Transcellular stomach absorption of a derivatized glucagon-like peptide-1 receptor agonist.

Oral administration of therapeutic peptides is hindered by poor absorption across the gastrointestinal barrier and extensive degradation by proteolytic enzymes. Here, we investigated the absorption of orally delivered semaglutide, a glucagon-like peptide-1 analog, coformulated with the absorption enhancer sodium N-[8-(2-hydroxybenzoyl) aminocaprylate] (SNAC) in a tablet. In contrast to intestinal absorption usually seen with small molecules, clinical and preclinical dog studies revealed that absorption of semaglutide takes place in the stomach, is confined to an area in close proximity to the tablet surface, and requires coformulation with SNAC. SNAC protects against enzymatic degradation via local buffering actions and only transiently enhances absorption. The mechanism of absorption is shown to be compound specific, transcellular, and without any evidence of effect on tight junctions. These data have implications for understanding how highly efficacious and specific therapeutic peptides could be transformed from injectable to tablet-based oral therapies.

Electrochemical oxidation of troglitazone: identification and characterization of the major reactive metabolite in liver microsomes.

Troglitazone (TGZ) was developed for the treatment of type 2 diabetes but was withdrawn from the market due to hepatotoxicity. The formation of reactive metabolites has been associated with the observed hepatotoxicity. Such reactive metabolites have been proposed to be formed via three different mechanisms. One of the proposed mechanisms involves the oxidation of the chromane moiety of TGZ to a reactive o-quinone methide. The two other mechanisms involve metabolic activation of the thiazolidinedione moiety of TGZ. In the present study, it is shown that electrochemical oxidations can be used to generate a reactive metabolite of TGZ, which can be trapped by GSH or N-acetylcysteine. From incubations of TGZ with rat and human liver microsomes in the presence of either GSH or N-acetylcysteine, it was shown that similar conjugates were formed in vitro as formed from electrochemical oxidations of TGZ. One- and two-dimensional NMR studies of the troglitazone- S-( N-acetyl)cysteine conjugate revealed that N-acetylcysteine was attached to a benzylic carbon in the chromane moiety, showing that the conjugate was formed via a reaction between the o-quinone methide of TGZ and N-acetylcysteine. From electrochemical oxidations of rosiglitazone, pioglitazone, and ciglitazone in the presence of GSH, no GSH conjugates could be identified. These three compounds all contain a thiazolidinedione moiety. In conclusion, it has been shown that the primary reactive metabolite of TGZ formed from electrochemical oxidation was the o-quinone methide, and this metabolite was similar to what was observed to be the primary reaction product in human and rat liver microsomes.

Bioactivation of diclofenac in vitro and in vivo: correlation to electrochemical studies.

Diclofenac is widely used in the treatment of, for example, arthritis and muscle pain. The use of diclofenac has been associated with hepatotoxicity, which has been linked to the formation of reactive metabolites. Diclofenac can be metabolized to 4'-OH- and 5-OH-diclofenac, both of which are able to form quinone imines capable of reacting with, for example, GSH and nucleophilic groups in proteins. Electrochemistry has been shown to be a suitable tool for mimicking some types of oxidative drug metabolism and for studying the formation of reactive metabolites. In these studies, the electrochemical oxidation of diclofenac to a +16 Da metabolite was shown to be identical to a synthetic standard of 5-OH-diclofenac. Furthermore, two different experimental designs were investigated with respect to the electrochemical oxidation of 4'-OH- and 5-OH-diclofenac. In the first approach, the oxidized sample was collected in an aqueous solution of GSH, whereas in the other approach, GSH was added to the sample before the oxidation was performed. From these electrochemical oxidations, a range of GSH conjugates of 4'-OH- and 5-OH-diclofenac were observed and characterized by MS/MS. This allowed the development of sensitive LC-MS methods in order to detect the GSH conjugates from in vivo (rat bile) and in vitro (human liver microsomes (HLM), rat liver microsomes (RLM), and rat hepatocytes) samples. A wide range of mono-, di-, and triglutathionyl conjugates were detected in the in vitro and in vivo samples. It was also observed that 5-OH-diclofenac formed GSH conjugates with RLM and HLM without addition of NADPH, whereas GSH conjugate formation of 4'-OH-diclofenac was NADPH-dependent. This indicated that 5-OH-diclofenac was prone to auto-oxidation. The oxidation potentials of the two hydroxy metabolites were determined by cyclic voltammetry. A difference of 69 mV was observed between the two oxidation potentials, which in part may explain the extent of auto-oxidation for 5-OH-diclofenac. In conclusion, it was shown that electrochemical oxidation was capable of mimicking the metabolic hydroxylation of diclofenac to 5-OH-diclofenac. Furthermore, electrochemical oxidation was used to generate a range of GSH conjugates of 4'-OH- and 5-OH-diclofenac and a number of these conjugates were also detected in metabolism studies with microsomes (HLM/RLM) and freshly isolated rat hepatocytes, and in vivo in rat bile.

Development and evaluation of an electrochemical method for studying reactive phase-I metabolites: correlation to in vitro drug metabolism.

A reactive metabolite may react covalently with proteins or DNA to form adducts that ultimately may lead to a toxic response. Reactive metabolites can be formed via, for example, cytochrome P450-mediated phase 1 reactions, and in this study, we report the development and evaluation of an electrochemical method for generating reactive metabolites. Paracetamol was used as a test compound to develop the method. The stability of the electrochemically generated N-acetyl-p-benzoquinoneimine (NAPQI) from paracetamol was investigated at 37 degrees C at pH 5.0, 7.4, and 9.0. The highest stability of NAPQI was observed at pH 7.4. The reaction rate between NAPQI and glutathione (GSH) was studied with cyclic voltammetry. NAPQI reacted quantitatively with GSH within 130 ms. The reactivity of NAPQI toward other nucleophiles was investigated, and for the reaction with N-acetyltyrosine, a time-dependent formation of a conjugate with N-acetyltyrosine was observed from 0 to 4 min. The applicability of the method was evaluated with compounds that were able to form quinone imines (amodiaquine), quinones (3-tert-butyl-4-hydroxyanisole and p-cresol), imine methides (3-methylindole; trimethoprim), quinone methides (3,5-di-tert-butyl-4-hydroxytoluene), and nitrenium ions (clozapine). The compounds were oxidized in an analytical electrochemical cell, and the formed reactive metabolites were trapped with GSH. The samples were then analyzed by LC-MS and LC-MS/MS. For comparison, all compounds were incubated with GSH in rat and human liver microsomes, and the formation of GSH conjugates was compared with that observed by electrochemical oxidation. Furthermore, the electrochemical method was used to synthesize a GSH conjugate of clozapine, which made it possible to obtain structural information by NMR. In summary, a high degree of similarity was observed between the conjugates identified from electrochemical oxidation and GSH conjugates identified from incubation with liver microsomes. In conclusion, we have developed a method that is useful for studies on reactive metabolites and furthermore can be scaled up for the synthesis of GSH conjugates for NMR.