Viral Kinetics Model of SARS-CoV-2 Infection Informs Drug Discovery, Clinical Dose, and Regimen Selection.

Quantitative systems pharmacology (QSP) has been an important tool to project safety and efficacy of novel or repurposed therapies for the SARS-CoV-2 virus. Here, we present a QSP modeling framework to predict response to antiviral therapeutics with three mechanisms of action (MoA): cell entry inhibitors, anti-replicatives, and neutralizing biologics. We parameterized three distinct model structures describing virus-host interaction by fitting to published viral kinetics data of untreated COVID-19 patients. The models were used to test theoretical behaviors and map therapeutic design criteria of the different MoAs, identifying the most rapid and robust antiviral activity from neutralizing biologic and anti-replicative MoAs. We found good agreement between model predictions and clinical viral load reduction observed with anti-replicative nirmatrelvir/ritonavir (Paxlovid®) and neutralizing biologics bamlanivimab and casirivimab/imdevimab (REGEN-COV®), building confidence in the modeling framework to inform a dose selection. Finally, the model was applied to predict antiviral response with ensovibep, a novel DARPin therapeutic designed as a neutralizing biologic. We developed a new in silico measure of antiviral activity, area under the curve (AUC) of free spike protein concentration, as a metric with larger dynamic range than viral load reduction. By benchmarking to bamlanivimab predictions, we justified dose levels of 75, 225, and 600 mg ensovibep to be administered intravenously in a Phase 2 clinical investigation. Upon trial completion, we found model predictions to be in good agreement with the observed patient data. These results demonstrate the utility of this modeling framework to guide the development of novel antiviral therapeutics.

Quantitative Proteogenomic Characterization of Inflamed Murine Colon Tissue Using an Integrated Discovery, Verification, and Validation Proteogenomic Workflow.

Chronic inflammation of the colon causes genomic and/or transcriptomic events, which can lead to expression of non-canonical protein sequences contributing to oncogenesis. To better understand these mechanisms, Rag2-/-Il10-/- mice were infected with Helicobacter hepaticus to induce chronic inflammation of the cecum and the colon. Transcriptomic data from harvested proximal colon samples were used to generate a customized FASTA database containing non-canonical protein sequences. Using a proteogenomic approach, mass spectrometry data for proximal colon proteins were searched against this custom FASTA database using the Galaxy for Proteomics (Galaxy-P) platform. In addition to the increased abundance in inflammatory response proteins, we also discovered several non-canonical peptide sequences derived from unique proteoforms. We confirmed the veracity of these novel sequences using an automated bioinformatics verification workflow with targeted MS-based assays for peptide validation. Our bioinformatics discovery workflow identified 235 putative non-canonical peptide sequences, of which 58 were verified with high confidence and 39 were validated in targeted proteomics assays. This study provides insights into challenges faced when identifying non-canonical peptides using a proteogenomics approach and demonstrates an integrated workflow addressing these challenges. Our bioinformatic discovery and verification workflow is publicly available and accessible via the Galaxy platform and should be valuable in non-canonical peptide identification using proteogenomics.

Engineered human blood-brain barrier microfluidic model for vascular permeability analyses.

The blood-brain barrier (BBB) greatly restricts the entry of biological and engineered therapeutic molecules into the brain. Due to challenges in translating results from animal models to the clinic, relevant in vitro human BBB models are needed to assess pathophysiological molecular transport mechanisms and enable the design of targeted therapies for neurological disorders. This protocol describes an in vitro model of the human BBB self-assembled within microfluidic devices from stem-cell-derived or primary brain endothelial cells, and primary brain pericytes and astrocytes. This protocol requires 1.5 d for device fabrication, 7 d for device culture and up to 5 d for downstream imaging, protein and gene expression analyses. Methodologies to measure the permeability of any molecule in the BBB model, which take 30 min per device, are also included. Compared with standard 2D assays, the BBB model features relevant cellular organization and morphological characteristics, as well as values of molecular permeability within the range expected in vivo. These properties, coupled with a functional brain endothelial expression profile and the capability to easily test several repeats with low reagent consumption, make this BBB model highly suitable for widespread use in academic and industrial laboratories.

P-glycoprotein Substrate Assessment in Drug Discovery: Application of Modeling to Bridge Differential Protein Expression Across In Vitro Tools.

P-glycoprotein (P-gp) efflux assay is an integral part of discovery screening, especially for drugs requiring brain penetration as P-gp efflux ratio (ER) inversely correlates with brain exposure. However, significant variability in P-gp ER generated across cell lines can lead to misclassification of a P-gp substrate and subsequently disconnect with brain exposure data. We hypothesized that the ER depends on P-gp protein expression level in the in vitro assay. Quantitative proteomics and immunofluorescence staining were utilized to characterize P-gp protein expression and localization in four recombinant cell lines, over-expressing human or mouse P-gp isoforms, followed by functional evaluation. Efflux data generated in each cell line was compared against available rodent brain distribution data. The results suggested that the cell line with highest P-gp expression (hMDCK-MDR1 sourced from NIH) led to greatest dynamic range for efflux; thus, proving to be the most sensitive model to predict brain penetration. Cell lines with lower P-gp expression exhibited the greatest tendency for compound-dependent in vitro efflux saturation leading to false negative results. Ultimately, P-gp kinetics were characterized using a compartmental model to generate system-independent parameters to resolve such discrepancy. This study highlights the need for careful choice of well characterized P-gp in vitro tools and utility of modeling techniques to enable appropriate interpretation of the data.

Low Doses of Arsenic in a Mouse Model of Human Exposure and in Neuronal Culture Lead to S-Nitrosylation of Synaptic Proteins and Apoptosis via Nitric Oxide.

BACKGROUND: Accumulating public health and epidemiological literature support the hypothesis that arsenic in drinking water or food affects the brain adversely. METHODS: Experiments on the consequences of nitric oxide (NO) formation in neuronal cell culture and mouse brain were conducted to probe the mechanistic pathways of nitrosative damage following arsenic exposure. RESULTS: After exposure of mouse embryonic neuronal cells to low doses of sodium arsenite (SA), we found that Ca2+ was released leading to the formation of large amounts of NO and apoptosis. Inhibition of NO synthase prevented neuronal apoptosis. Further, SA led to concerted S-nitrosylation of proteins significantly associated with synaptic vesicle recycling and acetyl-CoA homeostasis. Our findings show that low-dose chronic exposure (0.1-1 ppm) to SA in the drinking water of mice led to S-nitrosylation of proteomic cysteines. Subsequent removal of arsenic from the drinking water reversed the biochemical alterations. CONCLUSIONS: This work develops a mechanistic understanding of the role of NO in arsenic-mediated toxicity in the brain, incorporating Ca2+ release and S-nitrosylation as important modifiers of neuronal protein function.

Multi-Omics Characterization of Inflammatory Bowel Disease-Induced Hyperplasia/Dysplasia in the Rag2-/-/Il10-/- Mouse Model.

Epigenetic dysregulation is hypothesized to play a role in the observed association between inflammatory bowel disease (IBD) and colon tumor development. In the present work, DNA methylome, hydroxymethylome, and transcriptome analyses were conducted in proximal colon tissues harvested from the Helicobacter hepaticus (H. hepaticus)-infected murine model of IBD. Reduced representation bisulfite sequencing (RRBS) and oxidative RRBS (oxRRBS) analyses identified 1606 differentially methylated regions (DMR) and 3011 differentially hydroxymethylated regions (DhMR). These DMR/DhMR overlapped with genes that are associated with gastrointestinal disease, inflammatory disease, and cancer. RNA-seq revealed pronounced expression changes of a number of genes associated with inflammation and cancer. Several genes including Duox2, Tgm2, Cdhr5, and Hk2 exhibited changes in both DNA methylation/hydroxymethylation and gene expression levels. Overall, our results suggest that chronic inflammation triggers changes in methylation and hydroxymethylation patterns in the genome, altering the expression of key tumorigenesis genes and potentially contributing to the initiation of colorectal cancer.

The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity.

KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3-5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.

Application of Transmural Flow Across In Vitro Microvasculature Enables Direct Sampling of Interstitial Therapeutic Molecule Distribution.

In vitro prediction of physiologically relevant transport of therapeutic molecules across the microcirculation represents an intriguing opportunity to predict efficacy in human populations. On-chip microvascular networks (MVNs) show physiologically relevant values of molecular permeability, yet like most systems, they lack an important contribution to transport: the ever-present fluid convection through the endothelium. Quantification of transport through the MVNs by current methods also requires confocal imaging and advanced analytical techniques, which can be a bottleneck in industry and academic laboratories. Here, it is shown that by recapitulating physiological transmural flow across the MVNs, the concentration of small and large molecule therapeutics can be directly sampled in the interstitial fluid and analyzed using standard analytical techniques. The magnitudes of transport measured in MVNs reveal trends with molecular size and type (protein versus nonprotein) that are expected in vivo, supporting the use of the MVNs platform as an in vitro tool to predict distribution of therapeutics in vivo.

An on-chip model of protein paracellular and transcellular permeability in the microcirculation.

Recent therapeutic success of large-molecule biologics has led to intense interest in assays to measure with precision their transport across the vascular endothelium and into the target tissue. Most current in vitro endothelial models show unrealistically large permeability coefficients due to a non-physiological paracellular transport. Thus, more advanced systems are required to better recapitulate and discern the important contribution of transcellular transport (transcytosis), particularly of pharmaceutically-relevant proteins. Here, a robust platform technology for the measurement of transport through a human endothelium is presented, which utilizes in vitro microvascular networks (MVNs). The self-assembled MVNs recapitulate the morphology and junctional complexity of in vivo capillaries, and express key endothelial vesicular transport proteins. This results in measured permeabilities to large molecules comparable to those observed in vivo, which are orders of magnitude lower than those measured in transwells. The permeability of albumin and immunoglobulin G (IgG), biopharmaceutically-relevant proteins, is shown to occur primarily via transcytosis, with passage of IgG regulated by the receptor FcRn. The physiological relevance of the MVNs make it a valuable tool to assess the distribution of biopharmaceuticals into tissues, and may be used to prioritize candidate molecules from this increasingly important class of therapeutics.

Pharmacological Assessment of Sepiapterin Reductase Inhibition on Tactile Response in the Rat.

There is an unmet medical need for nonopioid pain therapies in human populations; several pathways are under investigation for possible therapeutic intervention. Tetrahydrobiopterin (BH4) has received attention recently as a mediator of neuropathic pain. Recent reports have implicated sepiapterin reductase (SPR) in this pain pathway as a regulator of BH4 production. To evaluate the role of SPR inhibition on BH4 reduction, we developed analytical methods to monitor the relationship between the plasma concentration of test article and endogenous pterins and applied these in the rat spinal nerve ligation pain model. Sepiapterin is an endogenous substrate, which accumulates upon inhibition of SPR. In response to a potent inhibitor of SPR, plasma concentrations of sepiapterin increased proportionally with exposure. An indirect-effect pharmacokinetic/pharmacodynamic model was developed to describe the relationship between the plasma pharmacokinetics of test article and plasma sepiapterin levels in the rat, which was used to determine an in vivo SPR IC50 value. SPR inhibition and mechanical allodynia were assessed coordinately with pterin biomarkers in plasma and at the site of neuronal injury (i.e., dorsal root ganglion). Upon daily oral administration for 3 consecutive days, unbound plasma concentrations of test article exceeded the unbound in vivo rat SPR IC90 throughout the dose intervals, leading to a 60% reduction in BH4 in the dorsal root ganglion. Despite evidence for pharmacological modulation of the BH4 pathway, there was no significant effect on the tactile paw withdrawal threshold relative to vehicle-treated controls.

Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease.

Inflammatory bowel disease (IBD) arises from inappropriate activation of the mucosal immune system resulting in a state of chronic inflammation with causal links to colon cancer. Helicobacter hepaticus-infected Rag2(-/-) mice emulate many aspects of human IBD, and our recent work using this experimental model highlights the importance of neutrophils in the pathology of colitis. To define molecular mechanisms linking colitis to the identity of disease biomarkers, we performed a translational comparison of protein expression and protein damage products in tissues of mice and human IBD patients. Analysis in inflamed mouse colons identified the neutrophil- and macrophage-derived damage products 3-chlorotyrosine (Cl-Tyr) and 3-nitrotyrosine, both of which increased with disease duration. Analysis also revealed higher Cl-Tyr levels in colon relative to serum in patients with ulcerative colitis and Crohn disease. The DNA chlorination damage product, 5-chloro-2'-deoxycytidine, was quantified in diseased human colon samples and found to be present at levels similar to those in inflamed mouse colons. Multivariate analysis of these markers, together with serum proteins and cytokines, revealed a general signature of activated innate immunity in human IBD. Signatures in ulcerative colitis sera were strongly suggestive of neutrophil activity, and those in Crohn disease and mouse sera were suggestive of both macrophage and neutrophil activity. These data point to innate immunity as a major determinant of serum and tissue profiles and provide insight into IBD disease processes.

Serum metabolomics in a Helicobacter hepaticus mouse model of inflammatory bowel disease reveal important changes in the microbiome, serum peptides, and intermediary metabolism.

Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disorder of the bowel. The etiology remains unknown, but IBD is immune-driven and multiple factors including genetic, environmental, and microbiological components play a role. Recombinase-activating gene-2-deficient (Rag2(-/-)) mice infected with Helicobacter hepaticus (H. hepaticus) have been developed as an animal model to imitate naturally occurring inflammatory events and associated key features of chronic inflammatory responses in humans. In this study, we have combined mass spectrometry-based metabolomics and peptidomics to analyze serum samples of Rag2(-/-) mice infected with H. hepaticus. Metabolomics profiling revealed that H. hepaticus infection dramatically changed numerous metabolite pathways, including tryptophan metabolism, glycerophospholipids, methionine-homocysteine cycle, citrate cycle, fatty acid metabolism and purine metabolism, with the majority of metabolites being down-regulated. In particular, there were notable effects of gut microflora on the blood metabolites in infected animals. In addition, the peptidomics approach identified a number of peptides, originating from proteins, including fibrinogen, complement C4, and alpha-2-macroglobulin, with diverse biological functions with potentially important implications for the progress of IBD. In summary, the strategy of integrating a relevant animal model and sensitive mass spectrometry-based profiling may offer a new perspective to explore biomarkers and provide mechanistic insights into IBD.

Infection-induced colitis in mice causes dynamic and tissue-specific changes in stress response and DNA damage leading to colon cancer.

Helicobacter hepaticus-infected Rag2(-/-) mice emulate many aspects of human inflammatory bowel disease, including the development of colitis and colon cancer. To elucidate mechanisms of inflammation-induced carcinogenesis, we undertook a comprehensive analysis of histopathology, molecular damage, and gene expression changes during disease progression in these mice. Infected mice developed severe colitis and hepatitis by 10 wk post-infection, progressing into colon carcinoma by 20 wk post-infection, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages. Transcriptional profiling revealed decreased expression of DNA repair and oxidative stress response genes in colon, but not in liver. Mass spectrometric analysis revealed higher levels of DNA and RNA damage products in liver compared to colon and infection-induced increases in 5-chlorocytosine in DNA and RNA and hypoxanthine in DNA. Paradoxically, infection was associated with decreased levels of DNA etheno adducts. Levels of nucleic acid damage from the same chemical class were strongly correlated in both liver and colon. The results support a model of inflammation-mediated carcinogenesis involving infiltration of phagocytes and generation of reactive species that cause local molecular damage leading to cell dysfunction, mutation, and cell death. There are strong correlations among histopathology, phagocyte infiltration, and damage chemistry that suggest a major role for neutrophils in inflammation-associated cancer progression. Further, paradoxical changes in nucleic acid damage were observed in tissue- and chemistry-specific patterns. The results also reveal features of cell stress response that point to microbial pathophysiology and mechanisms of cell senescence as important mechanistic links to cancer.

Site-specific and redox-controlled S-nitrosation of thioredoxin.

Protein S-nitrosation on cysteine residues has emerged as an important posttranslational modification in mammalian cells. Previous studies have suggested a primary role for thioredoxin (Trx) in controlling protein S-nitrosation reactions. Human Trx contains five conserved Cys, including two redox-active catalytic Cys (Cys32 and Cys35) and three non-active-site Cys (Cys62, Cys69, and Cys73), all of which have been reported as targets of S-nitrosation. Prior reports have studied thermodynamic end points of nitrosation reactions; however, the kinetics of Trx nitrosation has not previously been investigated. Using the transnitrosation agent, S-nitrosoglutathione, a kinetic analysis of the selectivity and redox dependence of Trx nitrosation at physiologically relevant concentrations and times was performed, utilizing a mass spectrometry-based method for the direct analysis of the nitrosated Trx. Reduced Trx (rTrx) was nitrosated 2.7-times faster than oxidized Trx (oTrx), and rTrx was nitrosated selectively on Cys62, whereas oTrx was nitrosated only on Cys73. These sites of nitrosation were confirmed at the peptide level using a novel modification of the biotin-switch technique called the reductive switch. These results suggest separate signaling pathways for Trx-SNO under different cellular redox states.

Oxidation and glycolytic cleavage of etheno and propano DNA base adducts.

Non-invasive strategies for the analysis of endogenous DNA damage are of interest for the purpose of monitoring genomic exposure to biologically produced chemicals. We have focused our research on the biological processing of DNA adducts and how this may impact the observed products in biological matrixes. Preliminary research has revealed that pyrimidopurinone DNA adducts are subject to enzymatic oxidation in vitro and in vivo and that base adducts are better substrates for oxidation than the corresponding 2'-deoxynucleosides. We tested the possibility that structurally similar exocyclic base adducts may be good candidates for enzymatic oxidation in vitro. We investigated the in vitro oxidation of several endogenously occurring etheno adducts [1,N(2)-epsilon-guanine (1,N(2)-epsilon-Gua), N(2),3-epsilon-Gua, heptanone-1,N(2)-epsilon-Gua, 1,N(6)-epsilon-adenine (1,N(6)-epsilon-Ade), and 3,N(4)-epsilon-cytosine (3,N(4)-epsilon-Cyt)] and their corresponding 2'-deoxynucleosides. Both 1,N(2)-epsilon-Gua and heptanone-1,N(2)-epsilon-Gua were substrates for enzymatic oxidation in rat liver cytosol; heteronuclear NMR experiments revealed that oxidation occurred on the imidazole ring of each substrate. In contrast, the partially or fully saturated pyrimidopurinone analogues [i.e., 5,6-dihydro-M(1)G and 1,N(2)-propanoguanine (PGua)] and their 2'-deoxynucleoside derivatives were not oxidized. The 2'-deoxynucleoside adducts, 1,N(2)-epsilon-dG and 1,N(6)-epsilon-dA, underwent glycolytic cleavage in rat liver cytosol. Together, these data suggest that multiple exocyclic adducts undergo oxidation and glycolytic cleavage in vitro in rat liver cytosol, in some instances in succession. These multiple pathways of biotransformation produce an array of products. Thus, the biotransformation of exocyclic adducts may lead to an additional class of biomarkers suitable for use in animal and human studies.

Gamma-glutamyltranspeptidase-mediated degradation of diclofenac-S-acyl-glutathione in vitro and in vivo in rat.

Diclofenac, a nonsteroidal antiinflammatory drug, is known to be metabolized to chemically reactive intermediates that transacylate GSH forming diclofenac-S-acyl-glutathione (D-SG) in vivo in rat and in vitro in rat and human hepatocytes. Recently, it was reported that the treatment of rats with diclofenac led to a substantial decrease in the activity of hepatic gamma-glutamyltranspeptidase (gamma-GT), an extracellular canalicular membrane enzyme. Because studies have indicated that D-SG is a chemically reactive transacylating species that is excreted into rat bile, we propose that D-SG formed in the liver may be a substrate for, and potential inhibitor of, hepatic gamma-GT. The present experiments were performed to investigate the ability of D-SG to be a substrate for gamma-GT in vivo in rat and in vitro with commercially available gamma-GT enzyme. We also examined the ability of D-SG to inhibit gamma-GT in vitro. Thus, LC-MS/MS analysis of bile extracts from diclofenac-dosed rats (200 mg/kg, iv) showed the presence of the gamma-GT-mediated D-SG degradation product diclofenac-N-acyl-cysteinylglycine (D- N-CG), where a total of approximately 8 microg was excreted 6 h postadministration. When D-SG (100 microM) was incubated with gamma-GT (1 unit/mL), the GSH adduct was degraded in a linear time-dependent fashion where approximately 94 microM D- N-CG was formed after 20 min of incubation. Dialysis studies showed that inhibition of gamma-GT by D-SG was completely reversible. Further inhibition studies showed that D-SG is a competitive inhibitor of the gamma-GT enzyme. Results from theses studies indicate that D-SG is a substrate for gamma-GT; however, the conjugate may not contribute significantly to the decrease in gamma-GT activity reported to occur in vivo in rat.

Monitoring in vivo metabolism and elimination of the endogenous DNA adduct, M1dG {3-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one}, by accelerator mass spectrometry.

Our laboratory is investigating the in vitro and in vivo metabolic processing of endogenously formed DNA adducts as a means of evaluating candidate urinary biomarkers. In particular, we have focused our studies on the metabolism and disposition of the peroxidation-derived pyrimidopurinone deoxyguanosine (dG) adduct, 3-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-R]purin-10(3H)-one (M1dG), and its principal metabolite, 6-oxo-M1dG. We now report the metabolic processing of M1dG at concentrations 4-8 orders of magnitude lower in concentration than previously analyzed, by the use of accelerator mass spectrometry analysis. Administration of 2.0 nCi/kg [14C]M1dG resulted in 49% of the 14C recovered in urine, whereas 51% was recovered in feces. In urine samples, approximately 40% of the 14C corresponded to the metabolite, 6-oxo-M1dG. Following iv administration of 0.5 and 54 pCi/kg [14C]M1dG, approximately 25% of the urinary recovery corresponded to the metabolite, 6-oxo-M1dG. Thus, upon administration of trace amounts of M1dG, a significant percentage of 6-oxo-M1dG was produced, suggesting that 6-oxo-M1dG maybe a useful urinary marker of exposure to endogenous oxidative damage.

"One-pot" syntheses of malondialdehyde adducts of nucleosides.

Short, "one-pot" syntheses of malondialdehyde adducts of deoxyguanosine, deoxyadenosine, and deoxycytidine are described. These syntheses proceed in improved yield and easier purification than previous syntheses and are well suited for the preparation of isotopically labeled nucleoside adducts for biomarker and metabolic studies.

Metabolism and elimination of the endogenous DNA adduct, 3-(2-deoxy-beta-D-erythropentofuranosyl)-pyrimido[1,2-alpha]purine-10(3H)-one, in the rat.

Endogenously occurring damage to DNA is a contributing factor to the onset of several genetic diseases, including cancer. Monitoring urinary levels of DNA adducts is one approach to assess genomic exposure to endogenous damage. However, metabolism and alternative routes of elimination have not been considered as factors that may limit the detection of DNA adducts in urine. We recently demonstrated that the peroxidation-derived deoxyguanosine adduct, 3-(2-deoxy-beta-D-erythropentofuranosyl)-pyrimido[1,2-alpha]purine-10(3H)-one (M1dG), is subject to enzymatic oxidation in vivo resulting in the formation of a major metabolite, 6-oxo-M1dG. Based on the administration of [14C]M1dG (22 microCi/kg) to Sprague-Dawley rats (n=4), we now report that 6-oxo-M1dG is the principal metabolite of M1dG in vivo representing 45% of the total administered dose. When [14C]6-oxo-M1dG was administered to Sprague-Dawley rats, 6-oxo-M1dG was recovered unchanged (>97% stability). These studies also revealed that M1dG and 6-oxo-M1dG are subject to biliary elimination. Additionally, both M1dG and 6-oxo-M1dG exhibited a long residence time following administration (>48 h), and the major species observed in urine at late collections was 6-oxo-M1dG.