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Urinary Biomarkers of Trimethoprim Bioactivation in Vivo Following Therapeutic Dosing in Children Leon van Haandel,† Jennifer. L. Goldman,†,‡ Robin E. Pearce,†,‡ and J. Steven Leeder†,‡ †

Division of Clinical Pharmacology and Therapeutic Innovation, Children’s Mercy Hospitals and Clinics, and ‡Department of Pediatrics, School of Medicine, University of Missouri Kansas City, 2401 Gillham Road, Kansas City, Missouri 64108, United States S Supporting Information *

ABSTRACT: The antimicrobial trimethoprim-sulfamethoxazole (TMP-SMX) is widely used for the treatment of skin and softtissue infections in the outpatient setting. Despite its therapeutic benefits, TMP-SMX has been associated with a number of adverse drug reactions, which have been primarily attributed to the formation of reactive metabolites from SMX. Recently, in vitro experiments have demonstrated that TMP may form reactive intermediates as well. However, evidence of TMP bioactivation in patients has not yet been demonstrated. In this study, we performed in vitro trapping experiments with N-acetylL-cysteine (NAC) to determine stable markers of reactive TMP intermediates, focusing on eight potential markers (NAC-TMP adducts), some of which were previously identified in vitro. We developed a specific and sensitive assay involving liquid chromatography followed by tandem mass spectrometry for measurement of these adducts in human liver microsomal samples and expanded the methodology toward the detection of these analytes in human urine. Urine samples from four patients receiving TMP-SMX treatment were analyzed, and all samples demonstrated the presence of six NAC-TMP adducts, which were also detected in vitro. These adducts are consistent with the formation of imino-quinone-methide and para-quinone-methide reactive intermediates in vivo. As a result, the TMP component of TMP-SMX should be considered as well when evaluating adverse drug reactions to TMP-SMX.



considered a causative agent as well.7−10 The mechanism behind TMP-associated idiosyncratic reactions is still not well understood but appears to be immunologic in nature.11 The metabolism of TMP in humans has been well-described. TMP is largely excreted unchanged in the urine; however, about 20% of the dose is metabolized by the liver (Scheme 1). ODemethylation is the dominant biotransformation pathway of TMP, yielding 3′-desmethyl-TMP and 4′-desmethyl-TMP. Oxidation of the nitrogen atoms in the pyrimidine ring to form 1-NO-TMP and 3-NO-TMP and oxidation of the methylene bridge to Cα-OH-TMP have been reported as well.12,13 Recent in vitro data have demonstrated other potential metabolic pathways including sequential O-demethylation reactions leading to formation of the catechol, 3′,4′-desmethyl-TMP.14,15 In general, these metabolites have been viewed as stable and have not been associated with idiosyncratic drug reactions to date. More recently, it has been shown that in addition to forming stable products, TMP can undergo bioactivation through a variety of pathways in vitro (Scheme 2). Lai et al. demonstrated that TMP is bioactivated to a reactive imino-quinone-methide

INTRODUCTION Trimethoprim-sulfamethoxazole (TMP-SMX) is a combined antimicrobial utilized for a variety of infections. In the era of methicillin-resistant Staphylococcus aureus (MRSA), it is frequently the drug of choice to treat skin and soft-tissue infections in the outpatient setting because there is minimal MRSA resistance to TMP-SMX. The antibiotic is also inexpensive and in general is well-tolerated. Thus, TMP-SMX will likely remain a primary agent to treat this type of infection because there are relatively few widely available oral antimicrobial options effective against MRSA.1 Despite its overall safety record, TMP-SMX has long been associated with rare, but serious, idiosyncratic drug reactions including Stevens Johnson Syndrome, toxic epidermal necrolysis, agranulocytosis, and hepatic necrosis.2 Historically, the SMX component has been viewed as the causative agent in these unpredictable reactions. It is believed that the initiating event is the formation of a highly reactive nitroso-metabolite in the liver, resulting in haptenization of (cellular) proteins that ultimately trigger an immunological reaction.3−6 Interestingly, idiosyncratic drug reactions, including toxic epidermal necrolysis, have also been reported when TMP was used as the sole agent, suggesting that TMP must be © 2013 American Chemical Society

Received: September 10, 2013 Published: December 31, 2013 211

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Scheme 1. Oxidative Biotransformation of Trimethoprim as Described in the Literature

Scheme 2. Overview of in Vitro Pathways to Trimethoprim Bioactivation as Described by the Literaturea

a Citations are denoted by superscript. Putative reactive intermediates are enclosed in brackets. Please note that although TMPT4−10 are shown as NAC adducts for consistency with the presented work, the original cited work utilized GSH as the trapping reagent, and trapped structures were the GSH analogues of the demonstrated structures.

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Table 1. Detected Analytesa analyte 3′-desmethyl-TMP 4′-desmethyl-TMP 3′,4′-Desmethyl-TMP 1-NO-TMP 3-NO-TMP Ca-OH-TMP TMP-trapped metabolites TMPT1 TMPT2 TMPT3 TMPT4,5 TMPT6,7 TMPT8 a

formula (M + H)+

exact mass

measured mass

error (mDa)

error (ppm)

tR

MS/MS transition

CE

HOCL

HLM

urine

C13H17N4O3+ C13H17N4O3+ C12H15N4O3+ C14H19N4O4+ C14H19N4O4+ C14H19N4O4+

277.1301 277.1301 263.1144 307.1406 307.1406 307.1406

277.1301 277.1302 263.1142 307.1404 307.1404 307.1409

0 0.1 −0.2 −0.2 −0.2 0.3

0.0 0.4 −0.8 −0.7 −0.7 1.0

2.38 2.12 1.73 3.05 3.21 2.47

277.1 → 261 277.1 → 261 263.1 → 247 307.1 → 259 307.1 → 259 307.1 → 277

22 22 20 20 20 20

× × × × × ×

× × × × × ×

× × − × × ×

C19H26N5O6S+ C19H26N5O6S+ C19H26N5O6S+ C18H24N5O6S+ C18H24N5O6S+ C18H24N5O6S+

452.1604 452.1604 452.1604 438.1447 438.1447 438.1447

452.1599 452.1596 452.1597 438.1442 438.1442 438.1447

0.5 0.8 0.7 0.5 0.5 0

1.0 1.7 1.4 1.1 11 0.0

3.31 2.82 2.9 2.09 2.37 2.33

452.2 → 323 452.2 → 289 452.2 → 289 438.2 → 275 438.2 → 275 438.2 → 309

25 20 20 20 20 25

× × × × × ×

− × × × × ×

− × × × × −

tR, retention time; ×, metabolite detected in matrix; and −, metabolite not detected in matrix.

species that can be trapped with N-acetyl-L-cysteine (NAC).16 The major product was the adduct of NAC to the pyrimidine ring (TMPT1), and minor products were diasteriomers of NAC attached to the α-carbon (TMPT2 and 3). Le Blanc et al. reported an O-demethylated glutathione-trapped TMP metabolite using mass-defect labeling strategies; however, no structure or site of addition were postulated (TMPT4−8).17 Damsten et al. further evaluated bioactivation of TMP in vitro, and in addition to formation of TMPT2 and 3, they reported the existence of a didemethylated TMP gluthathione (GSH) adduct TMPT9.14 The authors postulated that such a product may have been the result from the formation of a catechol intermediate, which can form reactive ortho-quinone and para-quinonemethide intermediates that are subject to trapping by nucleophiles. However, the authors were unable to assign the site of addition. Finally, low amounts of a TMP-GSH conjugate that was fully O-demethylated and hydroxylated were detected (TMPT10). Because imino-quinone, ortho-quinone, and paraquinone reactive intermediates have been implicated in various idiosyncratic reactions associated with other drugs, bioactivation of TMP should be considered as an alternative mechanism that might contribute to the development of idiosyncratic drug reactions of TMP-SMX.18 To date, the evidence for TMP bioactivation has been limited to in vitro systems and has not been confirmed in vivo. Therefore, the goal of this study was to identify stable urinary biomarkers that are indicative of TMP bioactivation in vivo and to develop sensitive and selective analytical methodology for the detection of candidate biomarkers in urine.



phosphate buffer (50 mM, pH 7.4), MgCl2 (3 mM), EDTA (1 mM), and TMP (5, 50, or 500 μM) with or without NAC (5 mM) at the final concentrations listed. Reactions were initiated by the addition of an NADPH-generating system (consisting of NADP (1 mM), glucose-6phosphate dehydrogenase (1 U/mL), and glucose-6-phosphate (5 mM)) placed in a shaking water bath at 37 ± 0.1 °C and terminated after 60 min by the addition of 200 μL of ice-cold methanol. Control incubations were conducted in the absence of either microsomal protein or the NADPH-generating system. Protein was precipitated by centrifugation at 10 000gmax for 10 min, and an aliquot of the supernatant was analyzed via direct injection by LC−MS. Synthesis of NAC Conjugates of TMP Using HOCl as Oxidant. TMP-NAC adducts with HOCl were generated as described by Lai et al., utilizing the same experimental procedure and concentrations of TMP and NAC. Urine Collection. After obtaining parental consent (and patient assent if ≥7 years of age), a single void urine sample was collected from four children ≤17 years of age who had taken a therapeutic dose of TMP-SMX for the treatment of a skin and soft-tissue infection within 12 h of urine collection. The median age of the study participants was 9.3 years (5.4−12.1). Doses of TMP-SMX ranged from 2.5 to 6.1 mg/kg, and the time between dosing and urine collection ranged from 1.67 to 10.6 h. Immediately following urine collection, samples were aliquoted into 15 mL capped test tubes and stored in a −80 °C freezer. The collection of urine samples was approved by the Children’s Mercy Hospitals and Clinics Pediatric Institutional Review Board. Urine Sample Preparation. Prior to analysis, urine samples were thawed, and 250 μL of each sample was transferred into a Microcon Ultracel YM-10 centrifugal filtration device with a molecular weight cut off of 10 kDa. The samples were centrifuged for 15 min at 13 000gmax to remove proteins and particulates. The filtrate was transferred into an autosampler vial and analyzed by LC−MS. Analytical Methods. Aliquots from reaction mixtures containing HLM (4 μL) and urine samples (10 μL) were analyzed on two LC−MS platforms at the injection volumes indicated. Metabolite identification was performed on a Xevo G2-QTOF time-of-flight mass spectrometer (Waters, Manchester, UK) that was connected to a classic Acquity ultraperformance liquid chromatograph (UPLC) (Waters, Milford, MA, USA). Analytes were ionized utilizing positive electrospray ionization (ESI+). N2 was used as the desolvation gas. The desolvation temperature was set at 400 °C with a flow rate of 800 L/h and a source temperature of 125 °C. The capillary and cone voltages were set to 3000 and 25 V, respectively. Data were collected in the sensitivity mode using MSe with collision energy ramp of 15−50 eV. Data were acquired between 50 and 1200 Da, with a scan time of 0.1 s and interscan delay of 0.01 s over an analysis time of 10 min. A solution of 2 ng/mL leucine enkaphalin was used as LockSpray and measured for 0.2 s at a 20 s interval, and three measurements were averaged. TOF data were interrogated by the Masslynx add-on, Metabolynx XS, for compounds

EXPERIMENTAL SECTION

Materials. LC−MS Optima grade water, methanol (MeOH), and acetonitrile (ACN) were purchased from Fisher Scientific (FairLawn, NJ, USA). LC−MS grade formic acid (FA), glucose-6-phosphate dehydrogenase, EDTA, magnesium chloride (MgCl2), N-acetyl-Lcysteine (NAC), NADP, potassium phosphate dibasic, potassium phosphate monobasic, and trimethoprim (TMP) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO) and were of reagent grade quality or higher. 1-NO-TMP and 3-NO-TMP were synthesized by Artis-Chem Co. Ltd. (Shanghai, China). 4′-Desmethyl-TMP was obtained through Toronto Research Chemicals (Canada). Pooled human adult liver microsomes (HLM) (n = 16) were purchased from XenoTech, LLC (Lenexa, KS, USA). Leucine enkephalin was obtained through Waters (Milford, MA, USA). In Vitro Incubation Conditions. In vitro incubation reactions (200 μL) contained pooled HLM (200 μg of microsomal protein), potassium 213

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Figure 1. LC−MS/MS chromatograms of NAC-trapped TMP reactive intermediates (TMPT1−8) generated in the in vitro systems HOCl, HLM, and human urine. TMPT1 was found in HOCl (A) but not HLM (B) or human urine (C). The stereoisomers TMPT2,3 could be generated in HOCl (D) and HLM (E) and were also detected in human urine (F). NAC-desmethyl-TMP adducts (TMPT4−7) could be generated by HOCl (G) and HLM (H) and were also detected in human urine (I). TMPT8 was generated in the HOCl system (J) and was also observed in HLM incubations (K) but was not detected in human urine (L). The insets in panel G demonstrate that the two peaks under the original chromatographic conditions could be separated into its two stereoisomers in a slower isocratic separation: TMPT4,5 in the left insert and TMPT6,7 on the right. eluting between 0.5 and 6 min. The mass-defect filter was populated with (combinations of) expected metabolites, and a window of 25 mDa was allowed. The dealkylation tool was allowed to break two bonds. Metabolynx was allowed to search for combinations of metabolites. Compounds were considered a metabolite if (a) they were absent in the control experiment, (b) the measured accurate mass versus calculated mass did not differ by more than 2.0 ppm, and (c) the MS/MS spectrum could be adequately assigned. The detection of metabolites in vivo was performed on a Xevo TQ-S triple quadrupole mass spectrometer (Waters, Manchester, UK) that was connected to an iClass Acquity UPLC with a flow-through needle (FTN) sample manager. The instrument was operated in ESI+ mode. The probe capillary was optimized at 3.0 kV, and the desolvation and source temperature was set to 500 and 150 °C, respectively. The source offset was 50 V. The following gas flows were used: desolvation gas, 800 L/h; collision gas flow, 0.15 mL/min; and nebulizer, 7.0 bar. Argon was used for collision-induced dissociation (CID). Quadrupole 1 and quadrupole 3 were set to transmit ions with a resolution of 0.8 amu at full width half-maximum (fwhm) during MRM experiments, and unit

resolution was utilized in all other experiments. Specific instrument parameters for MRM transitions utilized to measure metabolites are given in Table 1. Analytes were separated on a Waters Acquity UPLC C18 reversedphase column (BEH 1.7 μm, 2.1 × 100 mm) that was preceded by a Waters Acquity UPLC C18 VanGuard Precolumn (1.7 μm, 2.1 × 5 mm). The following gradient was used on both UPLC systems: initial conditions of 5% B, ramping up linearly to 40% B at 6.0 min; at 6.1 min, the composition was stepped up to 95% B and held for 1.5 min. At 8.6 min, the column was stepped down to initial conditions and reequilibrated for 2 min. Mobile phases A and B were H2O and ACN, both containing 0.1% formic acid. The flow rate was 0.3 mL/min, and the column temperature was 40 °C. The temperature of the sample compartment was held at 4 °C. For the classic Acquity, the strong needle wash solvent consisted of a 50:50 ACN/H2O mix, the weak needle wash solvent consisted of a 10:90 ACN/H2O mix, and wash volumes were 1.0 mL for each. The wash solvent of the FTN sample manager consisted of a 90:10 MeOH/H2O mix, the purge solvent was 90:10 H2O/ACN mix, and both were used for 6 s. 214

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Figure 2. LC−MS/MS spectra of the various NAC-trapped TMP metabolites. The spectra from TMPT1−3, metabolites that originate from the iminoquinone-methide intermediate, are depicted in panels A−C. MS/MS spectra from TMPT4−8, compounds that are consistent with the formation of a quinone-methide, are given in panels D−F, respectively.



RESULTS AND DISCUSSION The primary purpose of this study was to perform a qualitative assessment designed to identify and characterize stable markers of TMP bioactivation and detoxification in the urine of patients taking TMP-SMX. Because none of the potential metabolites proposed in Scheme 2 are commercially available, in vitro experiments were undertaken to generate and characterize TMP metabolites followed by the development of sensitive and selective methodology for their unambiguous detection in urine specimens from patients being actively treated with TMP-SMX. Phase I TMP Metabolites by Human Liver Microsomes. Initial experiments were aimed at a comprehensive identification of the oxidative formation of TMP metabolites in a HLM system. HLM incubations containing TMP were terminated and analyzed by the LC-QTOF method followed by mining of the data using Metabolynx to search for known and unknown metabolites of TMP (Scheme 1). In the absence of a trapping agent, six (stable) metabolites of TMP were identified in HLM incubations (Table 1). Two Odemethylated metabolites of TMP were identified on the basis of

their accurate mass-to-charge ratio (m/z) (277.1301 and 277.1302). The observed MS/MS spectra were in agreement with previous data from the literature for these compounds and matched our authentic 4′-desmethyl-TMP standard.15 Differentiation between the compounds was not possible based on the MS/MS spectra of each compound. However, the two compounds were significantly separated in the chromatographic dimension (retention time (tR): 2.12 vs 2.38 min). Utilizing an authentic standard of 4′-desmethyl-TMP, it was determined that the early eluting peak (tR 2.12 min) was 4′-desmethyl-TMP and thus, by exclusion, the peak at 2.38 min was assigned to 3′desmethyl-TMP (Figure S1). This elution order has also been earlier reported.19 A more polar compound (tR 1.73 min) with an accurate mass of m/z 263.1142 was observed. The 28 Da loss is consistent with two O-demethylation reactions, and the analyte was tentatively identified as 3′,4′-desmethyl-TMP. Three analytes demonstrated a m/z of 307.014, which is 16 Da larger than TMP, consistent with acquisition of an oxygen atom. The retention time of two compounds (3.05 and 3.21 min) was longer than the retention time of TMP (2.90 min), an indication 215

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that hydroxylation occurred on the pyrimidine ring.15 The MS/ MS spectra of both compounds were similar, indicating that these compounds were likely structural isomers. Furthermore, given that the pyrimidine ring fragment of TMP has a m/z of 123, the observation of a fragment ion with m/z 139 could be assigned to the formation of N-oxides on the 1 and 3 positions of the pyrimidine-ring fragment. The identity was later confirmed using authentic standards, with the early eluting compound being 1NO-TMP and the late eluting compound being 3-NO-TMP (Figure S1). The last oxidative metabolite had a retention time of 2.47 min and an m/z of 307.1409. Collision-induced dissociation produced a major fragment ion with an m/z of 289, indicative of the loss of H2O and thereby suggestive of an exocyclic hydroxylation. Therefore, this signal is consistent with a CαOH-TMP metabolite. Trapping of TMP Bioactivated Intermediates with NAC. A major pathway for detoxifying electrophilic species generated in vivo is via conjugation with endogenous GSH. GSH concentrations are extremely high in the liver (∼5 mM) and are believed to also protect the tissue against oxidative damage.20 Drug−GSH conjugates are typically further broken down to Nacetylcysteine derivatives prior to their elimination in urine.21−23 In an attempt to generate trapped electrophilic-reactive intermediates of TMP, the HLM incubations were repeated with the addition of 5 mM NAC as a trapping agent. The most direct route to TMP bioactivation is via oxidation to an imino-quinone-methide intermediate. This reactive intermediate could be trapped by a biological nucleophile on either the pyrimidine ring or the methylene bridge to generate TMPT1 or the exocyclic stereo isomers TMPT2 and TMPT3, respectively. Upon incubation of TMP with HOCl followed by addition of NAC, TMPT1 was the major product and was readily identified (m/z 452.1599 with a tR of 3.31 min), suggesting that the assay conditions were appropriate for analysis of TMPT1 (Figure 1A). The collision-induced dissociation spectrum of this compound also matched with earlier published data for this compound (Figure 2A). Interestingly, TMPT1 could not be detected when the experiment was performed with HLM incubations (Figure 1B). TMPT2 and TMPT3, m/z 452.1596 and 452.1597, respectively, were readily detected in incubations with HOCl and HLM incubations (Figure 1D,E). TMPT2 and TMPT3 eluted closely together (2.82 and 2.90 min) and showed a dominant fragment ion of 289 (loss of NAC), which is in agreement with earlier data for this adduct (Figure 2B,C).16 The formation of TMPT1 in HLM has been described by Lai et al. but was not formed in our incubations with pooled HLMs. Interestingly, Damsten et al. reported the detection of TMPT2 and TMPT3 in HLM incubations (observed as one peak) but did not report the formation of TMPT1 under their experimental conditions, which was unfortunately not discussed by the authors.14,16 LeBlanc et al. reported the presence of one TMPGSH product in HLM incubations; however, no structure (i.e., TMPT1, TMPT2, or TMPT3) was assigned to the reported product.17 Therefore, it is uncertain whether TMPT1 is formed in HLM incubations. Two analytes with an exact m/z of 438.1447, consistent with O-demethyl-TMP-NAC, were found (Figure 1G,H). Both analytes were easily separated in the chromatographic dimension (2.09 and 2.37 min), suggesting that they were structural isomers. The MS/MS spectra (Figure 2D,E) for both products were similar and indicated a loss of NAC to yield a fragment ion with an m/z 275.1. Because the MS/MS spectra did not reveal the identity of each compound, microsomal NAC incubations

were repeated with 4-desmethyl-TMP as a substrate (Figure S2). These incubations revealed that the peak with a retention time of 2.09 min could be assigned to Cα-NAC-4′-desmethyl-TMP (TMPT4) and thus the product with a retention time of 2.37 min was assigned as Cα-NAC-3′-desmethyl-TMP (TMPT6). As a result of NAC trapping on the Cα carbon, it was expected that TMPT4 and TMPT6 would also have diastereomers (similar to TMPT2 and 3). An isocratic separation using 5% mobile phase B was able to resolve TMPT4 and its diastereomer (TMPT5) and to resolve partially TMPT6 and the diastereomer (TMPT7) (Figure 1G). This evidence is strongly suggestive of NAC addition to the α-carbon. In addition to TMPT4−7, a product with an exact mass of 438.1447 Da and a tR of 2.32 was observed in both HOCl and microsomal incubations, which suggests formation of an additional O-demethyl-TMP NAC adduct. The MS/MS spectrum (Figure 2F) showed peaks that could be attributed to the release of an unaltered pyrimidine ring, implying that NAC addition had occurred on the benzylic side of the molecule. The dominant fragment, with an m/z of 308.9, could be assigned to a loss of NAC with sulfur still attached to the TMP molecule in a fragmentation pathway that is similar to that of TMPT1.24 The appearance of NAC adducts on the α-carbon and the benzyl group of O-demethyl-TMP is consistent with formation of a para-quinone-methide intermediate once 4′-Odemethylation has occurred. Interestingly, TMPT8 (the ring break-down product of the quinone-methide) was the dominant product in both HOCl and HLM incubations, whereas TMPT1 (the hetereocycle break-down product of the imino-quinone methide) was not formed by HLMs under the experimental conditions of the present study. TMP-NAC adducts involving two O-demethylation reactions, as described by Damsten et al., were not detected in our 1 h HLM incubations. This result is in agreement with our observation that only a small amount of the putative precursor (3′,4′-desmethylTMP) was generated. Formation of TMP-NAC adducts (TMPT9 and 10) that would require extensive biotransformation (i.e., exhaustive O-demethylation in addition to oxidation) were also not detected in our 1 h incubations. Precursors to these metabolites were not detected as well. Development of MRM Transitions. The sensitivity of the Waters Xevo G2 QTOF and Waters Xevo TQ-S triple quadrupole platforms were compared utilizing aqueous TMP standards. The Waters TQ-S (in MRM mode) was about 2 orders of magnitude more sensitive when compared to the Waters Xevo G2 QTOF (data not shown). Given that products of TMP bioactivation and detoxification may be present only in low abundance in vivo, assay methodology was transferred to the higher-sensitivity triple quadrupole platform. Optimal MRM conditions (Table 1) were established for each compound by identifying the dominant product ion in LC−-MS/MS spectra (Figure 2) utilizing a collision-energy ramp followed by maximizing the signal for this ion by repeated analysis of a HLM sample at various collision energies. Analysis of in Vivo Spot Urine Samples. Four spot urine samples obtained from patients treated with TMP-SMX for skin and soft-tissue infections were analyzed by LC−MS/MS to determine if bioactivation of TMP could be detected in vivo. The excretion of NAC-conjugates in urine of TMP-treated patients is shown in Figure 3. A representative urinary LC−MS/MS chromatogram for TMPT1−8 is presented in Figure 1C,F,I,L. Although TMPT1−3 may be formed from a common iminoquinone-methide reactive intermediate, only TMPT2 and 3 were detected in the urine samples, and TMPT1 was not detected in 216

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CONCLUSIONS

Clinically, TMP-SMX remains a mainstay of therapy for several types of disease processes including urinary tract infections, Pneumocystis jiroveci prophylaxis, and treatment of various highly resistant Gram-negative infections. National trends are demonstrating a significant increase in usage of TMP-SMX in this setting as well as an increase in TMP-SMX-associated adverse reactions.25,26 Although severe TMP-SMX idiosyncratic reactions are rare, they can result in significant morbidity and mortality. Thus, a further evaluation of potential mechanisms of these reactions is warranted. The aim of this study was to confirm that TMP bioactivation occurs in vitro and to demonstrate that the process also occurs in vivo. Several NAC derivatives of TMP were detected in patient urine, indicating that bioactivation also occurs in vivo under conditions of usual clinical practice. Our data imply that two distinct reactive intermediates are formed in vivo: an iminoquinone methide and a quinone-methide intermediate (following demethylation). These intermediates are subsequently trapped by GSH or NAC to form TMPT2−7 (Scheme 3) followed by excretion into the urine. These findings indicate that both the SMX and TMP components of the combination therapy should be considered in the evaluation of TMP-SMX adverse drug reactions, especially in mechanistic studies seeking to understand better the pathogenesis of the events as well as factors contributing to individual risk. The presented methodology has the potential to aid future studies designed to assess interindividual variation in TMP bioactivation with the potential of identifying those at greatest risk for developing these undesired and unpredictable reactions resulting from this commonly used antibiotic.

Figure 3. Four pediatric spot urine samples were analyzed for the presence of TMPT1−8. Instrument response values are given for TMPT1−8 showing that TMPT2−7 could be detected in all four samples. TMPT1 and 8 were not detected in the urine samples.

either urine or HLM incubations. TMPT4−7 were detected in the urine of all patients. TMPT8 was detected in HLMs, indicating that humans are capable of forming this metabolite, but it was not observed in any of the spot urine samples. A possible explanation is that TMPT8 is (partially) excreted in the bile or is excreted as cysteine- or S-methylconjugates that would have gone undetected by our approach. There was no evidence for formation of TMPT9 and 10 by the analysis of the urine samples. Finally, on the basis of the findings from our in vitro and in vivo analysis, we present proposed pathways of TMP bioactivation, including proposed reactive species and their stable markers found in urine, in Scheme 3.

Scheme 3. Proposed Scheme for TMP Bioactivation, Detoxification, and Elimination in Humans

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Chemical Research in Toxicology



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ASSOCIATED CONTENT

S Supporting Information *

LC−MS assignments of the primary TMP metabolites: 1-NOTMP, 3-NO-TMP, 3′-desmethyl-TMP, and 4′-desmethyl-TMP in a HLM incubation when compared to authentic standards and LC−MS data of a HLM incubation of 4′-desmethyl-TMP in the presence of NAC. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Funding

Funding for this study was provided by the Marion Merrell Dow Clinical Scholar Award. Notes

The authors declare no competing financial interest.



ABBREVIATIONS ACN, acetonitrile; CID, collision-induced dissociation; ESI+, positive electrospray ionization; FA, formic acid; GSH, gluthathione; HLM, human liver microsomes; MeOH, methanol; MRSA, methicillin-resistant Staphylococcus aureus; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NAC, N-acetylcysteine; QTOF, quadrupole time-of-flight mass spectrometer; SMX, sulfamethoxazole; TMP, trimethoprim; UPLC, ultra-performance liquid chromatography



REFERENCES

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dx.doi.org/10.1021/tx4003325 | Chem. Res. Toxicol. 2014, 27, 211−218

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Urinary biomarkers of trimethoprim bioactivation in vivo following therapeutic dosing in children.

Article pubs.acs.org/crt Urinary Biomarkers of Trimethoprim Bioactivation in Vivo Following Therapeutic Dosing in Children Leon van Haandel,† Jennife...

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