An Innovative, Collaborative, and Strategic Approach to Proactively Evaluate and Update Drug Interactions Based on Prescribing Information of Newly Approved Medicinal Products.

BACKGROUND: Drug-drug interaction (DDIs) are evaluated using pharmacokinetic (PK) simulation models, clinical studies, and scientific publications throughout drug development. DDIs with Norvir (ritonavir) and combination products (eg, Kaletra [lopinavir/ritonavir]) containing ritonavir as a PK enhancer are relevant, because these drugs could affect exposures of CYP3A4 substrates. Application of algorithms proactively identified recently approved drugs, which potentially cause adverse outcomes when given with drugs containing ritonavir. METHODS: An evidence-based medicine technology platform was used to identify newly approved products. PK-related information from the products' prescribing information was reviewed to identify DDIs with ritonavir. Algorithms were used to further evaluate PK, clinical, and postmarketing information pertinent to the interaction to determine if prescribing information required revision. RESULTS: From January 1, 2014, through December 31, 2015, 39 newly approved drugs were identified as having potential interactions with Norvir and/or Kaletra. Ten drugs were excluded, 19 drugs went through initial screening, and 10 drugs underwent in-depth algorithm-based analyses for DDIs. No changes to prescribing information for Norvir or Kaletra were recommended from evaluation of the DDIs. Regulatory concurrence with AbbVie decisions was 93.1% (27/29, 93.1%); in 6.9% (2/29, 6.9%) of the evaluated interactions, at least 1 local regulatory authority disagreed with recommendations, requiring label changes to incorporate the DDI information. CONCLUSIONS: This proactive algorithmic approach identifies and complements existing methods used to detect DDIs with newly approved products. Additionally, this approach facilitates timely communication of risks to patients and healthcare providers via label revisions, publications, or other regulatory communications.

Globin monoadducts and cross-links provide evidence for the presence of S-(1,2-dichlorovinyl)-L-cysteine sulfoxide, chlorothioketene, and 2-chlorothionoacetyl chloride in the circulation in rats administered S-(1,2-dichlorovinyl)-L-cysteine.

S-(1,2-Dichlorovinyl)-L-cysteine (DCVC), a mutagenic and nephrotoxic metabolite of trichloroethylene, is bioactivated to S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (DCVCS) and chlorothioketene and/or 2-chlorothionoacetyl chloride by cysteine conjugate S-oxidase (S-oxidase) and cysteine conjugate beta-lyase (beta-lyase), respectively. Previously, we identified DCVCS-globin monoadducts and cross-links upon treating rats with DCVCS or incubating erythrocytes with DCVCS. In this study, the formation of DCVC-derived reactive intermediates was investigated after rats were given a single (230 or 460 micromol/kg, i.p.) or multiple (3 or 30 micromol/kg daily for 5 days) DCVC doses. LC/ESI/MS of trypsin-digested globin peptides revealed both S-oxidase and beta-lyase-derived globin monoadducts and cross-links consistent with in vivo DCVC bioactivation by both pathways. MS/MS analyses of trypsin-digested fractions of globin from one of the rats treated with multiple 30 micromol/kg DCVC doses led to identification of beta-lyase-derived monoadducts on both Cys93 and Cys125 of the beta-chains. While rats dosed with the 230 micromol/kg DCVC dose exhibited beta-lyase-dependent monoadducts and cross-links only (four out of four rats), rats given the 460 micromol/kg DCVC dose (two out of four) and rats administered the multiple DCVC doses (two out of four) exhibited both beta-lyase- and S-oxidase-derived monoadducts and cross-links. Because previous incubations of erythrocytes with DCVC did not result in detection of DCVCS-derived monoadducts or cross-links and had only resulted in detection of beta-lyase-derived monoadducts and cross-links, the DCVCS-globin monoadducts and cross-links detected in this study are likely the result of DCVC bioactivation outside the circulation and subsequent translocation of DCVCS and N-acetylated DCVCS into the erythrocytes.

Cysteine conjugate beta-lyase activity of rat erythrocytes and formation of beta-lyase-derived globin monoadducts and cross-links after in vitro exposure of erythrocytes to S-(1,2-dichlorovinyl)-L-cysteine.

S-(1,2-Dichlorovinyl)-L-cysteine (DCVC), a mutagenic and nephrotoxic metabolite of trichloroethylene, can be bioactivated to reactive metabolites, S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (DCVCS) or chlorothioketene and/or 2-chlorothionoacetyl chloride, by cysteine conjugate S-oxidase (S-oxidase) and cysteine conjugate beta-lyase (beta-lyase), respectively. Previously, we characterized the reactivity of DCVCS with Hb upon incubation of erythrocytes with DCVCS and provided evidence for the formation of distinct DCVCS-Hb monoadducts and cross-links in both isolated erythrocytes and rats given DCVCS. In the present study, we investigated DCVC bioactivation and Hb adduct formation in isolated rat erythrocytes incubated with DCVC (9 and 450 microM) at 37 degrees C and pH 7.4. The results suggested that no DCVCS monoadducts or cross-links were formed; however, LC/electrospray ionization/MS and matrix-assisted laser desorption/ionization/MS of trypsin-digested globin peptides revealed the presence of beta-lyase-derived globin monoadducts and cross-links. Adducts and cross-links in which the sulfur atom of the reactive sulfur intermediates were replaced by oxygen have also been detected. Use of SDS-PAGE provided additional evidence for globin cross-link formation in the presence of DCVC. Interestingly, the MS results suggest that the observed peptide selectivity of the beta-lyase-derived reactive sulfur/oxygen-containing species was different than that previously observed with DCVCS. While these results suggested that erythrocytes have beta-lyase but not S-oxidase activity, further support for this hypothesis was obtained using S-(2-benzothiazolyl)-L-cysteine, an alternative substrate for beta-lyases. Collectively, the results demonstrate the utility of Hb adducts and cross-links to characterize the metabolic pathway responsible for DCVC bioactivation in erythrocytes and to provide distinct biomarkers for each reactive metabolite.

Mass spectral analyses of hydroxymethylvinyl ketone-hemoglobin adducts formed after in vivo exposure of Sprague-Dawley rats to 3-butene-1,2-diol.

3-Butene-1,2-diol (BDD), a known in vivo metabolite of 1,3-butadiene, is oxidized to a reactive Michael acceptor, hydroxymethylvinyl ketone (HMVK). Previously, we characterized the formation of three HMVK-amino acid monoadducts when HMVK was incubated in vitro with N-acetyl-l-cysteine, l-valinamide, and N-acetyl-l-lysine (NAL) at physiological conditions. One HMVK-NAL cyclic diadduct (cyclic diadduct 1) also formed by sequential Michael addition reactions of two HMVK molecules with the epsilon-amino group of NAL followed by enolization and cyclization. Loss of a water molecule and autoxidation convert cyclic diadduct 1 to a more stable cyclic diadduct 2. In the present study, we used multiple mass spectrometry techniques to investigate the formation of HMVK adducts with nucleophilic residues of Hb in vivo after dosing Sprague-Dawley rats with 25 and 200 mg/kg BDD. Trypsin-digested globin peptides with mass shifts consistent with the presence of HMVK monoadducts and cyclic diadducts were detected by LC/electrospray-quadrupole time-of-flight/MS with all rats given BDD. Use of matrix-assisted laser desorption ionization/Fourier transform ion cyclotron resonance provided further evidence for the formation of HMVK monoadducts and cyclic diadducts, and use of LC/MS/MS provided unequivocal evidence for adduction of HMVK with Cys125 of globin beta chains. Because BDD can also be oxidized to 1,2-dihydroxy-3,4-epoxybutane (EBD), the formation of N(2)-(2,3,4-trihydroxybutyl) (THB)-Hb adducts was also investigated in rats given BDD, and several peptides modified by THB were detected. However, because HMVK incubations with red blood cells in vitro also led to the detection of THB-Hb adducts, the THB adducts formed in vivo could be attributed to formation of HMVK, EBD, or both. Collectively, the results provide new insights into the reaction of HMVK with proteins.

Formation of mono- and bis-Michael adducts by the reaction of nucleophilic amino acids with hydroxymethylvinyl ketone, a reactive metabolite of 1,3-butadiene.

Previously, our laboratory has shown that hydroxymethylvinyl ketone (HMVK), a Michael acceptor oxidation product of the 1,3-butadiene metabolite, 3-butene-1,2-diol, readily reacts with hemoglobin at physiological conditions and that mass spectrometry of trypsin-digested peptides suggested adduct formation with various nucleophilic amino acids. In the present study, we characterized reactions ofHMVK (3 mM) with three model nucleophilic amino acids (6 and/or 15 mM): N-acetyl-L-cysteine (NAC),L-valinamide, and N-acetyl-L-lysine (NAL). NAC was the most reactive toward HMVK followed by L-valinamide and NAL. HMVK incubations with each amino acid at pH 7.4 and 37 degrees C resulted in the formation of a mono-Michael adduct. In addition, HMVK incubated with NAL gave rise to two additional bis-Michael adducts characterized by LC/MS, LC/MS/MS, 1H NMR, and 1H-detected heteronuclear single quantum correlation. The relative ratios of areas of NAL monoadduct (adduct 1) and diadducts (adducts 2 and 3) at 6 h were 49, 21, and 30% of total product area, respectively. The formation of adduct 2 was dependent upon the presence of both adduct 1 and HMVK, whereas the formation of adduct 3 was dependent upon the presence of adduct 2 only. Monoadducts were formed by a Michael addition reaction of one HMVK moiety with nucleophilic amino acid, whereas NAL diadducts were products of two Michael addition reactions of two HMVK moieties followed by enolization and formation of an octameric cyclic product. NAL diadduct (adduct 3) was formed by loss of a water molecule from adduct 2 followed by autoxidation of one of the hydroxy groups, yielding a diketone conjugated system. Collectively, our results provide strong evidence that HMVK can react with various nucleophilic residues and form different types of adducts, suggesting that a variety of proteins may be subjected to these modifications, which could result in loss of protein function.

Detection of multiple globin monoadducts and cross-links after in vitro exposure of rat erythrocytes to S-(1,2-dichlorovinyl)-L-cysteine sulfoxide and after in vivo treatment of rats with S-(1,2-dichlorovinyl)-L-cysteine sulfoxide.

S-(1,2-dichlorovinyl)- L-cysteine sulfoxide (DCVCS), a Michael acceptor produced by an FMO3-mediated oxidation of the trichloroethylene metabolite S-(1,2-dichlorovinyl)- L-cysteine (DCVC), is a more potent nephrotoxicant than DCVC. Because DCVCS incubations with N-acetyl- L-cysteine at pH 7.4, 37 degrees C resulted in the formation of three diastereomeric monoadducts and one diadduct, globin monoadducts and cross-links formed after in vitro incubations of rat erythrocytes with DCVCS (0.9-450 microM) for 2 h and those present at 30 min after in vivo treatment of rats with DCVCS (23 and 230 micromol/kg) were characterized. ESI/MS of intact globin chains revealed adduction of 1 DCVCS moiety on the beta2 chain at the three lowest DCVCS concentrations and on the beta1 chain after the in vivo treatment with 230 micromol/kg DCVCS. Interestingly, intact globin dimers and trimers were detectable by ESI/MS with all DCVCS concentrations in vitro (also by SDS-PAGE) and in vivo. LC/MS and MALDI/FTICR of trypsin digested peptides from globin samples obtained after in vitro (450 microM DCVCS) or in vivo exposure to DCVCS (230 micromol/kg) suggested the formation of DCVCS monoadducts not only with Cys93 and Cys125 of the beta chains but also with Cys13 of the alpha chains, whereas no monoadducted peptides were detected at lower DCVCS concentrations in vitro or in vivo. However, LC/MS and MALDI-TOF/TOF suggested the presence of several DCVCS-derived peptide cross-links both in vivo and in vitro at all DCVCS exposure levels. Collectively, the results indicate at least 4 out of the 5 cysteine moieties of the rat hemoglobin heterodimer may be alkylated by DCVCS, in reactions that could also lead to the formation of multiple cross-links. DCVCS- and N-acetyl-DCVCS (NA-DCVCS)-derived globin cross-links containing GSH and Cys were also detected by mass spectrometry, providing strong evidence for the reactivity and/or cross-linking ability of DCVCS, NA-DCVCS, and their GSH or Cys conjugates in both the in vitro and the in vivo. Thus, hemoglobin adducts and cross-links may be useful biomarkers to investigate the possible presence of DCVCS in circulation after DCVC or trichloroethylene exposure.

Formation of three N-acetyl-L-cysteine monoadducts and one diadduct by the reaction of S-(1,2-dichlorovinyl)-L-cysteine sulfoxide with N-acetyl-L-cysteine at physiological conditions: chemical mechanisms and toxicological implications.

Previously, our laboratory has shown that S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (DCVCS), a Michael acceptor produced by a flavin-containing monooxygenase 3 (FMO3)-mediated oxidation of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), is a more potent nephrotoxicant than DCVC. In the present study, we characterized reactions of DCVCS with nucleophilic amino acids. DCVCS incubations with N-acetyl-L-cysteine (NAC) at pH 7.4 and 37 degrees C for 1 h resulted in the formation of three monoadducts and one diadduct characterized by LC/MS, 1H NMR, and 1H-detected heteronuclear single quantum correlation. The formation of all adducts (with relative ratios of 29, 31, 24, and 12%, respectively) was rapid and time-dependent; the half-lives of the two DCVCS diastereomers in the presence of NAC were 13.8 (diastereomer I) and 9.4 min (diastereomer II). Adducts 1 and 2 were determined to be diastereomers of S-[1-chloro-2-(N-acetyl-L-cystein- S-yl)vinyl]-L-cysteine sulfoxide formed by Michael addition of NAC to the terminal vinylic carbon of DCVCS followed by loss of HCl. Adduct 4 was determined to be S-[2-chloro-2-(N-acetyl-L-cystein- S-yl)vinyl]-L-cysteine sulfoxide formed from the initial Michael addition product followed by a less favorable loss of HCl and/or by a rearrangement of adduct 2 through the formation of a cyclic chloronium ion. The addition of another molecule of NAC to monoadducts 1, 2, or 4 resulted in the formation of the novel diadduct, S-[2,2-( N-acetyl-L-cystein-S-yl)vinyl]-L-cysteine sulfoxide (adduct 3), whose detection in relatively large amount suggests that DCVCS could act as a cross-linking agent. DCVCS was not reactive with N-acetyl-L-lysine or L-valinamide at similar incubation conditions. Collectively, the results suggest selective reactivity of DCVCS toward protein sulfhydryl groups. Furthermore, the cross-linking properties of DCVCS may in part explain its high nephrotoxic potency.

Mass spectral analyses of hemoglobin adducts formed after in vitro exposure of erythrocytes to hydroxymethylvinyl ketone.

Hydroxymethylvinyl ketone (HMVK) is a reactive oxidation product of 3-butene-1,2-diol, a metabolite of 1,3-butadiene. The potential for HMVK (0.1 and 1mM) to form hemoglobin (Hb) adducts in erythrocytes from Sprague-Dawley rats was investigated at physiological conditions (pH 7.4, 37 degrees C) using electrospray ionization mass spectrometry (ESI/MS). With the 0.1mM HMVK globin samples, the results indicate HMVK adduction on the alpha2, beta2 and beta3 chains. With the 1.0mM HMVK globin samples, adducts were detected on the beta2 and beta3 chains. However, no correlation was observed between incubation time and the extent of adduct formation, and additional adducts were detected when globin samples were fractionated by HPLC before the ESI/MS analyses. For specific localizations of adducts on the globin chains, trypsin digested peptides from the 1mM HMVK globin samples were subjected to liquid chromatography/mass spectrometry analyses. The results, which are consistent with formation of HMVK adducts on several specific peptides within the alpha- and beta-chains, suggest selectivity in the interaction of HMVK with the different cysteine residues in Hb. Because adducts were also detected in peptides containing no cysteine residues and multiple HMVK moieties were detected on some of the cysteine-containing peptides, the results suggest other amino acids may be also reactive with HMVK. Adduct profiles and their relative intensities were consistent between the 1 and 2h samples providing evidence for the HMVK reactions being fast and selective. The finding that fewer peptides were adducted in the 0.1mM HMVK globin samples provides further evidence for selectivity of the HMVK reaction. Collectively, the results show HMVK readily and selectively forms adducts on Hb. Characterization of these adducts will facilitate development of useful biomarkers of exposure to HMVK and its precursor 1,3-butadiene.