Adapting decarbonylation chemistry for the development of prodrugs capable of in vivo delivery of carbon monoxide utilizing sweeteners as carrier molecules

Carbon monoxide as an endogenous signaling molecule exhibits pharmacological efficacy in various animal models of organ injury. To address the difficulty in using CO gas as a therapeutic agent for widespread applications, we are interested in developing CO prodrugs through bioreversible caging of CO in an organic compound. Specifically, we have explored the decarboxylation–decarbonylation chemistry of 1,2-dicarbonyl compounds. Examination and optimization of factors favorable for maximal CO release under physiological conditions led to organic CO prodrugs using non-calorific sweeteners as leaving groups attached to the 1,2-dicarbonyl core. Attaching a leaving group with appropriate properties promotes the desired hydrolysis–decarboxylation–decarbonylation sequence of reactions that leads to CO generation. One such CO prodrug was selected to recapitulate the anti-inflammatory effects of CO against LPS-induced TNF-α production in cell culture studies. Oral administration in mice elevated COHb levels to the safe and efficacious levels established in various preclinical and clinical studies. Furthermore, its pharmacological efficacy was demonstrated in mouse models of acute kidney injury. These studies demonstrate the potential of these prodrugs with benign carriers as orally active CO-based therapeutics. This represents the very first example of orally active organic CO prodrugs with a benign carrier that is an FDA-approved sweetener with demonstrated safety profiles in vivo.

1,2-Dicarbonyl compounds with FDA-approved sweeteners as leaving groups deliver CO for protection against acute kidney injury in mice.     

Activation and Delivery of Tetrazine-Responsive Bioorthogonal Prodrugs

Prodrugs, which remain inert until they are activated under appropriate conditions at the target site, have emerged as an attractive alternative to drugs that lack selectivity and show off-target effects. Prodrugs have traditionally been activated by enzymes, pH or other trigger factors associated with the disease. In recent years, bioorthogonal chemistry has allowed the creation of prodrugs that can be chemically activated with spatio-temporal precision. In particular, tetrazine-responsive bioorthogonal reactions can rapidly activate prodrugs with excellent biocompatibility. This review summarized the recent development of tetrazine bioorthogonal cleavage reaction and great promise for prodrug systems.     

Facile preparation of pH-responsive PEGylated prodrugs for activated intracellular drug delivery

The acid-cleavable amphiphilic prodrug DOX-PEG-DOX self-assemble to form nanoparticles and enter the cell by endocytosis for the pH-triggered intracellular delivery of DOX.     

Mechanochemical solid-state polymerization (X): the influence of copolymer structure in copolymeric prodrugs on the nature of drug release

From the standpoint of the mechanism of mechanochemical polymerization, two kinds of copolymeric prodrug, whose monomer sequence distribution (MSD) is different from each other, can be prepared by this polymerization under appropriate operational conditions: one is a random copolymer abundant in the longer block consisting of the same repeating units (multi-block copolymer), and the other is a block copolymer. To confirm the difference of MSD, the 13C-NMR spectra of poly(acrylamide-co-sodium acrylate) prepared by mechanochemical polymerization were measured and compared with the spectrum of that synthesized by a conventional radical-initiated solution polymerization, which produces the random copolymer normally. The results show that MSD in copolymers depends on the polymerization method (operational condition). We prepared three kinds of copolymeric prodrug consisting of acrylamide and vinyl monomer of 5-fluorouracil, whose MSD is different from one another. These copolymeric prodrugs had almost the same number average molecular weight, particle diameter and composition, and differed only in MSD. We compared the rate of drug release of these copolymeric prodrugs. The rate of drug release was the highest with the random copolymer, followed by the mechanochemically produced multi-block copolymer and the block copolymer. This result suggests that the rate of drug release depends on MSD of copolymeric prodrugs. These results are useful as they give a fundamental insight into the synthesis of copolymeric prodrugs having the desired rate of drug release.     

Facile preparation of pH-responsive PEGylated prodrugs for activated intracellular drug delivery

PEGylated prodrug, covalent attaching polyethylene glycol (PEG) polymer chains to therapeutic drugs, is one of the most promising techniques to improve the water-solubility, stability, and therapeutic effect of drugs. In this study, three PEGylated acid-sensitive prodrugs DOX-PEG-DOX with different molecular weights, were prepared via Schiff-base reaction between aldehyde-modified PEG and the amino groups of doxorubicin (DOX). This kind of amphiphilic polymeric prodrug could be self-assemble into nanoparticles in aqueous solution. The average particle size and morphologies of the prodrug nanoparticles under different pH conditions were observed by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. It turned out that the nanoparticles could be kept stable in the physiological environment, but degraded in acidic medium. Subsequently, we also investigated in vitro drug release behavior and found that the prodrug had acid-sensitive property. The cytotoxicity and intracellular uptake assays revealed that the prodrugs could rapidly internalized by HeLa or HepG2 cells to release DOX and effectively inhibited the proliferation of the tumor cells, which have the potential for use in cancer therapy.     

Self‐immolative dendrimers as novel drug delivery platforms

Self‐immolative dendrimers were recently developed and introduced as a potential platform for a single‐triggered multi‐prodrug. These unique structural dendrimers can release all of their tail units through domino‐like chain fragmentation, which is initiated by a single cleavage at the dendrimer core. The incorporation of drug molecules as the tail units and an enzyme substrate as the trigger generates a multi‐prodrug unit that is activated with a single enzymatic cleavage. We have demonstrated several examples of self‐immolative dendritic prodrug systems and have shown significant advantages with respect to the appropriate monomeric prodrug. We anticipate that single‐triggered, dendritic prodrugs will be exploited to further improve selective chemotherapeutic approaches in cancer therapy. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 1569–1578, 2006     

An Esterase‐Sensitive Prodrug Approach for Controllable Delivery of Persulfide Species

A strategy to deliver a well‐defined persulfide species in a biological medium is described. Under near physiological conditions, the persulfide prodrug can be activated by an esterase to generate a “hydroxymethyl persulfide” intermediate, which rapidly collapses to form a defined persulfide. Such persulfide prodrugs can be used either as chemical tools to study persulfide chemistry and biology or for future development as H₂S‐based therapeutic reagents. Using the persulfide prodrugs developed in this study, the reactivity between S ‐methyl methanethiosulfonate (MMTS) with persulfide was unambiguously demonstrated. Furthermore, a representative prodrug exhibited potent cardioprotective effects in a murine model of myocardial ischemia‐reperfusion (MI/R) injury with a bell shape therapeutic profile.     

Cyclization-activated prodrugs

Many drugs suffer from an extensive first-pass metabolism leading to drug inactivation and/or production of toxic metabolites, which makes them attractive targets for prodrug design. The classical prodrug approach, which involves enzyme-sensitive covalent linkage between the parent drug and a carrier moiety, is a well established strategy to overcome bioavailability/toxicity issues. However, the development of prodrugs that can regenerate the parent drug through non-enzymatic pathways has emerged as an alternative approach in which prodrug activation is not influenced by inter- and intraindividual variability that affects enzymatic activity. Cyclization-activated prodrugs have been capturing the attention of medicinal chemists since the middle-1980s, and reached maturity in prodrug design in the late 1990 s. Many different strategies have been exploited in recent years concerning the development of intramoleculary-activated prodrugs spanning from analgesics to anti-HIV therapeutic agents. Intramolecular pathways have also a key role in two-step prodrug activation, where an initial enzymatic cleavage step is followed by a cyclization-elimination reaction that releases the active drug. This work is a brief overview of research on cyclization-activated prodrugs from the last two decades.     

The effect of phenyl substituents on the release rates of esterase-sensitive coumarin-based prodrugs

A coumarin-based prodrug system has been recently developed in our laboratory for the preparation of esterase-sensitive prodrugs of amines, peptides, and peptidomimetics. The drug release rates from this prodrug system were found to be dependent on the structural features of the drug moiety. In certain cases, the release can be undesirably slow for drugs that are secondary amines with relatively high pKa's. Aimed at finding ways to manipulate the release rates to suit the need of different drugs, we have examined the effect of the phenyl ring substitutions on the release kinetics of such prodrugs and found that appropriately positioned alkyl substituents on the phenyl ring could help to facilitate the release by as much as 16-fold. Therefore, introduction of alkyl substituents on the phenyl ring should allow us to manipulate the release rates and, therefore, time profiles for different drugs.     

Long conjugated 2-nitrobenzyl derivative caged anticancer prodrugs with visible light regulated release: preparation and functionalizations

A series of anticancer prodrugs with different chemical functional groups were prepared, in which the styryl conjugated 2-nitrobenzyl derivatives were introduced as the phototrigger to regulate the drug (chlorambucil) release. Compared to the common 4,5-dimethoxy-2-nitrobenzyl caged compounds, most of the prodrugs exhibited large and redshifted one-photon absorption within the visible range. One-photon excitation for the drug release was studied by measuring UV-vis absorption, FT-IR, and HPLC spectra, which suggested that chlorambucil was released effectively and precisely by manipulating external light conditions. And the introduction of different functional groups made this type of prodrug a good platform to further react with some typical drug carriers and to further form excellent visible light responsive drug delivery systems. Moreover, the drug also could be effectively released under the excitation of two-photon at 800 nm with comparable photorelease efficiencies.     

Prodrugs in cardiovascular therapy

Prodrugs are biologically inactive derivatives of an active drug intended to solve certain problems of the parent drug such as toxicity, instability, minimal solubility and non-targeting capabilities. The majority of drugs for cardiovascular diseases undergo first-pass metabolism, resulting in drug inactivation and generation of toxic metabolites, which makes them appealing targets for prodrug design. Since prodrugs undergo a chemical reaction to form the parent drug once inside the body, this makes them very effective in controlling the release of a variety of compounds to the targeted site. This review will provide the reader with an insight on the latest developments of prodrugs that are available for treating a variety of cardiovascular diseases. In addition, we will focus on several drug delivery methodologies that have merged with the prodrug approach to provide enhanced target specificity and controlled drug release with minimal side effects.     

The feasibility of enzyme targeted activation for amino acid/dipeptide monoester prodrugs of floxuridine; cathepsin D as a potential targeted enzyme

The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5'-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0-105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5'-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5'-O-L-phenylalanyl-L-tyrosylfloxuridine and 5'-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enzymes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5'-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs.     

Synthesis of potential prodrug systems for reductive activation. Prodrugs for anti-angiogenic isoflavones and VEGF receptor tyrosine kinase inhibitory oxindoles

A number of potential prodrug systems for reductive activation have been investigated. The prodrug systems chosen for the study were the 2-nitrophenylacetyl, 3-methyl-3-(3,6-dimethyl-1,4-benzoquinon-2-yl)butanoyl and 4-nitrobenzyl groups, readily attached to acidic OH or NH groups in drug molecules, and released upon bioreductive activation. The drug molecules studied were the naturally occurring isoflavone biochanin A, an inhibitor of VEGF-induced angiogenesis, and the pyrrolylmethylidenyl oxindole SU5416 (semaxanib) and its 6-hydroxy derivative, inhibitors of VEGF receptor tyrosine kinase. Following coupling the prodrug system to the drug, the compounds were evaluated chemically and biologically. Under chemical reducing conditions, the 3-methyl-3-(3,6-dimethyl-1,4-benzoquinon-2-yl)butanoic acid based prodrugs appear to fragment the most efficiently, followed by the 2-nitrophenylacetate esters with the 4-nitrobenzyl ethers being the least efficient. The potentially pro-anti-angiogenic compounds were also assayed for their ability to block VEGF-induced angiogenesis in HUVECS in comparison to the free agents. Control compounds that cannot be activated under bioreductive conditions are less potent than the free drug, whereas many of the potential prodrugs not only exhibit a dose response, but appear at least equipotent with the free drug.     

Bioactivation of self-immolative dendritic prodrugs by catalytic antibody 38C2

Self-immolative dendrimers have recently been developed and introduced as a potential platform for a multi-prodrug. These unique structural dendrimers can release all of their tail units, through a self-immolative chain fragmentation, which is initiated by a single cleavage at the dendrimer's core. Incorporation of drug molecules as the tail units and an enzyme substrate as the trigger can generate a multi-prodrug unit that will be activated with a single enzymatic cleavage. We have synthesized the first generation of dendritic prodrugs with doxorubicin and camptothecin as tail units and a retro-aldol retro-Michael focal trigger, which can be cleaved by catalytic antibody 38C2. The bioactivation of the dendritic prodrugs was evaluated in cell-growth inhibition assay with the Molt-3 leukemia cell line in the presence and the absence of antibody 38C2. The dendritic unit was applied as a platform for a heterodimeric prodrug, which achieved a remarkable increase in toxicity with its bioactivation.     

H NMR studies on the reductively triggered release of heterocyclic and steroid drugs from 4,7-dioxoindole-3-methyl prodrugs

Hypoxia is a feature of several disease states, including cancer and rheumatoid arthritis. Prodrug systems which, after bioreduction, selectively release active drugs in these tissues may be important in therapy. An improved preparation of 1,2-dimethyl-3-hydroxymethyl-5-methoxyindole-4,7-dione was developed. Mitsunobu coupling with (5-substituted) isoquinolin-1-ones (potent inhibitors of poly(ADP-ribose)polymerase) gave 1-(1,2-dimethyl-4,7-dioxo-5-methoxyindol-3-ylmethoxy)isoquinolines and N-(1,2-dimethyl-4,7-dioxo-5-methoxyindol-3-ylmethyl)isoquinolin-1-ones. Similar coupling with the anticancer drug pentamethylmelamine gave its potential prodrug 1,2-dimethyl-3-(N-(4,6-bis(dimethylamino)-1,3,5-triazin-2-yl)-N-methylaminomethyl)-5-methoxyindole-4,7-dione. Treatment of sodium prednisolone hemisuccinate with 3-chloromethyl-1,2-dimethyl-5-methoxyindole-4,7-dione gave an analogous candidate prodrug of the anti-inflammatory drug prednisolone. In a chemical model system for bioreduction, SnCl₂ in CDCl₃/CD₃OD triggered rapid stoichiometric release of isoquinolin-1-ones from the O-linked prodrugs but not from the N-linked analogues. Use of this system allowed the release process to be monitored in situ by ¹H NMR spectroscopy. Diethyl hydrazine-1,2-dicarboxylate was found to reduce Sn^IV to Sn^II, making the overall reductive release catalytic in tin. The reduced (hydroquinone) prodrug may have a short lifetime under these reductive conditions, meaning that only good leaving groups are expelled. Thus 1-(1,2-dimethyl-4,7-dioxo-5-methoxyindol-3-ylmethoxy)isoquinolines and analogues may be useful as reductively triggered prodrugs.     

Mechanochemical solid-state polymerization (XI): effect of water-insoluble pharmaceutical aids on drug release from mechanically synthesized polymeric prodrugs

We discuss here the effect of water-insoluble pharmaceutical aids on the nature of drug release from composite polymeric prodrugs synthesized by mechanochemical solid-state polymerization. Magnesium stearate (Mgst) and hydrogen castor oil (HCO) were used as water-insoluble pharmaceutical aids. Composite polymeric prodrugs were synthesized by the mechanochemical solid-state polymerization of a vinyl monomer of 5-fluorouracil (I) in the presence of Mgst or HCO. The molecular weight of the resulting polymeric prodrugs increased with increasing the content of Mgst or HCO. Prodrug hydrolysis was carried out in a heterogeneous system in phosphate buffer at pH 6.8 and 37 degrees C. The rate of drug release from the composite polymeric prodrug containing Mgst (Poly-Mgst) was faster than that from polymeric prodrug containing no pharmaceutical aids (Poly-Non), while hydrolysis of the composite polymeric prodrug containing HCO (Poly-HCO) was slower than Poly-Non. Scanning electron microscope (SEM) photos showed the surface of Poly-HCO was smoother than that of Poly-Non and Poly-Mgst. It was suggested that the slower drug release from Poly-HCO may be responsible for the smaller specific surface area than that of Poly-Non. It was also shown that the rate of drug release from the composite polymeric prodrugs decreases with increasing the content of Mgst or HCO. Hence, novel composite polymeric prodrugs with a variety of drug release rates can be prepared by mechanochemical solid-state polymerization in a totally dry process.     

Monoclonal Antibody-conjugated Polyphosphoester-hyd-DOX Prodrug Nanoparticles for Targeted Chemotherapy of Liver Cancer Cells

In order to overcome the limitation of traditional active nano-therapeutic drugs on tumor targeting effciency which cannot reach the receptor/target in sufficient amount in the body,in this work,we developed a monoclonal antibody (mAb) and a polymer-hyd-doxorubicin prodrug conjugate,which enables the self-assembled nanoparticles to have precise targeting,tumor tissue aggregation and pH-sensitive drug release.We first prepared an amphiphilic polymer prodrug,abbreviated as H2N-PEEP-b-PBYP-hyd-DOX,via a combination of ring-opening polymerization (ROP) and "click" chemistry,in which PEEP and PBYP represent two kinds of phosphoester segmemts,-hyd-is hydrazone bond.After self-assembly into prodrug nanoparticles (PDNPs) with a diameter of about 93 nm,CD147 mAb was conjugated onto the PDNPs by EDC/NHS chemistry to form mAb-PDNPs.For the PDNPs and mAb-PDNPs,we also investigated their stability,in vitro drug release behavior and cellular uptake.The results showed that the pH-responsive PDNPs can remain relatively stable under the condition of PB 7.4 buffer solution.However,under acidic conditions or in the presence of phosphodiesterase I (PDE I),both the amount and rate of DOX release increased at the same incubation period.Cytotoxicity assay showed that mAb-PDNPs exhibited higher cytotoxicity (IC50:1.12 mg·L-1) against HepG2 cells than PDNPs (IC50:2.62 mg·L-1) without monoclonal antibody.The nanoparticles with antibodies mAb-PDNPs have relatively better stability and can directly achieve the targeting drug delivery through CD147 mAb.     

Dual Diomarkers Triggered Prodrugs for Precise Treatment of Melanoma:Design,Synthesis and Activities

Targeted prodrug strategy, which utilizes the endogenous biomarkers in cancer cells as activators to release the active drug, has been well established either in the fundamental research or the clinical treatment. However, many prodrugs suffer from safety concern due to "off-target activation". Dual or multiple biomarkers triggered prodrug may provide an effective strategy to overcoming the "off-target effect". Melanoma cells have both high levels of reactive oxygen species(ROS) and tyrosinase(TYR), which makes them significantly different from other tumor cells and normal cells. Here we reported a series of quinazolinone-aryl boronic acid/ester-based prodrugs, which can be activated by the cascade of ROS and TYR and selectively kill melanoma cells. The structure-activity relationship(SAR) analysis revealed that mitochondria-targeting property was vital for their cytotoxicity and the dual activated effector played a significant role in their selectivity towards melanoma cells. Among these candidates, compound 4b showed the highest toxicity to B16, leading to an imbalance of the redox system in melanoma cells, causing mitochondrial DNA damage, and then promoting melanoma cells death.     

A unique class of duocarmycin and CC-1065 analogues subject to reductive activation

N-Acyl O-amino phenol derivatives of CBI-TMI and CBI-indole2 are reported as prototypical members of a new class of reductively activated prodrugs of the duocarmycin and CC-1065 class of antitumor agents. The expectation being that hypoxic tumor environments, with their higher reducing capacity, carry an intrinsic higher concentration of "reducing" nucleophiles (e.g., thiols) capable of activating such derivatives (tunable N-O bond cleavage) and increasing their sensitivity to the prodrug treatment. Preliminary studies indicate the prodrugs effectively release the free drug in functional cellular assays for cytotoxic activity approaching or matching the activity of the free drug, yet remain essentially stable and unreactive to in vitro DNA alkylation conditions (<0.1-0.01% free drug release) and pH 7.0 phosphate buffer, and exhibit a robust half-life in human plasma (t1/2 = 3 h). Characterization of a representative O-(acylamino) prodrug in vivo indicates that they approach the potency and exceed the efficacy of the free drug itself (CBI-indole2), indicating that not only is the free drug effectively released from the inactive prodrug but also that they offer additional advantages related to a controlled or targeted release in vivo.     

A self-assembling prodrug nanosystem to enhance metabolic stability and anticancer activity of gemcitabine

Self-assembly is a powerful approach in molecular engineering for biomedical applications, in particular for creating self-assembling prodrugs. Here, we report a self-assembling prodrug of the anticancer drug gemcitabine(Gem) based on amphiphilic dendrimer approach. The prodrug reported in this study demonstrates high drug loading(40%) and robust ability to self-assemble into small nanomicelles, which increase the metabolic stability of Gem and enable entry into cells via endocytosis, hence bypa...