In pursuit of feedback activation: New insights into redox-responsive hydropersulfide prodrug combating oxidative stress.

Redox-responsive hydropersulfide prodrugs are designed to enable a more controllable and efficient hydropersulfide (RSSH) supply and to thoroughly explore their biological and therapeutic applications in oxidative damage. To obtain novel activation patterns triggered by redox signaling, we focused on NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1), a canonical antioxidant enzyme, and designed NQO1-activated RSSH prodrugs. We also performed a head-to-head comparison of two mainstream structural scaffolds with solid quantitative analysis of prodrugs, RSSH, and metabolic by-products by LC-MS/MS, confirming that the perthiocarbamate scaffold was more effective in intracellular prodrug uptake and RSSH production. The prodrug was highly potent in oxidative stress management against cisplatin-induced nephrotoxicity. Strikingly, this prodrug possessed potential feedback activation properties by which the delivered RSSH can further escalate the prodrug activation via NQO1 upregulation. Our strategy pushed RSSH prodrugs one step further in the pursuit of efficient release in biological matrices and improved druggability against oxidative stress.

Development and validation for bioanalysis of VK2809, its active metabolite VK2809A and glutathione-conjugated metabolite MB06588 in rat liver using LC-MS/MS.

VK2809 is a promising drug candidate in Phase II clinical trials for the treatment of non-alcoholic steatohepatitis (NASH). It is a prodrug with a HepDirect strategy, which can achieve selective hepatic metabolic activation, generating an active metabolite VK2809A as a potent and selective agonist for thyroid hormone receptor beta (TRß), a concomitant reactive metabolite VK2809B, and a glutathione (GSH) conjugate MB06588. Currently, there is no convenient and sensitive bioanalytical method for the simultaneous determination of the above three metabolites. Herein, we established an LC-MS/MS method to separate VK2809 and its metabolites on the XSelect HSS T3 column and quantified them in negative electrospray ionization mode. Subsequently, several factors were investigated such as the use of 60% acetonitrile for homogenization to stabilize the analytes, the addition of 20 mM glutathione for the derivation of VK2809B, and the protein precipitation with methanol containing Sobetirome as the internal standard (IS). The method exhibited good linearity for all compounds (19.4-388.4 nM for VK2809; 27.4-2744.4 nM for VK2809A and 10.6-211.0 nM for MB06588) with great correlation coefficients (r > 0.996). The method validation also demonstrated acceptable precision (RSD < 13.0% for VK2809, RSD < 7.9% for VK2809A, RSD < 14.4% for MB06588) and accuracy (92.7%-103% for VK2809, 91.2%-107.3% for VK2809A, 96%-106.7% for MB06588). The matrix effect, recovery, and stability were also suitable to determine all the analytes. This method is suitable for the bioanalysis of VK2809 and its metabolites and has been successfully applied to the study of intrahepatic exposure in rats. It is expected to be further practiced in drug design, optimization, and metabolism study in the following research.

Peroxynitrite-Promoted Persulfide Prodrugs with Protective Potential against Paracetamol Poisoning.

The newly emerging persulfide prodrugs provide additional options for the profound study of persulfide, a fascinating molecule expected to intervene in biological functions and even diseases. Peroxynitrite is often the culprit in pathological processes characterized by oxidative stress, while the persulfide prodrug responsive to it is still pending. To enrich the family of redox-activated prodrugs, we designed prodrugs with a 2-oxo-2-phenylacetamide trigger, which achieved the release of persulfide via 1, 6-N, S-relay. The degradation of prodrugs and the formation of persulfides were confirmed to be peroxynitrite-responsible by the qualitative and quantitative studies based on LC-MS/MS methods and a spectrophotometry-based tag-switch strategy. Furthermore, these prodrugs showed potent peroxynitrite scavenging activity, cellular therapeutic potential against paracetamol poisoning in HepG2 and oxidative stress in H9c2, as well as desirable in vitro metabolic properties.

Simultaneous determination of ZL-01, a novel nucleotide prodrug, and its metabolites in rat plasma by LC-MS/MS: Application to pharmacokinetic study.

ZL-01 is a novel dual-prodrug which shows promise to be an antiviral candidate for hepatitis C virus. Here we have established a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of ZL-01 and its four metabolites (M1, M7, M8, and M9) in rat plasma with special consideration of ex vivo ZL-01, M1, and M7 stability. Several factors affecting the stability were investigated. EDTA and citric acid solution (1 M) were added to plasma to maintain the stability of analytes. The protein-precipitation method was selected with acetonitrile containing sofosbuvir as internal standard (IS). Adequate separation of ZL-01 and its metabolites was achieved on XSelect HSS T3 (3.5 µm, 4.6 × 150 mm) column by a gradient-elution with a mobile phase consisting of 0.1% formic acid and acetonitrile at a flow rate of 0.5 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 599.2→418.5 for ZL-01, m/z 529.7→398.2 for M1, m/z 330.5→182.0 for M7, m/z 260.3→112.1 for M8, m/z 261.3→113.2 for M9 and m/z 530.4→243.4 for IS. The calibration curves exhibited good linearity (r＞0.997) for all components. The lower limit of quantitation (LLOQ) was in the range of 1-2 ng/mL. The intra-day and inter-day precisions (RSD) at three different levels were both less than 10.2% and the accuracies (RE) ranged from -3.7-7.6%. The matrix effect and extraction recovery of them ranged from 84% to 110.3% and 88.3-106.3%. This LC-MS/MS method for the simultaneous quantitation of ZL-01 and its metabolites was developed successfully and applied in the pharmacokinetic studies of these in rats. Pharmacokinetic results indicated ZL-01 would be metabolized rapidly and M8 might be the main metabolites after oral absorption.