QA-GSH

Definition

Quaternary ammonium glutathione analogue conjugating agent (QA-GSH), is a novel conjugating agent used for the detection of reactive metabolites.

Discovery

QA-GSH was described by Soglia in 2006. He described an in vitro semiquantitative reactive metabolite detection assay that incorporates NADPH-supplemented human liver microsomes, a novel quaternary ammonium glutathione analogue conjugating agent (QA-GSH), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for detection. The assay was developed to have high sample capacity and the potential for high sample throughput. MS/MS detection is selective and sensitive for the QA-GSH conjugating agent and semiquantitation of QA-GSH-reactive metabolite conjugates was performed using QA-GSH standards added to samples prior to analysis [i.e., internal standards (ISs)]. The reactive metabolite trapping capability of the free thiol group in QA-GSH was assessed using model drugs acetaminophen, clozapine, and flutamide, which were bioactivated to afford reactive metabolites. MS signal responses of equimolar amounts of QA-GSH standards were compared to assess the feasibility of using a QA-GSH IS approach to semiquantify reactive metabolite levels in vitro. The full scan Q1 MS response for each standard was within 3.3-fold of one another even though the "parent" moiety structure of each QA-GSH conjugate standard differed significantly. Standard curve analysis using selected reaction monitoring for each QA-GSH standard gave slope values that differed by only 1.5-fold 1.

Structural Characteristics

QA-GSH is a novel quaternary ammonium glutathione analogue conjugating agent, (QA-GSH) containing a fixed positive charge. MS/MS was selective and sensitive for the QA-GSH conjugating agent and successful quantitation of QA-GSH-reactive metabolic conjugates was performed using QA-GSH standards added to samples prior to analysis. The assay was tested with a number of model drugs including acetaminophen, clozapine, and flutamide, which were bioactivated to afford reactive metabolites 1,2.

Mode of Action

The QA-GSH is semiquantitation method tested by determining the level of QA-GS-acetaminophen conjugate formation at three different concentrations of acetaminophen and comparing the results to those from linear regression of authentic standards. The calculated levels of conjugate formed compared closely with those calculated from linear regression data of authentic standard curves. Results show that the QA-GSH semiquantitation assay described is a viable method for semiquantitatively assessing the bioactivation potential in vitro and is well-suited for use in early drug discovery high throughput screening paradigms 2.

Functions

Drug toxicity and QA-GSH, any unwanted effect of a drug aside from its expected therapeutic actions leads to major cause of patient morbidity and mortality. Toxicity is the major impediment to process of drug development. In 1998 more than $20 billion was spent on identification and development of drugs. More than 20% spent on screening methods and toxicity tests. Liver is common target due to its major role in metabolism of xenobiotics. More than 800 drugs have been implicated in causing hepatic injury or hepatotoxicity.  Drug-induced liver injury is most frequent reason for removal of an approved drug from the market. Drug-induced liver injury accounts for more than 50% of cases of acute liver failure in USA. Reactive drug metabolites could play a role in in-vivo toxicity events such as tissue necrosis, mutagenicity and teratogenicity. Although a direct link between reactive drug metabolite formation and the onset of certain toxicities has yet to be identified, a large number of cases are reported where the toxicity finding is accompanied by reactive drug metabolite formation by drug compounds. QA-GSH assay is a viable method for quantitatively assessing the bioactivation potential in vitro and is well-suited for use in early drug discovery high throughput screening.

Bioactivation of drug candidates resulting in chemically reactive, electrophilic metabolites have always presented a major challenge to pharmaceutical companies. Moreover, the measurement of these metabolites has been hampered by the lack of an effective quantitative method. QA-GSH is a novel new tool for the detection of reactive metabolites. QA-GSH can provide semiquantitative assessment of the bioactivation potential in vitro.

High throughput screening and QA-GSH, the methodology is well suited for use in early drug discovery high throughput screening paradigms. It can be used in LC-MS/MS and/or NMR spectroscopy for the detection of reactive metabolites. Semi-quantitative method for determining reactive metabolite levels using quaternary ammonium GSH analogue (QA-GSH) has following advantages,

-  Fixed positive charge significantly increased limit of detection.

-  Equalized MS response from equimolar amounts of different GSH conjugates.

-  m/z of QA-GSH conjugate determined for M+ or MH2+ ion.

-  Response factor for IS with same charge state determined (peak area/conc.)

-  Peak area of QA-GSH conjugate/response factor of IS provides semi-quantification.

References

1.Soglia JR, Contillo LG, Kalgutkar AS, Zhao S, Hop CECA, Boyd JG, Cole MJ (2006). A semiquantitative method for the determination of reactive metabolite conjugate levels in vitro utilizing liquid chromatography-tandem mass spectrometry and novel quaternary ammonium glutathione analogues. Chem. Res. Toxicol., 19:480-490.

2.Mozhaev V, Mozhaeva L (2006). Trip Report: American Chemical Society Meeting, San Francisco, California, September 9 -14, 2006. Technical Reports., 11(8):2-3.