Tissue distribution of the novel DPP‐4 inhibitor BI 1356 is dominated by saturable binding to its target in rats

BI 1356 (INN: linagliptin) is an inhibitor of dipeptidyl peptidase‐4 (DPP‐4). This study investigated whether saturable binding of BI 1356 to its target DPP‐4 occurs in tissues and whether drug accumulation occurs at these sites in vivo. In order to test these hypotheses, the tissue distribution of BI 1356 was determined in wild‐type and DPP‐4 deficient rats at different dose levels by means of whole body autoradiography and measurement of tissue radioactivity concentrations after single i.v. dosing of [¹⁴C]‐radio labeled BI 1356. The accumulation behavior of drug‐related radioactivity in tissues was further explored in an oral repeat dose study. Tissue levels of [¹⁴C]BI 1356 related radioactivity were markedly lower in all investigated tissues of the DPP‐4 deficient rats and the difference of the dose‐dependent increase of radioactivity tissue levels between both rat strains indicates that tissue distribution at low doses of BI 1356 is dominated by binding of BI 1356 to DPP‐4 in tissues. As the binding to DPP‐4 is strong but reversible, the tissue binding results in a long terminal half‐life in several tissues including plasma. The binding capacity to DPP‐4 is, however, limited. In the rat, saturation of DPP‐4 binding is suggested at an intravenous dose above 0.01–0.1 mg/kg [¹⁴C]BI 1356. As the DPP‐4 binding capacity is saturated already at low doses, accumulation of BI 1356 in tissues is unlikely, despite the long persistence of low amounts in the body. Copyright © 2009 John Wiley & Sons, Ltd.     

Concentration‐dependent plasma protein binding of the novel dipeptidyl peptidase 4 inhibitor BI 1356 due to saturable binding to its target in plasma of mice,rats and humans

Objectives The purpose of this study was to characterise the plasma protein binding of BI 1356. Methods BI 1356 (proposed trade name ONDERO) is a novel dipeptidyl peptidase 4 (DPP‐4) inhibitor, which is under clinical development for the treatment of type 2 diabetes. DPP‐4 is expressed in various tissues but soluble DPP‐4 is also present in plasma. Therefore, binding to soluble DPP‐4 may influence the pharmacokinetics of BI 1356. Plasma protein binding of BI 1356 was determined in vitro for wild type mice and rats and the results compared with those for DPP‐4 knockout mice and DPP‐4 deficient Fischer rats. In addition, protein binding of BI 1356 was examined in plasma from healthy human volunteers and renal excretion of the compound in the DPP‐4 knockout mice was compared with that occurring in wild type mice. Key findings The results showed that BI 1356 exhibited a prominent concentration‐dependent plasma protein binding due to a saturable high affinity binding to the DPP‐4 target in plasma. Differences in renal excretion of BI 1356 between DPP‐4 knockout mice and wild type mice suggested that saturable binding of BI 1356 to DPP‐4 in the body also influenced elimination. Conclusions High affinity, but readily saturable binding of BI 1356 to its target DPP‐4 accounted primarily for the concentration‐dependent plasma protein binding at therapeutic plasma concentrations of BI 1356.     

Tissue distribution of teneligliptin in rats and comparisons with data reported for other dipeptidyl peptidase‐4 inhibitors

The tissue distribution of teneligliptin, a dipeptidyl peptidase (DPP)‐4 inhibitor, was investigated in rats, and compared with tissue distributions previously reported for other DPP‐4 inhibitors. Following the oral administration of [¹⁴C]teneligliptin to Sprague–Dawley rats, it was predominantly distributed to the kidney and liver, followed by the lung, spleen and pituitary gland. The elimination half‐life (t_1/2) of [¹⁴C]teneligliptin was 68.3 and 69.0 h in the kidney and liver, respectively; these values were about 10 times greater than the plasma t_1/2. Of note, the elimination of [¹⁴C]teneligliptin from tissues with high DPP‐4 activity (kidney, liver and lung) was slower in wild‐type rats than in DPP‐4‐deficient rats, especially in the kidney. By contrast, in the heart and pancreas, which weakly express DPP‐4, no difference was observed in [¹⁴C]teneligliptin concentrations between the two animal strains. In the kidney, most radioactivity was attributable to unchanged teneligliptin from 0.5 to 72 h after administration. The marked difference in the distribution of [¹⁴C]teneligliptin between the two strains suggests that the high binding affinity of teneligliptin for DPP‐4 is involved in its tissue distribution. The currently marketed DPP‐4 inhibitors are highly distributed to the liver, kidney and lung, but the extent of tissue distribution varies greatly among the drugs. The differences in the tissue distributions of DPP‐4 inhibitors might be related to differences in their pleiotropic effects. © 2016 The Authors Biopharmaceutics & Drug Disposition Published by John Wiley & Sons Ltd.     

Evaluation of drug efficacy of DPP‐4 inhibitors based on theoretical analysis with pharmacokinetics and pharmacodynamics

Dipeptidyl peptidase‐4 (DPP‐4) inhibitors are used clinically as therapeutic agents for the treatment of diabetes. To determine the rate of DPP‐4 inhibition induced by these inhibitors, pharmacokinetic and pharmacodynamic parameters were used to theoretically examine the relationship between the rate of DPP‐4 inhibition and clinical efficacy following the administration of four different DPP‐4 inhibitors (sitagliptin, vildagliptin, alogliptin, linagliptin) by focusing on the increase in the level of glucagon‐like peptide‐1 (GLP‐1) induced by their administration. On the basis of the relationship shown, changes in clinical efficacy in association with dose change were examined in order to discuss clinical dosage from the standpoint of proper usage. The results indicate that a high rate of DPP‐4 inhibition is necessary for the onset of the effect of an administered the DPP‐4 inhibitor and that the average value for the DPP‐4 inhibition rate can be utilized as a common parameter of clinical efficacy. Furthermore, the efficacy profiles of the present DPP‐4 inhibitors could be demonstrated on the basis of an increase in the GLP‐1 level. It is considered that the present findings provide useful information for promoting the proper clinical use of DPP‐4 inhibitors. Copyright © 2016 John Wiley & Sons, Ltd.     

Pharmacokinetics of tolbutamide and its metabolite 4‐hydroxy tolbutamide in poloxamer 407‐induced hyperlipidemic rats

Under hyperlipidemic conditions, there are likely to be alterations in the pharmacokinetics of CYP2C11 substrates following decreased expression of CYP2C11, which is homologous to human CYP2C9. The pharmacokinetics of tolbutamide (TB) and its metabolite 4‐hydroxy tolbutamide (4‐OHTB) were evaluated as a CYP2C11 probe after intravenous and oral administration of 10 mg/kg tolbutamide to poloxamer 407‐induced hyperlipidemic rats (HL rats). Changes in the expression and metabolic activity of hepatic CYP2C11 and the plasma protein binding of tolbutamide in HL rats were also evaluated. The total area under the plasma concentration–time curve (AUC) of tolbutamide in HL rats after intravenous administration was comparable to that in controls due to their comparable non‐renal clearance (CL_NR). The free fractions of tolbutamide in plasma were comparable between the control and HL rats. The 4‐hydroxylated metabolite formation ratio (AUC_4‐OHTB/AUC_TB) in HL rats was significantly smaller than that in the control rats as a result of the reduced expression of hepatic CYP2C11 (by 15.0%) and decreased hepatic CL_int (by 28.8%) for metabolism of tolbutamide to 4‐OHTB via CYP2C11. Similar pharmacokinetic changes were observed in HL rats after oral administration of tolbutamide. These findings have potential therapeutic implications, assuming that the HL rat model qualitatively reflects similar changes in patients with hyperlipidemia. Since other sulfonylureas in clinical use are substrates of CYP2C9, their hepatic CL_int changes have the potential to cause clinically relevant pharmacokinetic changes in a hyperlipidemic state. Copyright © 2014 John Wiley & Sons, Ltd.     

Prediction of liver volume – a population‐based approach to meta‐analysis of paediatric,adult and geriatric populations – an update

Liver volume is a critical scaling factor for predicting drug clearance in physiologically based pharmacokinetic modelling and for both donor/recipient graft size estimation in liver transplantation. The accurate and precise estimation of liver volume is therefore essential. The objective here was to extend an existing meta‐analysis using a non‐linear mixed effects modelling approach for the estimation of liver volume to other race groups and paediatric and geriatric populations. Interrogation of the PubMed® database was undertaken using a text string query to ensure as objective a retrieval of liver volume data for the modelling exercise as possible. Missing body size parameters were estimated using simulations from the Simcyp Simulator V13R1 for an age and ethnically appropriate population. Non‐linear mixed effect modelling was undertaken in Phoenix 1.3 (Certara) utilizing backward deletion and forward inclusion of covariates from fully parameterized models. Existing liver volume models based on body surface area (BSA) and body weight and height were implemented for comparison. The extension of a structural model using a BSA equation and incorporating the Japanese race and age as covariates and exponents on LV0 (θ _Baseline) and body surface area (θ _BSA), respectively, delivered a comparatively low objective function value. Bootstrapping of the original dataset revealed that the confidence intervals (2.5–97.5%) for the fitted (theta) parameter estimates were bounded by the bootstrapped estimates of the same. In conclusion, extension and re‐parameterization of the existing Johnson model adequately describes changes in liver volume using the body surface area in all investigated populations. Copyright © 2017 John Wiley & Sons, Ltd.     

Effects of poloxamer 407‐induced hyperlipidemia on hepatic multidrug resistance protein 2 (Mrp2/Abcc2) and the pharmacokinetics of mycophenolic acid in rats

Hepatic multidrug resistance‐associated protein 2 (Mrp2) is responsible for the majority of the biliary elimination of endogenous and exogenous substances, therefore it is important to evaluate possible functional changes in Mrp2 activity under conditions of hyperlipidemia (HL). Thus, the present study assessed the protein expression and transporting activity of hepatic Mrp2 based on the in vivo biliary excretion of phenolsulfonphthalein (PSP) as a model anionic substrate for Mrp2 in poloxamer 407‐induced hyperlipidemic rats (HL rats) and compared these values with those for control rats. The pharmacokinetics of mycophenolic acid (MPA) and mycophenolic acid‐7‐O‐glucuronide (MPAG) were evaluated after the intravenous (5 mg/kg) and oral (10 mg/kg) administration of MPA to control and HL rats. In HL rats, the protein expression of hepatic Mrp2 and its biliary transporting activity exhibited significant reductions (by 24.3% and 24.6%, respectively) in the absence of a change in bile flow rate. Unexpectedly, HL and control rats showed comparable biliary excretion rates of MPAG due to the counter effects of the reduced expression and activity of Mrp2 and a 484% increase in the free fraction of MPAG in HL rats. The estimated biliary clearance value of free MPAG in HL rats was considerably slower (by 77.1%) than that in control rats. Although significant pharmacokinetic changes in total MPA and MPAG levels were not observed in HL rats, there was a marked increase in free MPA and MPAG levels. Clinically relevant pharmacokinetic changes in subjects with HL that are related to MRP2 could not be ruled out. Copyright © 2016 John Wiley & Sons, Ltd.     

A validated,sensitive HPLC‐MS/MS method for quantification of cis‐para‐methyl‐4‐methylaminorex (cis‐4,4'‐DMAR) in rat and human plasma: application to pharmacokinetic studies in rats

4,4′‐DMAR is an analogue of the known psychostimulants 4‐methylaminorex and aminorex. In the light of reports of deaths associated with its abuse, and the easy access from Internet vendors, the EU Council recently decided on control measures across member states. Here we describe a validated method for measuring plasma levels of cis ‐4,4′‐DMAR, crucial for preclinical studies and analysis in human plasma. Chromatographic separation was done by gradient elution on a Kinetex C18 column with 0.1% formic acid in water and 0.1% formic acid in acetonitrile at 0.2 mL/min. Detection was by positive electrospray ionization (ESI+) in multiple reaction monitoring mode monitoring the quantifier transitions m /z 191.4 → m /z 148.3 for cis ‐4,4'‐DMAR and m /z 259.3 → m /z 194.2 for carbamazepine (internal standard). Protein precipitation with 1% of formic acid in acetonitrile was used in cis ‐4,4'‐DMAR extraction from plasma; recovery was high (>93%) with a negligible matrix effect. This method provides an accurate, precise, and sensitive method for cis ‐4,4’‐DMAR quantification in human and rat plasma, following European Medicine Agency guidelines for bioanalytical method validation. Pharmacokinetic studies were conducted in rats. After an intravenous dose of 1 mg/kg, plasma levels declined rapidly (≥80% in 4 h), followed by a slow elimination phase (t_1/2 of 5.14 ± 0.65 h). Absorption was rapid after intraperitoneal injection (t_max = 15 min) with a rapid decline thereafter; C_max and AUC_0‐240min showed dose‐proportionality over the dose range 1–10 mg/kg. This method was successfully applied to investigate pharmacokinetic properties in rats and could be used to quantify cis ‐4,4’‐DMAR levels in human plasma. Copyright © 2016 John Wiley & Sons, Ltd.     

Binding of a metabolite of triflusal (2-hydroxy-4-trifluoromethylbenzoic acid) to serum proteins in rat and man

Summary 2-hydroxy-4-trifluoromethylbenzoic acid (HTB) is the main active metabolite of the platelet anti-aggregant drug triflusal. Its binding to plasma proteins of rats and healthy volunteers in vitro and in vivo has been studied.Rats were given a single oral dose of 50 mg·kg^–1 triflusal and the healthy volunteers received 300 mg as a single oral dose or a multiple dose regimen of 600 mg every 24 h and 300 mg every 8 h, both for 13 days. Protein-free HTB was obtained by ultrafiltration. Unbound and total HTB concentrations were determined by HPLC.HTB was primarily bound to albumin in plasma. The Scatchard plots suggested two types of binding sites for HTB on the albumin molecule. In rats, the binding constants (K=intrinsic affinity constant, n=number of binding sites) were K₁=1.4×10⁵ l·mol^–1, n₁=1.23, and K₂=4.1×10³ l·mol^–1 and n₂=3.77. The mean plasma concentration in rats after oral administration was 185 (37) g·ml^–1 (protein-free HTB: 2.44 (0.77)%). The binding constants in human plasma were K₁=4.7×10⁵ l·mol^–1, n₁=1.93, K₂=4.3 l·mol^–1 and n₂=4.28.The plasma HTB concentration in man (n=8) was 35 g·ml^–1 (C_max) after a single oral dose of triflusal 300 mg, 172.96 g·ml^–1 (C_max·ss) during the multiple dosage regimen of 300 mg every 8 h, and 131 g·ml^–1 (C_max·ss) during the multiple oral dose regimen of 600 mg every 24 h. Unbound HTB ranged from 0.27 to 0.43%, depending on dose. HTB had high affinity for plasma albumin, which was not saturable after therapeutic doses. It showed linear elimination.     

Synthesis,radiosynthesis and in vivo evaluation of [123I]‐4‐(2‐(bis(4‐fluorophenyl)methoxy)ethyl)‐1‐(4‐iodobenzyl)piperidine as a selective tracer for imaging the dopamine transporter

Dopamine transporter (DAT) neuroimaging is a useful tool in Parkinson's disease diagnosis, staging and follow‐up providing information on the integrity of the dopaminergic neurotransmitter system in vivo. 4‐(2‐(Bis(4‐fluorophenyl)methoxy)ethyl)‐1‐(4‐iodobenzyl)piperidine (7) has nanomolar affinity for DAT and better selectivity over the other monoamine transporters compared with the existing SPECT radioligands for DAT. The aim of this study was to synthesize and evaluate [¹²³I]‐7 as an in vivo tracer for DAT. The tributylstannyl precursor was synthesized with an overall yield of 25%. [¹²³I]‐7 was synthesized by electrophilic destannylation with a yield of 40±10%. Radiochemical purity appeared to be >98%, whereas specific activity was at least 667 GBq/µmol. Biodistribution studies in mice showed brain uptake of 0.96±0.53%ID/g at 30 s post injection (p.i.) and 0.26±0.02%ID/g at 3 h p.i. High blood activity was observed at all time points. Pretreatment with Cyclosporin A raised brain uptake indicating that [¹²³I]‐7 is transported by P‐glycoprotein (P‐gp) pumps. In rats, regional brain distribution of [¹²³I]‐7 was not in agreement with DAT distribution. These results indicate that [¹²³I]‐7 is not suitable for mapping DAT in vivo but could be a useful tracer for the P‐gp transporter. Copyright © 2009 John Wiley & Sons, Ltd.     

In‐situ absorption,protein binding and pharmacokinetic studies of S002‐853, a novel antidiabetic and antidyslipidaemic flavone derivative in rats

Objectives The aim of the study was to investigate the in‐situ absorption kinetics, plasma protein binding and pharmacokinetic characteristics of a novel synthetic flavone derivative, S002‐853, which shows pronounced antidiabetic and antidyslipidaemic activity. Methods Quantification of S002‐853 in plasma was performed by the LC‐MS/MS method and in‐situ sample analysis was carried out by the HPLC‐UV method. Key findings The absorption rate constant was 0.274/h in a mild alkaline environment, which S002‐853 experiences in the intestine following oral dose administration. Plasma protein binding was found to be 26.37 ± 2.58% at a concentration of 1 μg/ml. The pharmacokinetic parameters were determined in male rats after administration of a single 40 mg/kg oral dose and 10 mg/kg intravenous dose. The peak plasma concentration (C_max) was found to be 60.93 ng/ml at 8 h after oral administration. Irregular concentration–time profiles with secondary peaks were observed after oral dose administration. The elimination half‐life of the compound was 19.56 h and 16.30 h after oral and intravenous doses, respectively. Comparison of the AUC after oral and intravenous dosing of S002‐853 indicates that only about 29.48% (bioavailability) of the oral dose reaches the systemic circulation. Conclusions In‐situ study of S002‐853 shows slow absorption from the gastrointestinal tract. S002‐853 also shows low plasma protein binding. The pharmacokinetic parameters after oral and intravenous dose reveal low oral bioavailability and high mean residence time.     

Enantioselective disposition of (R)‐salmeterol and (S)‐salmeterol in urine following inhaled dosing and application to doping control

Salmeterol (USAN, INN, BAN) is a long‐acting beta2‐adrenoceptor agonist (LABA) widely used in the treatment of airways disease. Although salmeterol is permitted via inhalation by athletes and supratherapeutic dosing may enhance performance, no urine threshold has been established by the World Anti‐Doping Agency (WADA). Salmeterol is a chiral compound consisting of (R)‐ and (S)‐enantiomers, normally administered as racemic (rac‐ ) mixture via inhalation. Levels of rac‐ salmeterol in urine are often below detectable levels and there is surprisingly little information regarding the enantioselectivity of salmeterol pharmacokinetics. In this study, subjects inhaled either 50 (n = 6) or 200 µg (n = 4; generally regarded as maximum therapeutic dose) of salmeterol and urine was then collected for 24 h and analyzed by enantioselective ultra performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS). Maximum rac‐ salmeterol urine concentrations were obtained at 2 h for both doses with medians of 0.084 ng/mL after the 50 µg dose and 2.1 ng/mL after the 200 µg dose, with an individual maximum of 5.7 ng/mL. Levels were detectable at 24 h for both doses. Salmeterol displayed enantioselective pharmacokinetics, with a mean ± SD log (S):(R) = 0.055 ± 0.025 (P < 0.0001) equivalent to (S):(R) of 1.13. In conclusion, rac‐ salmeterol by inhalation exhibits modest enantioselectivity in urine following single dose administration and can be detected following a single 50 µg dose for up to 24 h after inhalation. The present findings are of relevance if a urine threshold limit is to be introduced for salmeterol on the list of prohibited substances. The application of an enantiomer ratio analysis may offer improved discriminatory detection capability for doping control analysis applications. Copyright © 2016 John Wiley & Sons, Ltd.     

Enantioselective disposition of (R/S)‐albuterol in skeletal and cardiac muscle

Significant enhancement of skeletal muscle function has been observed with racemic albuterol (salbutamol). There is now general acceptance that the R‐albuterol enantiomer elicits the pharmacological response, both in the lungs and extrapulmonary, while S‐albuterol is pharmacologically inert. The objective of this study was to investigate the distribution of (R/S)‐albuterol enantiomers into skeletal and cardiac muscle. Initially oral dosing was undertaken in neonatal mice administered a maximum tolerable dose of racemic albuterol. An in vivo infusion rat model was employed for the investigation of albuterol uptake into skeletal and cardiac muscle over 4 h. Tissue concentrations were determined using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). From the oral dosing model, mean (±SD) levels of racemic albuterol after 5 days were 915 (±293) ng/mL in plasma, 2574 (±196) ng/g in muscle, and 53 (±6.6) ng/g in brain with enantioselective partitioning (muscle:plasma ratio of 5.7 and 1.7 for R‐ and S‐albuterol, respectively). In the infusion model, enantioselective disposition was observed in skeletal muscle (muscle:plasma ratio of 1.2–1.7 and 0.6–0.7 for R‐ and S‐albuterol, respectively) and in cardiac muscle (4.1 and 0.5, respectively). In conclusion, there is greater partitioning of active (R)‐albuterol than inactive (S)‐albuterol into both skeletal and cardiac muscle compared to plasma. These findings have relevance for albuterol sports doping, cardiac effects, and therapeutic use in muscle wasting diseases. Furthermore, the greater muscle partitioning of the active R‐albuterol, and the availability of pure R‐albuterol formulations highlight shortcomings in doping control measures using non‐enantioselective assays. Copyright © 2013 John Wiley & Sons, Ltd.     

Antifibrotic effects of D‐limonene (5(1‐methyl‐4‐[1‐methylethenyl]) cyclohexane) in CCl4 induced liver toxicity in Wistar rats

This study was designed to assess the potential antifibrotic effect of D‐Limonene—a component of volatile oils extracted from citrus plants. D‐limonene is reported to have numerous therapeutic properties. CCl₄‐intduced model of liver fibrosis in Wistar rats is most widely used model to study chemopreventive studies. CCl₄‐intoxication significantly increased serum aminotransferases and total cholesterol these effects were prevented by cotreatment with D‐Limonene. Also, CCl₄‐intoxication caused depletion of glutathione and other antioxidant enzymes while D‐Limonene preserved them within normal values. Hydroxyproline and malondialdehyde content was increased markedly by CCl₄ treatment while D‐Limonene prevented these alterations. Levels of TNF‐α, TGF‐β, and α‐SMA were also assessed; CCl₄ increased the expression of α‐SMA, NF‐κB and other downstream inflammatory cascade while D‐Limonene co‐treatment inhibited them. Collectively these findings indicate that D‐Limonene possesses potent antifibrotic effect which may be attributed to its antioxidant and anti‐inflammatory properties.     

Synthesis of N‐(2‐diethylamino‐ethyl)‐4‐(4‐fluoro‐benzamido)‐2‐methoxybenzamide (desiodo‐MIP‐1145) by coupling technique and its radioiodination: a potential melanoma imaging agent

Radioiodinated MIP‐1145, which specifically targets melanin, is an ideal candidate for targeted therapy of melanoma. An analogue of MIP‐1145 lacking the iodo‐substituent (desiodo‐MIP‐1145) was synthesized as a labeling precursor in three simple steps. The radioiodination of desiodo‐MIP‐1145 by iodine‐125 was carried out via an electrophilic substitution reaction. An optimization study for the iodination reaction was carried out. The labeled compound was isolated and purified by means of electrophoresis and HPLC. The maximum radiochemical yield, 76%, was obtained with radiochemical purity greater than 99%. The log P value for [¹²⁵I]MIP‐1145 was measured as 4.5.     

Synthesis of deuterium‐labeled analogs of the lipid hydroperoxide‐derived bifunctional electrophile 4‐oxo‐2(E)‐nonenal

Lipid hydroperoxides undergo homolytic decomposition into the bifunctional 4‐hydroxy‐2(E)‐nonenal and 4‐oxo‐2(E)‐nonenal (ONE). These bifunctional electrophiles are highly reactive and can readily modify intracellular molecules including glutathione (GSH), deoxyribonucleic acid (DNA) and proteins. Lipid hydroperoxide‐derived bifunctional electrophiles are thought to contribute to the pathogenesis of a number of diseases. ONE is an α,β ‐unsaturated aldehyde that can react in multiple ways and with glutathione, proteins and DNA. Heavy isotope‐labeled analogs of ONE are not readily available for conducting mechanistic studies or for use as internal standards in mass spectrometry (MS)‐based assays. An efficient one‐step cost‐effective method has been developed for the preparation of C‐9 deuterium‐labeled ONE. In addition, a method for specific deuterium labeling of ONE at C‐2, C‐3 or both C‐2 and C‐3 has been developed. This latter method involved the selective reduction of an intermediate alkyne either by lithium aluminum hydride or lithium aluminum deuteride and quenching with water or deuterium oxide. The availability of these heavy isotope analogs will be useful as internal standards for quantitative studies employing MS and for conducting mechanistic studies of complex interactions between ONE and DNA bases as well as between ONE and proximal amino acid residues in peptides and proteins. Copyright © 2011 John Wiley & Sons, Ltd.