ABSTRACT
SIGNIFICANCE: The combined preclinical features of NVL-520 that include potent targeting of ROS1 and diverse ROS1 resistance mutations, high selectivity for ROS1 G2032R over TRK, and brain penetration mark the development of a distinct ROS1 TKI with the potential to surpass the limitations of earlier-generation TKIs for ROS1 fusion-positive patients. This article is highlighted in the In This Issue feature, p. 517.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Carcinoma, Non-Small-Cell Lung/genetics , Protein-Tyrosine Kinases/genetics , Aminopyridines , Lactams, Macrocyclic/pharmacology , Lactams , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins/genetics , Pyrazoles , Lung Neoplasms/genetics , Brain , MutationABSTRACT
Optimization of isoquinolinone PI3K inhibitors led to the discovery of a potent inhibitor of PI3K-γ (26 or IPI-549) with >100-fold selectivity over other lipid and protein kinases. IPI-549 demonstrates favorable pharmacokinetic properties and robust inhibition of PI3K-γ mediated neutrophil migration in vivo and is currently in Phase 1 clinical evaluation in subjects with advanced solid tumors.
ABSTRACT
The synthesis and structure-activity relationship studies on 5-trifluoromethylpyrido[4,3-d]pyrimidin-4(3H)-ones as antagonists of the human calcium receptor (CaSR) have been recently disclosed [ Didiuk et al. ( 2009 ) Bioorg. Med. Chem. Lett. 19 , 4555 - 4559 ). On the basis of its pharmacology and disposition attributes, (R)-2-(2-hydroxyphenyl)-3-(1-phenylpropan-2-yl)-5-(trifluoromethyl)pyrido[4,3-d]pyrimidin-4(3H)-one (1) was considered for rapid advancement to first-in-human (FIH) trials to mitigate uncertainty surrounding the pharmacokinetic/pharmacodynamic (PK/PD) predictions for a short-acting bone anabolic agent. During the course of metabolic profiling, however, glutathione (GSH) conjugates of 1 were detected in human liver microsomes in an NADPH-dependent fashion. Characterization of the GSH conjugate structures allowed insight(s) into the bioactivation pathway, which involved CYP3A4-mediated phenol ring oxidation to the catechol, followed by further oxidation to the electrophilic ortho-quinone species. While the reactive metabolite (RM) liability raised concerns around the likelihood of a potential toxicological outcome, a more immediate program goal was establishing confidence in human PK predictions in the FIH study. Furthermore, the availability of a clinical biomarker (serum parathyroid hormone) meant that PD could be assessed side by side with PK, an ideal scenario for a relatively unprecedented pharmacologic target. Consequently, progressing 1 into the clinic was given a high priority, provided the compound demonstrated an adequate safety profile to support FIH studies. Despite forming identical RMs in rat liver microsomes, no clinical or histopathological signs prototypical of target organ toxicity were observed with 1 in in vivo safety assessments in rats. Compound 1 was also devoid of metabolism-based mutagenicity in in vitro (e.g., Salmonella Ames) and in vivo assessments (micronuclei induction in bone marrow) in rats. Likewise, metabolism-based studies (e.g., evaluation of detoxicating routes of clearance and exhaustive PK/PD studies in animals to prospectively predict the likelihood of a low human efficacious dose) were also conducted, which mitigated the risks of idiosyncratic toxicity to a large degree. In parallel, medicinal chemistry efforts were initiated to identify additional compounds with a complementary range of human PK predictions, which would maximize the likelihood of achieving the desired PD effect in the clinic. The back-up strategy also incorporated an overarching goal of reducing/eliminating reactive metabolite formation observed with 1. Herein, the collective findings from our discovery efforts in the CaSR program, which include the incorporation of appropriate derisking steps when dealing with RM issues are summarized.
Subject(s)
Anabolic Agents/chemistry , Anabolic Agents/metabolism , Osteoporosis/drug therapy , Pyridines/chemistry , Pyridines/metabolism , Pyrimidinones/chemistry , Pyrimidinones/metabolism , Receptors, Calcium-Sensing/antagonists & inhibitors , Anabolic Agents/adverse effects , Animals , Crystallography, X-Ray , Humans , Pyridines/adverse effects , Pyrimidinones/adverse effects , RatsABSTRACT
Propranolol is a widely used quality control and validation compound for liver microsome and hepatocyte metabolism studies. A multitude of literature reports describing the identification of propranolol metabolites exists today. However, no literature reports currently exist showing hepatocyte metabolism across the five species commonly used during pre-clinical drug discovery, namely mouse, rat, dog, monkey, and human. Herein, we present full metabolic profiles of propranolol in mouse, rat, dog, monkey and human hepatocytes. As expected, extensive phase I and phase II metabolism was observed across all five species and species-specific metabolites were detected in monkey and dog hepatocytes. Of particular interest was the detection of an N-hydroxylamine glucuronide metabolite in monkey and dog hepatocytes.
Subject(s)
Hepatocytes/chemistry , Hepatocytes/metabolism , Mass Spectrometry/methods , Propranolol/pharmacokinetics , Animals , Cryopreservation , Dogs , Haplorhini , Hepatocytes/drug effects , Humans , Inactivation, Metabolic , Metabolic Clearance Rate , Mice , Propranolol/chemistry , RatsABSTRACT
Evaluation and optimization of drug metabolism and pharmacokinetic data plays an important role in drug discovery and development and several reliable in vitro ADME models are available. Recently higher throughput in vitro ADME screening facilities have been established in order to be able to evaluate an appreciable fraction of synthesized compounds. The ADME screening process can be dissected in five distinct steps: (1) plate management of compounds in need of in vitro ADME data, (2) optimization of the MS/MS method for the compounds, (3) in vitro ADME experiments and sample clean up, (4) collection and reduction of the raw LC-MS/MS data and (5) archival of the processed ADME data. All steps will be described in detail and the value of the data on drug discovery projects will be discussed as well. Finally, in vitro ADME screening can generate large quantities of data obtained under identical conditions to allow building of reliable in silico models.
Subject(s)
Drug Evaluation, Preclinical/standards , Drug-Related Side Effects and Adverse Reactions , Pharmaceutical Preparations/metabolism , Animals , Computer Simulation , Drug Evaluation, Preclinical/statistics & numerical data , Humans , Pharmacokinetics , Quality ControlABSTRACT
The synthesis and SAR for a series of diaminopyrimidines as PYK2 inhibitors are described. Using a combination of library and traditional medicinal chemistry techniques, a FAK-selective chemical series was transformed into compounds possessing good PYK2 potency and 10- to 20-fold selectivity against FAK. Subsequent studies found that the majority of the compounds were positive in a reactive metabolite assay, an indicator for potential toxicological liabilities. Based on the proposed mechanism for bioactivation, as well as a combination of structure-based drug design and traditional medicinal chemistry techniques, a follow-up series of PYK2 inhibitors was identified that maintained PYK2 potency, FAK selectivity and HLM stability, yet were negative in the RM assay.
Subject(s)
Focal Adhesion Kinase 2/antagonists & inhibitors , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Animals , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Disease Models, Animal , Drug Design , Focal Adhesion Protein-Tyrosine Kinases/antagonists & inhibitors , Humans , Molecular Conformation , Molecular Structure , Osteoporosis/drug therapy , Pyrimidines/chemistry , Rats , Structure-Activity RelationshipABSTRACT
In vitro covalent binding assessments of drugs have been useful in providing retrospective insights into the association between drug metabolism and a resulting toxicological response. On the basis of these studies, it has been advocated that in vitro covalent binding to liver microsomal proteins in the presence and the absence of NADPH be used routinely to screen drug candidates. However, the utility of this approach in predicting toxicities of drug candidates accurately remains an unanswered question. Importantly, the years of research that have been invested in understanding metabolic bioactivation and covalent binding and its potential role in toxicity have focused only on those compounds that demonstrate toxicity. Investigations have not frequently queried whether in vitro covalent binding could be observed with drugs with good safety records. Eighteen drugs (nine hepatotoxins and nine nonhepatotoxins in humans) were assessed for in vitro covalent binding in NADPH-supplemented human liver microsomes. Of the two sets of nine drugs, seven in each set were shown to undergo some degree of covalent binding. Among hepatotoxic drugs, acetaminophen, carbamazepine, diclofenac, indomethacin, nefazodone, sudoxicam, and tienilic acid demonstrated covalent binding, while benoxaprofen and felbamate did not. Of the nonhepatotoxic drugs evaluated, buspirone, diphenhydramine, meloxicam, paroxetine, propranolol, raloxifene, and simvastatin demonstrated covalent binding, while ibuprofen and theophylline did not. A quantitative comparison of covalent binding in vitro intrinsic clearance did not separate the two groups of compounds, and in fact, paroxetine, a nonhepatotoxin, showed the greatest amount of covalent binding in microsomes. Including factors such as the fraction of total metabolism comprised by covalent binding and the total daily dose of each drug improved the discrimination between hepatotoxic and nontoxic drugs based on in vitro covalent binding data; however, the approach still would falsely identify some agents as potentially hepatotoxic.
Subject(s)
Drug Evaluation, Preclinical , Hepatocytes/drug effects , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Toxicity Tests/methods , Acetaminophen/chemistry , Acetaminophen/metabolism , Acetaminophen/pharmacology , Binding Sites , Buspirone/chemistry , Buspirone/metabolism , Buspirone/pharmacology , Carbamazepine/chemistry , Carbamazepine/metabolism , Carbamazepine/pharmacology , Diclofenac/chemistry , Diclofenac/metabolism , Diclofenac/pharmacology , Diphenhydramine/chemistry , Diphenhydramine/metabolism , Diphenhydramine/pharmacology , Dose-Response Relationship, Drug , Hepatocytes/metabolism , Humans , Indomethacin/chemistry , Indomethacin/metabolism , Indomethacin/pharmacology , Meloxicam , Microsomes, Liver/drug effects , Molecular Structure , Paroxetine/chemistry , Paroxetine/metabolism , Paroxetine/pharmacology , Piperazines , Propranolol/chemistry , Propranolol/metabolism , Propranolol/pharmacology , Raloxifene Hydrochloride/chemistry , Raloxifene Hydrochloride/metabolism , Raloxifene Hydrochloride/pharmacology , Simvastatin/chemistry , Simvastatin/metabolism , Simvastatin/pharmacology , Structure-Activity Relationship , Thiazines/chemistry , Thiazines/metabolism , Thiazines/pharmacology , Thiazoles/chemistry , Thiazoles/metabolism , Thiazoles/pharmacology , Ticrynafen/chemistry , Ticrynafen/metabolism , Ticrynafen/pharmacology , Triazoles/chemistry , Triazoles/metabolismABSTRACT
The primary pathway of clearance of the methylenedioxyphenyl-containing compound and selective serotonin reuptake inhibitor paroxetine in humans involves P450 2D6-mediated demethylenation to a catechol intermediate. The process of demethylenation also results in the mechanism-based inactivation of the P450 isozyme. While the link between P450 2D6 inactivation and pharmacokinetic interactions of paroxetine with P450 2D6 substrates has been firmly established, there is a disconnect in terms of paroxetine's excellent safety record despite the potential for bioactivation. In the present study, we have systematically assessed the NADPH-dependent covalent binding of [(3)H]paroxetine to human liver microsomes and S-9 preparations in the absence and presence of cofactors of the various phase II drug-metabolizing enzymes involved in the downstream metabolism/detoxification of the putative paroxetine-catechol intermediate. Incubation of [(3)H]paroxetine with human liver microsomes and S-9 preparations resulted in irreversible binding of radioactive material to macromolecules by a process that was NADPH-dependent. The addition of reduced glutathione (GSH) to the microsomal and S-9 incubations resulted in a dramatic reduction of covalent binding. Following incubations with NADPH- and GSH-supplemented human liver microsomes and S-9, three sulfydryl conjugates with MH(+) ions at 623 Da (GS1), 779 Da (GS2), and 928 Da (GS3), respectively, were detected by LC-MS/MS. The collision-induced dissociation spectra allowed an insight into the structure of the GSH conjugates, based on which, bioactivation pathways were proposed. The formation of GS 1 was consistent with Michael addition of GSH to the quinone derived from two-electron oxidation of paroxetine-catechol. GS 3 was formed by the addition of a second molecule of GSH to the quinone species obtained via the two-electron oxidation of GS 1. The mechanism of formation of GS 2 can be rationalized via (i) further two-electron oxidation of the catechol motif in GS 3 to the ortho-quinone, (ii) loss of a glutamic acid residue from one of the adducted GSH molecules, and (iii) condensation of a cysteine-NH 2 with an adjacent carbonyl of the ortho-quinone to yield an ortho-benzoquinoneimine structure. Inclusion of the catechol-O-methyltransferase cofactor S-adenosylmethionine (SAM) in S-9 incubations also dramatically reduced the covalent binding of [(3)H]paroxetine, a finding that was consistent with O-methylation of the paroxetine-catechol metabolite to the corresponding guaiacol regioisomers in S-9 incubations. While the NADPH-dependent covalent binding was attenuated by GSH and SAM, these reagents did not alter paroxetine's ability to inactivate P450 2D6, suggesting that the reactive intermediate responsible for P450 inactivation did not leave the active site to react with other proteins. The results of our studies indicate that in addition to the low once-a-day dosing regimen (20 mg) of paroxetine, efficient scavenging of the catechol and quinone metabolites by SAM and GSH, respectively, serves as an explanation for the excellent safety record of paroxetine despite the fact that it undergoes bioactivation.
Subject(s)
Microsomes, Liver/metabolism , NADP/metabolism , Paroxetine/metabolism , Quinones/metabolism , Biotransformation , Cytochrome P-450 CYP2D6 Inhibitors , Glutathione/metabolism , Humans , Quinones/chemistry , TritiumABSTRACT
The current study examined the bioactivation potential of a nonpeptidyl thrombopoietin receptor agonist, 1-(3-chloro-5-((4-(4-fluoro-3-(trifluoromethyl)phenyl)thiazol-2-yl)carbamoyl)pyridine-2-yl)piperidine-4-carboxylic acid (1), containing a 2-carboxamido-4-arylthiazole moiety in the core structure. Toxicological risks arising from P450-catalyzed C4-C5 thiazole ring opening in 1 via the epoxidation-->diol sequence were alleviated, since mass spectrometric analysis of human liver microsome and/or hepatocyte incubations of 1 did not reveal the formation of reactive acylthiourea and/or glyoxal metabolites, which are prototypic products derived from thiazole ring scission. However, 4-(4-fluoro-3-(trifluoromethyl)phenyl)thiazol-2-amine (2), the product of hydrolysis of 1 in human liver microsomes, hepatocytes, and plasma, underwent oxidative bioactivation in human liver microsomes, since trapping studies with glutathione led to the formation of two conjugates derived from the addition of the thiol nucleophile to 2 and a thiazole- S-oxide metabolite of 2. Mass spectral fragmentation and NMR analysis indicated that the site of attachment of the glutathionyl moiety in both conjugates was the C5 position in the thiazole ring. Based on the structures of the glutathione conjugates, two bioactivation pathways are proposed, one involving beta-elimination of an initially formed hydroxylamine metabolite and the other involving direct two-electron oxidation of the electron-rich 2-aminothiazole system to electrophilic intermediates. This mechanistic insight into the bioactivation process allowed the development of a rational chemical intervention strategy that involved blocking the C5 position with a fluorine atom or replacing the thiazole ring with a 1,2,4-thiadiazole group. These structural changes not only abrogated the bioactivation liability associated with 1 but also resulted in compounds that retained the attractive pharmacological and pharmacokinetic attributes of the prototype agent.
Subject(s)
Pyridines/pharmacology , Receptors, Thrombopoietin/agonists , Thiazoles/chemistry , Animals , Biological Availability , Biotransformation , Cell Line , Drug Stability , Glutathione/metabolism , Hepatocytes/metabolism , Humans , Male , Mice , Microsomes, Liver/metabolism , Molecular Structure , Pyridines/blood , Pyridines/chemistry , Pyridines/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Thrombopoietin/genetics , Thiazoles/blood , Thiazoles/metabolism , Thiazoles/pharmacology , TransfectionABSTRACT
N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide (14, PHA-543,613), a novel agonist of the alpha7 neuronal nicotinic acetylcholine receptor (alpha7 nAChR), has been identified as a potential treatment of cognitive deficits in schizophrenia. Compound 14 is a potent and selective alpha7 nAChR agonist with an excellent in vitro profile. The compound is characterized by rapid brain penetration and high oral bioavailability in rat and demonstrates in vivo efficacy in auditory sensory gating and, in an in vivo model to assess cognitive performance, novel object recognition.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Cognition Disorders/drug therapy , Nicotinic Agonists/chemical synthesis , Nootropic Agents/chemical synthesis , Quinuclidines/chemical synthesis , Receptors, Nicotinic/metabolism , Schizophrenia/drug therapy , Animals , Biological Availability , Brain/metabolism , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Drug Stability , Ether-A-Go-Go Potassium Channels/drug effects , Evoked Potentials, Auditory/drug effects , Humans , In Vitro Techniques , Learning/drug effects , Male , Memory/drug effects , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Neurons/drug effects , Neurons/physiology , Nicotinic Agonists/pharmacokinetics , Nicotinic Agonists/pharmacology , Nootropic Agents/pharmacokinetics , Nootropic Agents/pharmacology , Patch-Clamp Techniques , Quinuclidines/chemistry , Quinuclidines/pharmacology , Radioligand Assay , Rats , Rats, Sprague-Dawley , Receptors, Nicotinic/physiology , Recognition, Psychology/drug effects , Stereoisomerism , Structure-Activity Relationship , alpha7 Nicotinic Acetylcholine ReceptorABSTRACT
An in vitro semiquantitative reactive metabolite detection assay is described 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 is 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 are 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. The QA-GSH IS semiquantitation method was 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. These results show that the QA-GSH semiquantitation assay described herein 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.
Subject(s)
Biotransformation , Glutathione/chemistry , Quaternary Ammonium Compounds/chemistry , Acetaminophen/chemistry , Acetaminophen/metabolism , Chromatography, High Pressure Liquid , Clozapine/chemistry , Clozapine/metabolism , Female , Flutamide/chemistry , Flutamide/metabolism , Glutathione/analogs & derivatives , Humans , In Vitro Techniques , Male , Mass Spectrometry , Microsomes, Liver/metabolism , Peptides/chemical synthesis , Spectrometry, Mass, Electrospray IonizationABSTRACT
We report herein, facile metabolite identification workflow on the anti-depressant nefazodone, which is derived from accurate mass measurements based on a single run/experimental analysis. A hybrid LTQ/orbitrap mass spectrometer was used to obtain accurate mass full scan MS and MS/MS in a data-dependent fashion to eliminate the reliance on a parent mass list. Initial screening utilized a high mass tolerance ( approximately 10 ppm) to filter the full scan MS data for previously reported nefazodone metabolites. The tight mass tolerance reduces or eliminates background chemical noise, dramatically increasing sensitivity for confirming or eliminating the presence of metabolites as well as isobaric forms. The full scan accurate mass analysis of suspected metabolites can be confirmed or refuted using three primary tools: (1) predictive chemical formula and corresponding mass error analysis, (2) rings-plus-double bonds, and (3) accurate mass product ion spectra of parent and suspected metabolites. Accurate mass characterization of the parent ion structure provided the basis for assessing structural assignment for metabolites. Metabolites were also characterized using parent product ion m/z values to filter all tandem mass spectra for identification of precursor ions yielding similar product ions. Identified metabolite parent masses were subjected to chemical formula calculator based on accurate mass as well as bond saturation. Further analysis of potential nefazodone metabolites was executed using accurate mass product ion spectra. Reported mass measurement errors for all full scan MS and MS/MS spectra was <3 ppm, regardless of relative ion abundance, which enabled the use of predictive software in determining product ion structure. The ability to conduct biotransformation profiling via tandem mass spectrometry coupled with accurate mass measurements, all in a single experimental run, is clearly one of the most attractive features of this methodology.
Subject(s)
Microsomes, Liver/metabolism , Spectrometry, Mass, Electrospray Ionization/methods , Triazoles/pharmacokinetics , Antidepressive Agents/analysis , Antidepressive Agents/pharmacokinetics , Cells, Cultured , Humans , Piperazines , Reproducibility of Results , Sensitivity and Specificity , Triazoles/analysisABSTRACT
Therapy with the antidepressant trazodone has been associated with several cases of idiosyncratic hepatotoxicity. While the mechanism of hepatotoxicity remains unknown, it is possible that reactive metabolites of trazodone play a causative role. Studies were initiated to determine whether trazodone undergoes bioactivation in human liver microsomes to electrophilic intermediates. LC/MS/MS analysis of incubations containing trazodone and NADPH-supplemented microsomes or recombinant P4503A4 in the presence of glutathione revealed the formation of conjugates derived from the addition of the sulfydryl nucleophile to mono-hydroxylated- and hydrated-trazodone metabolites. Product ion spectra suggested that mono-hydroxylation and sulfydryl conjugation occurred on the 3-chlorophenyl-ring, whereas hydration and subsequent sulfydryl conjugation had occurred on the triazolopyridinone ring system. These findings are consistent with bioactivation sequences involving: (1) aromatic hydroxylation of the 3-chlorophenyl-ring in trazodone followed by the two-electron oxidation of this metabolite to a reactive quinone-imine intermediate, which reacts with glutathione in a 1,4-Michael fashion and (2) oxidation of the pyridinone ring to an electrophilic epoxide, ring opening of which, by glutathione or water generates the corresponding hydrated-trazodone-thiol conjugate or the stable diol metabolite, respectively. The pathway involving trazodone bioactivation to the quinone-imine has also been observed with many para-hydroxyanilines including the structurally related antidepressant nefazodone. It is proposed that the quinone-imine and/or the epoxide intermediate(s) may represent a rate-limiting step in the initiation of trazodone-mediated hepatotoxicity.
Subject(s)
Antidepressive Agents, Second-Generation/pharmacokinetics , Benzoquinones/metabolism , Cytochrome P-450 Enzyme System/metabolism , Epoxy Compounds/metabolism , Microsomes, Liver/metabolism , Trazodone/pharmacokinetics , Biotransformation , Chromatography, High Pressure Liquid , Cytochrome P-450 CYP3A , Humans , Imines/metabolism , Liver/drug effects , Liver/metabolism , Liver/pathology , Microsomes, Liver/drug effects , Recombinant Proteins , Spectrometry, Mass, Electrospray IonizationABSTRACT
Cigarette smoking has been associated with various cancers including bladder and pancreas. 4-Aminobiphenyl has been isolated as a constituent of cigarette smoke and has been established as a carcinogen in various animal models and humans. In rodents and humans, 4-aminobiphenyl is N-hydroxylated and forms adducts to DNA, the predominant one being N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP). In this study, we report a micro-electrospray mass spectrometric (muESI-MS) isotope dilution method for the detection and quantification of dG-C8-ABP in human pancreatic tissue. A reverse phase capillary column (320 microm ID) was connected to a triple quadrupole mass spectrometer via a commercially available micro-ESI source. The system was operated in the selected reaction monitoring mode transmitting the [M + H]+ --> [M + H - 116]+ transitions for both the analyte and the isotopically labeled internal standard. Twelve human pancreas samples were analyzed, where six were current smokers (three male and three female) and six were considered nonsmokers (three female and three male). Of the samples analyzed, six showed dG-C8-ABP levels above the limit of quantification for the method, five were considered to have levels that were undetectable, and one was discarded due to inconsistent internal standard signal. The age of the human subjects ranged from 17 to 63, and, in samples where adduct was present, levels ranged anywhere from 1 to 60/10(8) nucleotides. Although no correlation between smoking preference, age, or gender was proven with this particular sample pool, this report demonstrates that capillary LC-muESI-MS can provide a sensitive and definitive method for DNA adduct analysis in human tissue.
Subject(s)
Aminobiphenyl Compounds/metabolism , Carcinogens/metabolism , DNA Adducts/analysis , Pancreas/metabolism , Adolescent , Adult , Chromatography, Liquid , Female , Humans , Male , Middle Aged , Spectrometry, Mass, Electrospray IonizationABSTRACT
Investigations into the role of bioactivation in the pathogenesis of xenobiotic-induced toxicity have been a major area of research since the link between reactive metabolites and carcinogenesis was first reported in the 1930s. Circumstantial evidence suggests that bioactivation of relatively inert functional groups to reactive metabolites may contribute towards certain drug-induced adverse reactions. Reactive metabolites, if not detoxified, can covalently modify essential cellular targets. The identity of the susceptible biomacromolecule(s), and the physiological consequence of its covalent modification, will dictate the resulting toxicological response (e.g., covalent modification of DNA by reactive intermediates derived from procarcinogens that potentially leads to carcinogenesis). The formation of drug-protein adducts often carries a potential risk of clinical toxicities that may not be predicted from preclinical safety studies. Animal models used to reliably predict idiosyncratic drug toxicity are unavailable at present. Furthermore, considering that the frequency of occurrence of idiosyncratic adverse drug reactions (IADRs) is fairly rare (1 in 1000 to 1 in 10,000), it is impossible to detect such phenomena in early clinical trials. Thus, the occurrence of IADRs during late clinical trials or after a drug has been released can lead to an unanticipated restriction in its use and even in its withdrawal. Major themes explored in this review include a comprehensive cataloguing of bioactivation pathways of functional groups commonly utilised in drug design efforts with appropriate strategies towards detection of corresponding reactive intermediates. Several instances wherein replacement of putative structural alerts in drugs associated with IADRs with a latent functionality eliminates the underlying liability are also presented. Examples of where bioactivation phenomenon in drug candidates can be successfully abrogated via iterative chemical interventions are also discussed. Finally, appropriate strategies that aid in potentially mitigating the risk of IADRs are explored, especially in circumstances in which the structural alert is also responsible for the primary pharmacology of the drug candidate and cannot be replaced.
Subject(s)
Drug Design , Pharmaceutical Preparations/metabolism , Technology, Pharmaceutical/methods , Animals , Biotransformation/drug effects , Biotransformation/physiology , Drug Evaluation, Preclinical/methods , Drug-Related Side Effects and Adverse Reactions , HumansABSTRACT
An in vitro reactive intermediate screening assay, incorporating the use of the close analog of glutathione, glutathione ethyl ester (GSH-EE) as a conjugating agent, was developed to identify compounds that form reactive intermediates in an in vitro metabolite generating system. The biological assay consisted of substrate [s] = 10 microM, human liver microsomes, an NADPH generating system and glutathione ethyl ester. Conjugates were extracted from the biological matrix using a combination of protein precipitation and a semi-automated 96-well plate solid phase extraction (SPE) procedure. A micro-bore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry (microLC-microESI-MS/MS) method detected glutathione ethyl ester conjugates using selected reaction monitoring (SRM) to simultaneously monitor for multiple MH+ to [MH - 129]+ transitions, where the 129 mass unit (Da) represents the neutral loss of the pyroglutamate moiety from GSH-EE. The multiple MH+ to [MH - 129]+ transitions (SRM mass table) were generated for potential reactive intermediates of each compound. Glutathione (GSH) and GSH-EE conjugate standards were used to evaluate MS detection sensitivity. Based on direct comparison of standard curve data, an approximate 10-fold increase in sensitivity was observed for conjugates containing GSH-EE moiety versus GSH. In vitro experiments were conducted using literature substrates acetaminophen, rosiglitazone, clozapine, diclofenac and either GSH-EE or GSH as a reactive intermediate conjugating agent. An increase in detection sensitivity was observed for each GSH-EE conjugate and in the case of acetaminophen-GSH-EE the peak area increase was approximately 80-fold. Twelve drug compounds, each having known biotransformation mechanisms, were used to further test the detection capabilities of the assay and establish a concordance to literature data. When GSH was used in the assay, conjugates were detected for 4 out of the 12 test compounds (33%). When GSH-EE was used in the assay, conjugates were detected for 10 out of the 12 test compounds (83%).