,8-eliminations, photodegradable triggers, multiple triggers, among others - are described in this ten-year review, along with their application as theranostic agents. We intend that the examples presented in this review inspire researchers working on drug delivery systems to further explore their application.

Prodrugs in medicinal chemistry and enzyme prodrug therapies.

Prodrugs are cunning derivatives of therapeutic agents designed to improve the pharmacokinetics profile of the drug. Within a prodrug, pharmacological activity of the drug is masked and is recovered within the human body upon bioconversion of the prodrug, a process that is typically mediated by enzymes. This concept is highly successful and a significant fraction of marketed therapeutic formulations is based on prodrugs. An advanced subset of prodrugs can be engineered such as to achieve site-specific bioconversion of the prodrug - to comprise the highly advantageous "enzyme prodrug therapy", EPT. Design of prodrugs for EPT is similar to the prodrugs in general medicinal use in that the pharmacological activity of the drug is masked, but differs significantly in that site-specific bioconversion is a prime consideration, and the enzymes typically used for EPT are non-mammalian and/or with low systemic abundance in the human body. This review focuses on the design of prodrugs for EPT in terms of the choice of an enzyme and the corresponding prodrug for bioconversion. We also discuss the recent success of "self immolative linkers" which significantly empower and diversify the prodrug design, and present methodologies for the design of prodrugs with extended blood residence time. The review aims to be of specific interest for medicinal chemists, biomedical engineers, and pharmaceutical scientists.

Self-Immolative Linkers Literally Bridge Disulfide Chemistry and the Realm of Thiol-Free Drugs.

The ultimate goal of controlled, intracellulardrug delivery is to get the drug to the target cell without spilling the contents in transit and then release the entire payload upon cell entry. One of the most powerful platforms to achieve this relies on the intracellular disulfide reshuffling as a trigger for drug release form the engineered prodrugs. However, utility of disulfide reshuffling for drug release is naturally applicable only to the thiol containing molecules-ultimately leaving nearly all commercialized drugs beyond the scope of this platform. This is a drastic limitation. A cunning new tool of organic chemistry is fast entering the mainstream of prodrug design: the self-immolative linkers. This platform allows overcoming the natural chemical barrier and makes it possible to link virtually any drug to its carrier via a disulfide bond and engineer a specific intracellular release. It is a game-changing accomplishment of modern organic chemistry. The scope and limitations of this novel opportunity for medicinal chemistry and nanomedicine are outlined.

ß-Glucuronidase-responsive prodrugs for selective cancer chemotherapy: an update.

The design of novel antitumor agents allowing the destruction of malignant cells while sparing healthy tissues is one of the major challenges in medicinal chemistry. In this context, the use of non-toxic prodrugs programmed to be selectively activated by beta-glucuronidase present at high concentration in the microenvironment of most solid tumors has attracted considerable attention. This review summarizes the major progresses that have been realized in this field over the past ten years. This includes the new prodrugs that have been designed to target a wide variety of anticancer drugs, the prodrugs employed in the course of a combined therapy, the dendritic glucuronide prodrugs and the concept of ß-glucuronidase-responsive albumin binding prodrugs.