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ion at m/z 582 corresponds to a neutral loss of the phenylalanine moiety (165 Da). The ion at m/z 470 is derived from a neutral loss of dehydrated DCL (277 Da). Consecutive losses of water from m/z 470 generated ions at m/z 452,434, and 416. The ion at m/z 294 is the protonated molecular ion of (KF: Lys-phe) (Figure 10.15C and 10.15D).

The amount of AG that is covalently bound to protein is dependent both on the concentration of AG and the degree of reactivity to form protein and peptide adducts. Historically, AG reactivity has been expressed as the percentage of total AG that is involved in the reaction with protein [26,27]. For this technique, the AG reactivity is calculated as reactivity index, "C%," which is the ratio of ion current peak areas of AG peptide adducts (peak area "a" + "b" in Figure 10.15A) to those that correspond to AGs from the same sample (peak area "a" +"b"+ "c" in Figure 10.14A) multiplied by 100. The C% for DCL-AG was determined to be 0.88%. The AG reactivities of the seven drugs were ranked based on their C% (Table 10.3).

As major metabolites of most carboxylic acid-bearing compounds, AGs have been shown to be labile electrophiles that covalently bind to the nucle-ophilic functional groups of tissue macromolecules. The chemical reactivity of these conjugates corresponds well with the toxicity observed for drugs that contain a carboxylic acid group [35-38]. It has been suggested that long-lived, drug-altered proteins may act as immunogens and produce cytotoxic T-cell-mediated or antibody-dependent, cell-mediated toxicity in susceptible patients (Boelsterli et al.) [39]. It has also been demonstrated with this method that Schiff base adducts of AGs showed a linear increase over the entire time range of 0-67 hours at physiological pH.

The formation of adducts through a "Schiff base mechanism" was the basis to assess the reactivity of the seven model compounds with this new technique. The reactivity index generated with our method was consistent with those reported by Benet et al. [26] and Bolze et al. [27] (Table 10.3), which validated this technique to evaluate AG reactivity. Schiff base adducts of AGs and proteins were obtained from the literature for TOL, ZOM, and DCL

250 ' ' 300' ' ^ 35o' ' 1 400 ' ' 45^ 1 500 ' ' 550 ' ' 600 ' ' 650 ' ' 700 ' ' 750

FIGURE 10.15 The LC-MS/MS analysis of DCL-AGP in the second incubation at a 67-h time point. (A) Ion chromatogram of the deprotonated molecular ion of DCL-AGP; m /z 745. (B) Positive-ion full-scan mass spectrum of the LC-MS peak at 18.07 min. (C) Product-ion MS/MS spectrum of m/z 747 at 18.07 min. (D) Product-ion spectrum of ions at m/z 747 at (RT) 18.9 min.

250 ' ' 300' ' ^ 35o' ' 1 400 ' ' 45^ 1 500 ' ' 550 ' ' 600 ' ' 650 ' ' 700 ' ' 750

FIGURE 10.15 The LC-MS/MS analysis of DCL-AGP in the second incubation at a 67-h time point. (A) Ion chromatogram of the deprotonated molecular ion of DCL-AGP; m /z 745. (B) Positive-ion full-scan mass spectrum of the LC-MS peak at 18.07 min. (C) Product-ion MS/MS spectrum of m/z 747 at 18.07 min. (D) Product-ion spectrum of ions at m/z 747 at (RT) 18.9 min.

TABLE 10.3 Comparison of the Reactivity Ranking Obtained with This Method and Reported from the Literature (reactivity degree 7 > 6 > 5 > 4 > 3 > 2 > 1)

Compound Name Our Result Bolze et al. Benet et al.

Tolmetin 7 7 7

Diclofenac 6 5 6

Ketoprofen 3 2 3

Furosemide 1 1 1

[40-42]. Smith et al. [40] and Kretz-Rommel et al. [41] have confirmed the occurrence of Schiff base formation by the observation of increased covalent binding with the addition of NaCN to the reaction mixture of ZOM- and DCL-AGs in plasma.

The work described here demonstrated that the seven model compounds form AG peptide Schiff base adducts, and the yield is correlated with the rate of AG rearrangement. As shown in Figure 10.12, the rearrangement of AGs is a prerequisite for Schiff base-peptide adduct formation. The extent of the formation of Schiff base-peptide adducts from AGs should be proportional to the rearrangement rate of the primary AG. A correlation (R2 = 0.95) between the reactivity index and percentage of rearrangement of the primary acyl glucuronide of the seven compounds is shown in Figure 10.16. Based on

FIGURE 10.16 The correlation of the reactivity index (C%) with the rearrangement percentage (AGr%).

Rearrangement (AGr%)

FIGURE 10.16 The correlation of the reactivity index (C%) with the rearrangement percentage (AGr%).

these results, it appears that the covalent binding reactivity of AGs to proteins can be predicted based on their rearrangement rate.

Further studies investigated the relationship between the structure of the aglycone and the rearrangement of AGs. In addition to well-known environmental factors, such as pH and temperature, the rate of AG rearrangement can be affected by the structure of the aglycone. Chemically, the rearrangement of an AG is an intramolecular transesterification process, where the acyl group migrates between the hydroxyl groups of the glucuronic acid moiety, driven by the nucleophilic attack from each adjacent -OH group. This migration process can be affected by the inherent electronic and steric properties of the aglycone. Although the number of compounds tested was limited to only seven, the order of rate of rearrangement observed was acetic acid > isopropionic acid > benzoic acid, which implies that inherent electronic and steric properties may play an important role in affecting the rate of primary AG rearrangement. It could be hypothesized that the drug containing the car-boxylic acid group bound to an aromatic ring showed the lowest extent of rearrangement due to resonance stabilization provided by the aromatic moiety. Isopropionic acid derivatives display a slower rearrangement rate than those of acetic acid derivatives, possibly due to the higher steric hindrance capacity of the isopropyl group over the acetyl group.

Due to their low rate of covalent binding to protein, it might be suggested that compounds bearing benzoic acid or isopropionic acid substituents at the a-carbon of the carboxylic acid moiety should be considered when designing new chemical entities, provided these functional groups do not undermine desirable pharmacological activity.

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