During human development impressive changes in drug disposition occur. An important determinant of drug clearance is metabolism, something that is not only determined by ontogenic regulation but also by genetic processes which add to the variability of drug metabolism during different stages of childhood. Therefore, an understanding of the developmental regulation of different metabolic pathways, together with information on the genetic determinants of drug metabolism, will increase the knowledge of inter- and intraindividual variability in drug disposition during childhood.

Glucuronidation in humans. Pharmacogenetic and developmental aspects

Publisher Summary The role of plasma protein binding is recognized as an important factor in drug disposition and efficacy. This chapter discusses the structures of drug-binding proteins, particularly human serum albumin (HSA) and alpha-1-acid glycoprotein (AAG), the relationship between ligand structure and protein binding and the effect of plasma protein binding on drug disposition and pharmacological activity. Human serum albumin (HSA) binds a wide variety of endogenous and exogenous ligands with association constants typically in the range of 10 4 to 10 6 M –1 . As the most abundant protein in plasma, it plays an important role in the maintenance of blood pH and colloidal osmotic pressure and accounts for most of the thiol content of plasma. Numerous competition and spectroscopic studies, using various ligands, have identified two sites (I and II) most relevant for drug binding. The interaction of various nonsteroidal anti-inflammatory drugs (NSAIDs) with HSA has been studied extensively. The interaction of other drug classes with HSA has also been investigated. Covalent modification of plasma proteins can be produced by direct attachment of an electrophilic moiety of the parent molecule to nucleophilic protein residues or by metabolic activation of the parent to a more reactive species. These reactions are important as their potential to form immunogenic adducts, with the possibility of producing idiosyncratic hypersensitivity reactions. Microdialysis is a potent technique to measure the extent of binding in vivo or in vitro in plasma. high-performance liquid chromatography (HPLC) methods, utilizing protein stationary phases, offer the potential for rapid screening, especially for those molecules bound to albumin. The blood brain barrier (BBB) effectively restricts the access of polar, hydrophilic drugs into the central nervous system (CNS), thus establishing a significant drug delivery impediment.

Chapter 33. Plasma Protein Binding of Drugs

A number of therapeutic drugs with different structures and mechanisms of action have been reported to undergo metabolic activation by Phase I or Phase II drug-metabolizing enzymes. The bioactivation gives rise to reactive metabolites/intermediates, which readily confer covalent binding to various target proteins by nucleophilic substitution and/or Schiff's base mechanism. These drugs include analgesics (e.g., acetaminophen), antibacterial agents (e.g., sulfonamides and macrolide antibiotics), anticancer drugs (e.g., irinotecan), antiepileptic drugs (e.g., carbamazepine), anti-HIV agents (e.g., ritonavir), antipsychotics (e.g., clozapine), cardiovascular drugs (e.g., procainamide and hydralazine), immunosupressants (e.g., cyclosporine A), inhalational anesthetics (e.g., halothane), nonsteroidal anti-inflammatory drugs (NSAIDSs) (e.g., diclofenac), and steroids and their receptor modulators (e.g., estrogens and tamoxifen). Some herbal and dietary constituents are also bioactivated to reactive metabolites capable of binding covalently and inactivating cytochrome P450s (CYPs). A number of important target proteins of drugs have been identified by mass spectrometric techniques and proteomic approaches. The covalent binding and formation of drug-protein adducts are generally considered to be related to drug toxicity, and selective protein covalent binding by drug metabolites may lead to selective organ toxicity. However, the mechanisms involved in the protein adduct-induced toxicity are largely undefined, although it has been suggested that drug-protein adducts may cause toxicity either through impairing physiological functions of the modified proteins or through immune-mediated mechanisms. In addition, mechanism-based inhibition of CYPs may result in toxic drug-drug interactions. The clinical consequences of drug bioactivation and covalent binding to proteins are unpredictable, depending on many factors that are associated with the administered drugs and patients. Further studies using proteomic and genomic approaches with high throughput capacity are needed to identify the protein targets of reactive drug metabolites, and to elucidate the structure-activity relationships of drug's covalent binding to proteins and their clinical outcomes.

Drug Bioactivation Covalent Binding to Target Proteins and Toxicity Relevance

Although glucuronidation is generally considered a detoxification route of drug metabolism, the chemical reactivity of acyl glucuronides has been linked with the toxic properties of drugs that contain carboxylic acid moieties. It is now well documented that such metabolites can reach appreciable concentrations in blood. Furthermore, they are labile, undergo hydrolysis and pH-dependent intramolecular acyl migration to isomeric conjugates of glucuronic acid, and may react irreversibly with plasma proteins, tissue proteins, and with nucleic acids. This stable binding causes chemical alterations that are thought to contribute to drug toxicity either through changes in the functional properties of the modified molecules or through antigen formation with subsequent hypersensitivity and other immune reactions. Whereas in vitro data on the toxicity of acyl glucuronides have steadily accumulated, direct evidence for their toxicity in vivo is scarce. Acyl glucuronides display limited stability, which is dependent on pH, temperature, nature of the aglycon, and so on. Therefore, careful sample collection, handling, and storage procedures are critical to ensure generation of reliable pharmacologic and toxicologic data during clinical studies. Acyl glucuronides can be directly quantified in biologic specimens using chromatographic procedures. Their adducts with plasma or cell proteins can be determined after electrophoretic separation, followed by blotting. ELISA techniques have been used to assess the presence of antibodies against acyl glucuronide-protein adducts. This review summarizes the most recent evidence concerning biologic and toxicologic effects of acyl glucuronide metabolites of various drugs and discusses their relevance for drug monitoring. A critical evaluation of the available methodology is included.

Acyl glucuronide drug metabolites: toxicological and analytical implications.

Structure, enzymatic activities, glycation and therapeutic potential of human serum albumin: A natural cargo

Acyl glucuronide metabolites of bilirubin and many drugs can react with serum albumin in vivo to form covalent adducts. Such adducts may be responsible for some toxic effects of carboxylic nonsteroidal antiinflammatory agents. The mechanism of formation of the adducts and their chemical structures are unknown. In this paper we describe the use of tandem mass spectrometry to locate binding sites and elucidate the binding mechanism involved in the formation of covalent adducts from tolmetin glucuronide and albumin in vitro. Human serum albumin and excess tolmetin glucuronide were coincubated in the presence of sodium cyanoborohydride to trap imine intermediates. The total protein product was reduced, carboxymethylated, and digested with trypsin. Six tolmetin-containing peptides (indicated by absorbance at 313 nm) were isolated by high-pressure liquid chromatography and analyzed by liquid secondary-ion mass spectrometry and collision-induced dissociation, using a four-sector tandem mass spectrometer. All six peptides contained tolmetin linked covalently via a glucuronic acid to protein lysine groups. Major attachment sites on the protein were Lys-195, -199, and -525; minor sites were identified as Lys-137, -351, and -541. Our results show unambiguously that the glucuronic acid moiety of acyl glucuronides can be retained within the structure when these reactive metabolites bind covalently to proteins, and they suggest that acyl migration followed by Schiff base (imine) formation is a credible mechanism for the generation of covalent adducts in vivo.

https://www.pnas.org/content/pnas/90/9/3797.full.pdf

Andreas Ding

Papers

Evidence for covalent binding of acyl glucuronides to serum albumin via an imine mechanism as revealed by tandem mass spectrometry.