Absolute oral bioavailability of milvexian spray-dried dispersion formulation under fasted and fed conditions in healthy adult participants: An intravenous microtracer approach.

Milvexian is an oral, small-molecule factor XIa inhibitor being developed to prevent thromboembolic events. This study assessed the absolute bioavailability (F) of milvexian following single doses of milvexian spray-dried dispersion (SDD) formulation under fed and fasted conditions, and milvexian solution, in healthy adult participants using an intravenous microtracer approach. This was a phase I, open-label, partially randomized, 4-sequence, 5-period crossover study. After fasting for ≥10 h, participants received milvexian 200-mg oral solution with a 100-µg 14C milvexian intravenous microtracer at the time of maximum observed plasma concentration. Following a 3-day washout, participants were randomized to 1 of 4 milvexian SDD treatment sequences in a crossover fashion: 25 mg fasted, 25 mg fed, 200 mg fasted, or 200 mg fed. Pharmacokinetic data were collected up to 72 h postdose. Seventeen participants were dosed, and 14 completed treatment. Under fasted conditions, milvexian F was ~100%, 58.2%, and 54.2% following administration of the oral solution, 25 mg SDD, and 200 mg SDD, respectively. Under fed conditions, milvexian F following 25 mg and 200 mg SDD was 44.3% and 75.6%, respectively. The milvexian SDD formulation at 25 mg and 200 mg resulted in similar F in a fasted state; under fed conditions, milvexian F decreased at 25 mg and increased at 200 mg. These findings clarify pharmacokinetic-related gaps observed in previous studies.

The Importance of Tracking "Missing" Metabolites: How and Why?

Technologies currently employed to find and identify drug metabolites in complex biological matrices generally yield results that offer a comprehensive picture of the drug metabolite profile. However, drug metabolites can be missed or are captured only late in the drug development process. This could be due to a variety of factors, such as metabolism that results in partial loss of the molecule, covalent bonding to macromolecules, the drug being metabolized in specific human tissues, or poor ionization in a mass spectrometer. These scenarios often draw a great deal of attention from chemistry, safety assessment, and pharmacology. This review will summarize scenarios of missing metabolites, why they are missing, and associated uncovering strategies from deeper investigations. Uncovering previously missed metabolites can have ramifications in drug development with toxicological and pharmacological consequences, and knowledge of these can help in the design of new drugs.

Metabolomic Profiling and Drug Interaction Characterization Reveal Riboflavin As a Breast Cancer Resistance Protein-Specific Endogenous Biomarker That Demonstrates Prediction of Transporter Activity In Vivo.

Advancement of endogenous biomarkers for drug transporters as a tool for assessing drug-drug interactions (DDIs) depends on initial identification of biomarker candidates and relies heavily on biomarker validation and its response to reference inhibitors in vivo. To identify endogenous biomarkers of breast cancer resistance protein (BCRP), we applied metabolomic approaches to profile plasma from Bcrp-/-, multidrug resistance protein (Mdr)1a/1b-/-, and Bcrp/Mdr1a/1b-/- mice. Approximately 130 metabolites were significantly altered in Bcrp and P-glycoprotein (P-gp) knockout mice, indicating numerous metabolite-transporter interactions. We focused on BCRP-specific substrates and identified riboflavin, which was significantly elevated in the plasma of Bcrp single- and Bcrp/P-gp double- but not P-gp single-knockout mice. Dual BCRP/P-gp inhibitor elacridar caused a dose-dependent increase of the area under the plasma concentration-time curve (AUC) of riboflavin in mice (1.51- and 1.93-fold increases by 30 and 150 mg/kg elacridar, respectively). In three cynomolgus monkeys, we observed approximately 1.7-fold increases in the riboflavin concentrations caused by ML753286 (10 mg/kg), which correlated well with the increase of sulfasalazine, a known BCRP probe in monkeys. However, the BCRP inhibitor had no effect on isobutyryl carnitine, arginine, or 2-arachidonoyl glycerol levels. Additionally, clinical studies on healthy volunteers indicated low intrasubject and intermeal variability of plasma riboflavin concentrations. In vitro experiments using membrane vesicles demonstrated riboflavin as a select substrate of monkey and human BCRP over P-gp. Collectively, this proof-of-principle study indicates that riboflavin is a suitable endogenous probe for BCRP activity in mice and monkeys and that future investigation of riboflavin as a blood-based biomarker of human BCRP is warranted. SIGNIFICANCE STATEMENT: Our results identified riboflavin as an endogenous biomarker candidate of BCRP. Its selectivity, sensitivity, and predictivity regarding BCRP inhibition have been explored. The findings of this study highlight riboflavin as an informative BCRP plasma biomarker in animal models. The utility of this biomarker requires further validation by evaluating the effects of BCRP inhibitors of different potencies on riboflavin plasma concentrations in humans. Ultimately, riboflavin may shed light on the risk assessment of BCRP DDIs in early clinical trials.

Effects of rifampin on the pharmacokinetics and pharmacodynamics of milvexian, a potent, selective, oral small molecule factor XIa inhibitor.

Milvexian (BMS-986177/JNJ-70033093) is a potent, oral small molecule that inhibits the active form of factor XI with high affinity and selectivity. This study assessed the single-dose pharmacokinetic and pharmacodynamic properties of milvexian co-administered with rifampin, an organic anion transport protein (OATP) inhibitor and potent cytochrome P450 (CYP) 3A and P-glycoprotein (P-gp) inducer. In this open-label, nonrandomized, single-sequence study, healthy participants (N = 16) received single doses of milvexian on Day 1 (100 mg), milvexian and rifampin (600 mg) on Day 4, rifampin on Days 5-11, milvexian and rifampin on Day 12, and rifampin on Days 13-14. Pharmacokinetic data were summarized using descriptive statistics. Administration of milvexian, alone or in combination with rifampin, was generally safe and well tolerated. Single-dose co-administration of rifampin and milvexian demonstrated no meaningful changes in milvexian exposure versus milvexian alone (Cmax, 110%; AUC[0-T], 102%; AUC[INF], 101%). After multiple doses of rifampin and milvexian, peak and total milvexian exposure substantially decreased versus milvexian alone (Cmax, 22%; AUC[0-T], 15%; AUC[INF], 15%). Results were consistent with preclinical data, indicating that milvexian is a substrate for CYP3A4/5 and P-gp but not OATP. The implications of these results on the need for dose adjustment of milvexian will be further elucidated following the completion of phase 2 and 3 trials.Trial registration The study was registered with ClinicalTrials.gov (NCT02959060; submitted 7/11/2016, first posted 8/11/2016).

Effects of Itraconazole and Diltiazem on the Pharmacokinetics and Pharmacodynamics of Milvexian, A Factor XIa Inhibitor.

INTRODUCTION: Modulation of Factor XIa (FXIa) may provide a novel mechanism for systemic anticoagulation with the potential to improve the risk-benefit profile observed with existing anticoagulants through greater efficacy or a safer bleeding profile. This study assessed the effects of co-administration with strong and moderate CYP3A inhibitors itraconazole and diltiazem, respectively, on the pharmacokinetic and pharmacodynamic properties of milvexian, a Factor XIa inhibitor. METHODS: This was an open-label, non-randomized, two-period crossover study in healthy participants. In period 1, participants received a single oral dose of milvexian (30 mg) on day 1, followed by a washout on days 2 and 3. In period 2, participants received multiple oral doses of itraconazole (200 mg) or diltiazem (240 mg) with a single dose of milvexian. RESULTS: A total of 28 participants entered the treatment period. Following itraconazole co-administration, milvexian exposure was increased; AUC(0-T), AUC(INF), and C24 were 2.5-, 2.5-, and 3.8-fold higher, while mean Cmax was 28% higher versus milvexian alone. Diltiazem co-administration also increased milvexian exposure; AUC(0-T), AUC(INF), and C24 were 38, 38, and 64% higher, and mean Cmax was 9.6% higher versus milvexian alone. Prolongation of activated partial thromboplastin time was observed with milvexian in a concentration-dependent fashion irrespective of co-administration with itraconazole or diltiazem. Administration of a single dose of milvexian, alone or in combination with itraconazole or diltiazem, was generally safe and well tolerated; there were no deaths or serious adverse events. CONCLUSIONS: A moderate increase in milvexian exposure was observed following co-administration of itraconazole while a minimal increase was seen with diltiazem, consistent with the involvement of CYP3A metabolism and P-glycoprotein in drug absorption/elimination. Milvexian was generally safe and well tolerated in healthy participants. TRIAL REGISTRATION: The study was registered with ClinicalTrials.gov (NCT02807909; submitted June 17, 2016).

Prediction of drug-drug interaction potential mediated by transporters between dasatinib and metformin, pravastatin, and rosuvastatin using physiologically based pharmacokinetic modeling.

PURPOSE: Recent in vitro studies demonstrated that dasatinib inhibits organic cation transporter 2 (OCT2), multidrug and toxin extrusion proteins (MATEs), and organic anion transporting polypeptide 1B1/1B3 (OATP1B1/1B3). We developed a physiologically based pharmacokinetic (PBPK) model to assess drug-drug interaction (DDI) potential between dasatinib and known substrates for these transporters in a virtual population. METHODS: The dasatinib PBPK model was constructed using Simcyp® Simulator by combining its physicochemical properties, in vitro data, in silico predictions, and pharmacokinetic (PK) results from clinical studies. Model validation against three independent clinical trials not used for model development included dasatinib DDI studies with ketoconazole, rifampin, and simvastatin. The validated model was used to simulate DDIs of dasatinib and known substrates for OCT2 and MATEs (metformin) and OATP1B1/1B3 (pravastatin and rosuvastatin). RESULTS: Simulations of metformin PK in the presence and absence of dasatinib, using inhibitor constant (Ki) values measured in vitro, produced estimated geometric mean ratios (GMRs) of the maximum observed concentration (Cmax) and area under the concentration-time curve (AUC) of 1.05 and 1.06, respectively. Sensitivity analysis showed metformin exposure increased < 30% in both AUC and Cmax when dasatinib Ki was reduced by tenfold for OCT2 and MATEs simultaneously, and < 40% with a 20-fold Ki reduction. The estimated GMRs of Cmax and AUC for pravastatin and rosuvastatin with co-administration of dasatinib were unity (1.00). CONCLUSIONS: This PBPK model accurately described the observed PK profiles of dasatinib. The validated PBPK model predicts low risk of clinically significant DDIs between dasatinib and metformin, pravastatin, or rosuvastatin.

Interindividual Variability and Differential Tissue Abundance of Mitochondrial Amidoxime Reducing Component Enzymes in Humans.

Mitochondrial amidoxime-reducing component (mARC) enzymes are molybdenum-containing proteins that metabolize a number of endobiotics and xenobiotics. The interindividual variability and differential tissue abundance of mARC1 and mARC2 were quantified using targeted proteomics in three types of tissue fractions: 1) pediatric liver tissue homogenates, 2) total membrane fraction of the paired liver and kidney samples from pediatric and adult donors, and 3) pooled S9 fractions of the liver, intestine, kidney, lung, and heart. The absolute levels of mARC1 and mARC2 in the pediatric liver homogenate were 40.08 ± 4.26 and 24.58 ± 4.02 pmol/mg homogenate protein, respectively, and were independent of age and sex. In the total membrane fraction of the paired liver and kidney samples, the abundance of hepatic mARC1 and mARC2 was comparable, whereas mARC2 abundance in the kidney was approximately 9-fold higher in comparison with mARC1. The analysis of the third set of samples (i.e., S9 fraction) revealed that mARC1 abundance in the kidney, intestine, and lung was 5- to 13-fold lower than the liver S9 abundance, whereas mARC2 abundance was approximately 3- and 16-fold lower in the intestine and lung than the liver S9, respectively. In contrast, the kidney mARC2 abundance in the S9 fraction was approximately 2.5-fold higher as compared with the hepatic mARC2 abundance. The abundance of mARC enzymes in the heart was below the limit of quantification (∼0.6 pmol/mg protein). The mARC enzyme abundance data presented here can be used to develop physiologically based pharmacokinetic models for the prediction of in vivo pharmacokinetics of mARC substrates. SIGNIFICANCE STATEMENT: A precise targeted quantitative proteomics method was developed and applied to quantify newly discovered drug-metabolizing enzymes, mARC1 and mARC2, in pediatric and adult tissue samples. The data suggest that mARC enzymes are ubiquitously expressed in an isoform-specific manner in the human liver, kidney, intestine, and lung, and the enzyme abundance is not associated with age and sex. These data are important for developing physiologically based pharmacokinetic models for the prediction of in vivo pharmacokinetics of mARC substrates.

Preclinical metabolism and disposition of an orally bioavailable macrocyclic FXIa inhibitor.

FXIa-6f is a high affinity, orally bioavailable macrocyclic FXIa inhibitor with antithrombotic activity in preclinical species.The objectives of this study were to characterize the in vitro metabolism, determine circulating metabolites in pre-clinical species, and examine the disposition of the compound in a bile duct-cannulated rat study (BDC) study to inform clinical development of the compound and the medicinal chemistry approach to identify molecules with improved properties.Across species, metabolic pathways included several oxidative metabolites, including hydroxylated metabolites on the macrocycle or P1 region, descarbamoylation of the methyl carbamate side chain, and a glutathione conjugate on the 2,6-difluoro-3-chlorophenyl ring.In BDC rat, the absorbed dose of [3H]FXIa-6f was cleared mainly by metabolism, with excretion of drug-related material in the bile, mostly as metabolites.In all preclinical species, the parent drug was the primary drug-related component in circulation, but the species differences in the metabolic pathways observed in vitro were reflected in the plasma, where M6, a descarbamoylated metabolite, was more prominent in rat plasma, and M9, a hydroxylated metabolite, was more prominent in monkey plasma. Based on the available data, the human metabolism appears to be most similar to monkey.

Hepatocyte spheroids as a viable in vitro model for recapitulation of complex in vivo metabolism pathways of loratadine in humans.

Accurate prediction of in vivo metabolic pathways in humans can be challenging because in vitro liver matrices may fail to produce certain in vivo metabolites.Rat and human spheroids, generated from cryopreserved hepatocytes in media that contained minimal amount of serum, maintained morphology, viability and cytochrome P450 (CYP) activities for at least a week without media exchange.With spheroid cultures, multiple Phase I and Phase II metabolites were observed in rat and human spheroid cultures that were incubated with loratadine (LOR) for multiple days. Consistent with in vivo observations, 3-hydroxydesloratadine, (3-OH-DL), along with its glucuronide, were observed in human spheroids, but not in rat spheroids. Interestingly, the putative intermediate metabolite leading to 3-OH-DL, DL-N-glucuronide, was observed in incubations with both rat and human spheroids. In conclusion, hepatocyte spheroid were capable of recapitulating the inter-species differences in metabolism between human and rat for LOR, therefore, it may represent a viable model for studying complex metabolic pathways.

UHPLC-MS/MS bioanalysis of urinary DHEA, cortisone and their hydroxylated metabolites as potential biomarkers for CYP3A-mediated drug-drug interactions.

AIM: A UHPLC-MS/MS assay was developed to quantify urinary dehydroepiandrosterone (DHEA), 7ß-hydroxy-DHEA, cortisone and 6ß-hydroxycortisone as potential biomarkers to predict CYP3A activity. RESULTS: A sensitive assay at LLOQ of 0.500 ng/ml with good accuracy and precision was developed for the four analytes in human urine. This UHPLC-MS/MS assay was optimized by eliminating nonspecific loss of the analytes in urine, ensuring complete hydrolysis of the conjugates to unconjugated forms and use of the product ions of [M+H-H2O]+ for multiple reaction monitoring detection of DHEA and 7ß-hydroxy-DHEA. CONCLUSION: This assay was successfully applied to a pilot clinical study. It is also suitable for future drug-drug interaction studies to continue evaluating the potential of these steroids as biomarkers for CYP3A inhibition and induction.

A validated LC-MS/MS method for the quantitative measurement of creatinine as an endogenous biomarker in human plasma.

BACKGROUND: Creatinine is an endogenous compound generated from creatine by normal muscular metabolism. It is an important indicator of renal function and the serum level is routinely monitored in clinical labs. Results & methodology: Surrogate analyte (d3-creatinine) was used for calibration standard and quality control preparation and the relative instrument response ratio between creatinine and d3-creatinine was used to calculate the endogenous creatinine concentrations. CONCLUSION: A fit-for-purpose strategy of using a surrogate analyte and authentic matrix was adopted for this validation. The assay was the first human plasma assay using such strategy and was successfully applied to a clinical study to confirm a transient elevation of creatinine observed using an existing clinical assay.

Characterization of Organic Anion Transporter 2 (SLC22A7): A Highly Efficient Transporter for Creatinine and Species-Dependent Renal Tubular Expression.

The contribution of organic anion transporter OAT2 (SLC22A7) to the renal tubular secretion of creatinine and its exact localization in the kidney are reportedly controversial. In the present investigation, the transport of creatinine was assessed in human embryonic kidney (HEK) cells that stably expressed human OAT2 (OAT2-HEK) and isolated human renal proximal tubule cells (HRPTCs). The tubular localization of OAT2 in human, monkey, and rat kidney was characterized. The overexpression of OAT2 significantly enhanced the uptake of creatinine in OAT2-HEK cells. Under physiologic conditions (creatinine concentrations of 41.2 and 123.5 µM), the initial rate of OAT2-mediated creatinine transport was approximately 11-, 80-, and 80-fold higher than OCT2, multidrug and toxin extrusion protein (MATE)1, and MATE2K, respectively, resulting in approximately 37-, 1850-, and 80-fold increase of the intrinsic transport clearance when normalized to the transporter protein concentrations. Creatinine intracellular uptake and transcellular transport in HRPTCs were decreased in the presence of 50 µM bromosulfophthalein and 100 µM indomethacin, which inhibited OAT2 more potently than other known creatinine transporters, OCT2 and multidrug and toxin extrusion proteins MATE1 and MATE2K (IC50: 1.3 µM vs. > 100 µM and 2.1 µM vs. > 200 µM for bromosulfophthalein and indomethacin, respectively) Immunohistochemistry analysis showed that OAT2 protein was localized to both basolateral and apical membranes of human and cynomolgus monkey renal proximal tubules, but appeared only on the apical membrane of rat proximal tubules. Collectively, the findings revealed the important role of OAT2 in renal secretion and possible reabsorption of creatinine and suggested a molecular basis for potential species difference in the transporter handling of creatinine.

Reflecting on a decade of metabolite screening and monitoring.

The last 10 years have witnessed robust debate within the bioanalytical community and regulatory authorities on the topic of metabolite monitoring and safety assessment. Of particular interest to regulated bioanalytical laboratories was the acceptance by the US FDA and other major regulatory bodies of a tiered approach to bioanalytical assay validation. The tiered approach defines a sliding scale of regulatory rigor for the evaluation of significant human metabolites that encompasses a range of assessments from semi-quantitative assays to fully validated assays, all of which can be used in support of regulatory submissions. This article describes the utilization of a tiered approach at Bristol-Myers Squibb and the decision trees guiding the selection of the appropriate level of assay qualification. Case studies illustrate how decisions are made, how different scientific situations influence the assay choice, and what criteria may be set to continue or discontinue metabolite monitoring in later drug development.

Sensitivity-based analytical approaches to support human absolute bioavailability studies.

The characterization of absolute bioavailability (BA) is useful for non-intravenous (iv.) formulations during drug development and is required by some health authorities. A study design of co-administrating an iv. isotopically labeled microdose with a therapeutic oral dose is a viable approach for the determination of human PK and has been accepted by regulatory agencies. The implementation of an iv.-microdose with oral therapeutic dose in absolute BA studies speeds up clinical development. In recent years, AMS to measure a radiolabeled microdose has been utilized to support several clinical absolute BA studies. An alternative approach for conducting microdose studies is using LC-MS/MS alone to quantitate both the iv. drug and the oral drug. Because both labeled and unlabeled drugs can be measured simultaneously with LC-MS/MS, it is cost effective. However, for compounds with high volume of distribution and/or poor LC-MS/MS response, AMS still provides a superior LLOQ. In this Perspective, we discuss a paradigm for selecting either an LC-MS/MS or AMS-based approach for generating concentration data in absolute BA studies dependent on the required sensitivity.

The synthesis of a carbon-14 labeled pegylated Adnectin™ for placental transfer studies in guinea pigs.

Adnectins™ are novel fibronectin-based proteins containing domains engineered to bind to targets of therapeutic interest. The molecular weights of adnectins are less than conventional monoclonal antibodies but larger than traditional small molecules. Until now, there has been no information on the placental transfer of adnectins. To assess placental permeability to adnectins in pregnant guinea pigs, a radiolabeled adnectin, ATI-1072, bound to polyethylene glycol through a [(14) C]Maleimide linker, was synthesized from [1,4-(14) C]Maleic acid. This publication describes the synthesis and analysis of PEG-[(14) C]Maleimide-adnectin ([(14) C]ATI-1072).

Inhibitory effect of ketoconazole on the pharmacokinetics of a multireceptor tyrosine kinase inhibitor BMS-690514 in healthy participants: assessing the mechanism of the interaction with physiologically-based pharmacokinetic simulations.

BMS-690514, a selective inhibitor of the ErbB and vascular endothelial growth factor receptors, has shown antitumor activity in early clinical development. The compound is metabolized by multiple enzymes, with CYP3A4 responsible for the largest fraction (34%) of metabolism. It is also a substrate of P-glycoprotein (P-gp) in vitro. To assess the effect of ketoconazole on BMS-690514 pharmacokinetics, 17 healthy volunteers received 200 mg BMS-690514 alone followed by 100 mg BMS-690514 with ketoconazole (400 mg once daily for 4 days). The AUC(∞) of 100 mg BMS-690514 concomitantly administered with ketoconazole was similar to that of 200 mg BMS-690514 alone. The dose-normalized C(max) and AUC(∞) of BMS-690514 from the 100-mg BMS-690514/400-mg ketoconazole treatment increased by 55% and 127%, respectively, relative to those from 200 mg BMS-690514 alone. Prediction of the drug-drug interaction (DDI) using a population-based simulator (Simcyp) indicated that, in addition to CYP3A4 inhibition, the inhibition of P-gp by ketoconazole in the intestine, liver, and kidneys must be invoked to fully account for the DDI observed. This finding suggests that the inhibition of P-gp by ketoconazole, along with its effect on CYP3A4, needs to be considered when designing a DDI study of ketoconazole with a victim drug that is a dual substrate.

Simultaneous oral therapeutic and intravenous ¹4C-microdoses to determine the absolute oral bioavailability of saxagliptin and dapagliflozin.

AIM: To determine the absolute oral bioavailability (F(p.o.) ) of saxagliptin and dapagliflozin using simultaneous intravenous ¹4C-microdose/therapeutic oral dosing (i.v.micro + oraltherap). METHODS: The F(p.o.) values of saxagliptin and dapagliflozin were determined in healthy subjects (n = 7 and 8, respectively) following the concomitant administration of single i.v. micro doses with unlabelled oraltherap doses. Accelerator mass spectrometry and liquid chromatography-tandem mass spectrometry were used to quantify the labelled and unlabelled drug, respectively. RESULTS: The geometric mean point estimates (90% confidence interval) F(p.o) . values for saxagliptin and dapagliflozin were 50% (48, 53%) and 78% (73, 83%), respectively. The i.v.micro had similar pharmacokinetics to oraltherap. CONCLUSIONS: Simultaneous i.v.micro + oraltherap dosing is a valuable tool to assess human absolute bioavailability.

Overcoming bioanalytical challenges in an Onglyza(®) intravenous [(14)C]microdose absolute bioavailability study with accelerator MS.

BACKGROUND: An absolute bioavailability study that utilized an intravenous [(14)C]microdose was conducted for saxagliptin (Onglyza(®)), a marketed drug product for the treatment of Type 2 diabetes mellitus. Concentrations of [(14)C]saxagliptin were determined by accelerator MS (AMS) after protein precipitation, chromatographic separation by UPLC and analyte fraction collection. A series of investigative experiments were conducted to maximize the release of the drug from high-affinity receptors and nonspecific adsorption, and to determine a suitable quantitation range. RESULTS: A technique-appropriate validation demonstrated the accuracy, precision, specificity, stability and recovery of the AMS methodology across the concentration range of 0.025 to 15.0 dpm/ml (disintegration per minute per milliliter), the equivalent of 1.91-1144 pg/ml. Based on the study sample analysis, the mean absolute bioavailability of saxagliptin was 50% in the eight subjects with a CV of 6.6%. Incurred sample reanalysis data fell well within acceptable limits. CONCLUSION: This study demonstrated that the optimized sample pretreatment and chromatographic separation procedures were critical for the successful implementation of an UPLC plus AMS method for [(14)C]saxagliptin. The use of multiple-point standards are useful, particularly during method development and validation, to evaluate and correct for concentration-dependent recovery, if observed, and to monitor and control process loss and operational variations.

Characterization of the in vitro and in vivo metabolism and disposition and cytochrome P450 inhibition/induction profile of saxagliptin in human.

Saxagliptin is a potent dipeptidyl peptidase-4 inhibitor approved for the treatment of type 2 diabetes mellitus. The pharmacokinetics and disposition of [(14)C]saxagliptin were investigated in healthy male subjects after a single 50-mg (91.5 µCi) oral dose. Saxagliptin was rapidly absorbed (T(max), 0.5 h). Unchanged saxagliptin and 5-hydroxy saxagliptin (M2), a major, active metabolite, were the prominent drug-related components in the plasma, together accounting for most of the circulating radioactivity. Approximately 97% of the administered radioactivity was recovered in the excreta within 7 days postdose, of which 74.9% was eliminated in the urine and 22.1% was excreted in the feces. The parent compound and M2 represented 24.0 and 44.1%, respectively, of the radioactivity recovered in the urine and feces combined. Taken together, the excretion data suggest that saxagliptin was well absorbed and was subsequently cleared by both urinary excretion and metabolism; the formation of M2 was the major metabolic pathway. Additional minor metabolic pathways included hydroxylation at other positions and glucuronide or sulfate conjugation. Cytochrome P450 (P450) enzymes CYP3A4 and CYP3A5 metabolized saxagliptin and formed M2. Kinetic experiments indicated that the catalytic efficiency (V(max)/K(m)) for CYP3A4 was approximately 4-fold higher than that for CYP3A5. Therefore, it is unlikely that variability in expression levels of CYP3A5 due to genetic polymorphism will impact clearance of saxagliptin. Saxagliptin and M2 each showed little potential to inhibit or induce important P450 enzymes, suggesting that saxagliptin is unlikely to affect the metabolic clearance of coadministered drugs that are substrates for these enzymes.

Liquid chromatography and tandem mass spectrometry method for the quantitative determination of saxagliptin and its major pharmacologically active 5-monohydroxy metabolite in human plasma: method validation and overcoming specific and non-specific binding at low concentrations.

A liquid chromatography and tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine the concentrations of saxagliptin (Onglyza™, BMS-477118) and its major active metabolite, 5-hydroxy saxagliptin to support pharmacokinetic analyses in clinical studies. The dynamic range of the assay was 0.1-50 ng/mL for saxagliptin and 0.2-100 ng/mL for 5-hydroxy saxagliptin. Protein precipitation (PPT) with acetonitrile was used to extract the analytes from plasma matrix before injecting on an Atlantis(®) dC18 column (50 mm × 2.1 mm, 5 µm) for LC-MS/MS analysis. The sample pre-treatment process was carefully controlled to disrupt DPP4-specific binding and non-specific binding observed at lower concentrations. The recoveries for both analytes were >90%. The assay was selective, rugged and reproducible; storage stability of at least 401 days at -20°C was demonstrated. Under these chromatographic conditions, the isomers of saxagliptin and 5-hydroxy saxagliptin were chromatographically separated from saxagliptin and 5-hydroxy saxagliptin. The assay has been used to support multiple clinical studies and regulatory approvals.