ESHRE guideline: number of embryos to transfer during IVF/ICSI†.

STUDY QUESTION: Which clinical and embryological factors should be considered to apply double embryo transfer (DET) instead of elective single embryo transfer (eSET)? SUMMARY ANSWER: No clinical or embryological factor per se justifies a recommendation of DET instead of eSET in IVF/ICSI. WHAT IS KNOWN ALREADY: DET is correlated with a higher rate of multiple pregnancy, leading to a subsequent increase in complications for both mother and babies. These complications include preterm birth, low birthweight, and other perinatal adverse outcomes. To mitigate the risks associated with multiple pregnancy, eSET is recommended by international and national professional organizations as the preferred approach in ART. STUDY DESIGN, SIZE, DURATION: The guideline was developed according to the structured methodology for development and update of ESHRE guidelines. Literature searches were performed in PUBMED/MEDLINE and Cochrane databases, and relevant papers published up to May 2023, written in English, were included. Live birth rate, cumulative live birth rate, and multiple pregnancy rate were considered as critical outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Based on the collected evidence, recommendations were discussed until a consensus was reached within the Guideline Development Group (GDG). A stakeholder review was organized after the guideline draft was finalized. The final version was approved by the GDG and the ESHRE Executive Committee. MAIN RESULTS AND THE ROLE OF CHANCE: The guideline provides 35 recommendations on the medical and non-medical risks associated with multiple pregnancies and on the clinical and embryological factors to be considered when deciding on the number of embryos to transfer. These recommendations include 25 evidence-based recommendations, of which 24 were formulated as strong recommendations and one as conditional, and 10 good practice points. Of the evidence-based recommendations, seven (28%) were supported by moderate-quality evidence. The remaining recommendations were supported by low (three recommendations; 12%), or very low-quality evidence (15 recommendations; 60%). Owing to the lack of evidence-based research, the guideline also clearly mentions recommendations for future studies. LIMITATIONS, REASONS FOR CAUTION: The guideline assessed different factors one by one based on existing evidence. However, in real life, clinicians' decisions are based on several prognostic factors related to each patient's case. Furthermore, the evidence from randomized controlled trials is too scarce to formulate high-quality evidence-based recommendations. WIDER IMPLICATIONS OF THE FINDINGS: The guideline provides health professionals with clear advice on best practice in the decision-making process during IVF/ICSI, based on the best evidence currently available, and recommendations on relevant information that should be communicated to patients. In addition, a list of research recommendations is provided to stimulate further studies in the field. STUDY FUNDING/COMPETING INTEREST(S): The guideline was developed and funded by ESHRE, covering expenses associated with the guideline meetings, the literature searches, and the dissemination of the guideline. The guideline group members did not receive payment. DPB declared receiving honoraria for lectures from Merck, Ferring, and Gedeon Richter. She is a member of ESHRE EXCO, and the Mediterranean Society for reproductive medicine and the president of the Croatian Society for Gynaecological Endocrinology and Reproductive Medicine. CDG is the past Chair of the ESHRE EIM Consortium and a paid deputy member of the Editorial board of Human Reproduction. IR declared receiving reimbursement from ESHRE and EDCD for attending meetings. She holds an unpaid leadership role in OBBCSSR, ECDC Sohonet, and AER. KAR-W declared receiving grants for clinical researchers and funding provision to the institution from the Swedish Cancer Society (200170F), the Senior Clinical Investigator Award, Radiumhemmets Forskningsfonder (Dnr: 201313), Stockholm County Council FoU (FoUI-953912) and Karolinska Institutet (Dnr 2020-01963), NovoNordisk, Merck and Ferring Pharmaceuticals. She received consulting fees from the Swedish Ministry of Health and Welfare. She received honoraria from Roche, Pfizer, and Organon for chairmanship and lectures. She received support from Organon for attending meetings. She participated in advisory boards for Merck, Nordic countries, and Ferring. She declared receiving time-lapse equipment and grants with payment to institution for pre-clinical research from Merck pharmaceuticals and from Ferring. SS-R received research funding from Roche Diagnostics, Organon/MSD, Theramex, and Gedeo-Richter. He received consulting fees from Organon/MSD, Ferring Pharmaceuticals, and Merck Serono. He declared receiving honoraria for lectures from Ferring Pharmaceuticals, Besins, Organon/MSD, Theramex, and Gedeon Richter. He received support for attending Gedeon Richter meetings and participated in the Data Safety Monitoring Board of the T-TRANSPORT trial. He is the Deputy of ESHRE SQART special interest group. He holds stock options in IVI Lisboa and received equipment and other services from Roche Diagnostics and Ferring Pharmaceuticals. KT declared receiving payment for honoraria for giving lectures from Merck Serono and Organon. She is member of the safety advisory board of EDQM. She holds a leadership role in the ICCBBA board of directors. ZV received reimbursement from ESHRE for attending meetings. She also received research grants from ESHRE and Juhani Aaltonen Foundation. She is the coordinator of EHSRE SQART special interest group. The other authors have no conflicts of interest to declare. DISCLAIMER: This guideline represents the views of ESHRE, which were achieved after careful consideration of the scientific evidence available at the time of preparation. In the absence of scientific evidence on certain aspects, a consensus between the relevant ESHRE stakeholders has been obtained. Adherence to these clinical practice guidelines does not guarantee a successful or specific outcome, nor does it establish a standard of care. Clinical practice guidelines do not replace the need for application of clinical judgement to each individual presentation, nor variations based on locality and facility type. ESHRE makes no warranty, express or implied, regarding the clinical practice guidelines and specifically excludes any warranties of merchantability and fitness for a particular use or purpose (full disclaimer available at https://www.eshre.eu/Guidelines-and-Legal).

Exploring the Micro-Mosaic Landscape of FGFR3 Mutations in the Ageing Male Germline and Their Potential Implications in Meiotic Differentiation.

Advanced paternal age increases the risk of transmitting de novo germline mutations, particularly missense mutations activating the receptor tyrosine kinase (RTK) signalling pathway, as exemplified by the FGFR3 mutation, which is linked to achondroplasia (ACH). This risk is attributed to the expansion of spermatogonial stem cells carrying the mutation, forming sub-clonal clusters in the ageing testis, thereby increasing the frequency of mutant sperm and the number of affected offspring from older fathers. While prior studies proposed a correlation between sub-clonal cluster expansion in the testis and elevated mutant sperm production in older donors, limited data exist on the universality of this phenomenon. Our study addresses this gap by examining the testis-expansion patterns, as well as the increases in mutations in sperm for two FGFR3 variants-c.1138G>A (p.G380R) and c.1948A>G (p.K650E)-which are associated with ACH or thanatophoric dysplasia (TDII), respectively. Unlike the ACH mutation, which showed sub-clonal expansion events in an aged testis and a significant increase in mutant sperm with the donor's age, as also reported in other studies, the TDII mutation showed focal mutation pockets in the testis but exhibited reduced transmission into sperm and no significant age-related increase. The mechanism behind this divergence remains unclear, suggesting potential pleiotropic effects of aberrant RTK signalling in the male germline, possibly hindering differentiation requiring meiosis. This study provides further insights into the transmission risks of micro-mosaics associated with advanced paternal age in the male germline.

Characterization of Enzymes Involved in Nintedanib Metabolism in Humans.

Nintedanib, which is used to treat idiopathic pulmonary fibrosis and non-small cell lung cancer, is metabolized to a pharmacologically inactive carboxylate derivative, BIBF1202, via hydrolysis and subsequently by glucuronidation to BIBF1202 acyl-glucuronide (BIBF1202-G). Since BIBF1202-G contains an ester bond, it can be hydrolytically cleaved to BIBF1202. In this study, we sought to characterize these metabolic reactions in the human liver and intestine. Nintedanib hydrolysis was detected in human liver microsomes (HLMs) (Clearance [CL int]: 102.8 ± 18.9 µL/min per mg protein) but not in small intestinal preparations. CES1 was suggested to be responsible for nintedanib hydrolysis according to experiments using recombinant hydrolases and hydrolase inhibitors as well as proteomic correlation analysis using 25 individual HLM. BIBF1202 glucuronidation in HLM (3.6 ± 0.3 µL/min per mg protein) was higher than that in human intestinal microsomes (1.5 ± 0.06 µL/min per mg protein). UGT1A1 and gastrointestinal UGT1A7, UGT1A8, and UGT1A10 were able to mediate BIBF1202 glucuronidation. The impact of UGT1A1 on glucuronidation was supported by the finding that liver microsomes from subjects homozygous for the UGT1A1*28 allele showed significantly lower activity than those from subjects carrying the wild-type UGT1A1 allele. Interestingly, BIBF1202-G was converted to BIBF1202 in HLS9 at 70-fold higher rates than the rates of BIBF1202 glucuronidation. An inhibition study and proteomic correlation analysis suggested that ß-glucuronidase is responsible for hepatic BIBF1202-G deglucuronidation. In conclusion, the major metabolic reactions of nintedanib in the human liver and intestine were quantitatively and thoroughly elucidated. This information could be helpful to understand the inter- and intraindividual variability in the efficacy of nintedanib. SIGNIFICANCE STATEMENT: To our knowledge, this is the first study to characterize the enzymes responsible for each step of nintedanib metabolism in the human body. This study found that ß-glucuronidase may contribute to BIBF1202-G deglucuronidation.

A Novel Artificial Intelligence-Based Approach for Quantitative Assessment of Angiogenesis in the Ex Ovo CAM Model.

Angiogenesis is a highly regulated process. It promotes tissue regeneration and contributes to tumor growth. Existing therapeutic concepts interfere with different steps of angiogenesis. The quantification of the vasculature is of crucial importance for research on angiogenetic effects. The chorioallantoic membrane (CAM) assay is widely used in the study of angiogenesis. Ex ovo cultured chick embryos develop an easily accessible, highly vascularised membrane on the surface. Tumor xenografts can be incubated on this membrane enabling studies on cancer angiogenesis and other major hallmarks. However, there is no commonly accepted gold standard for the quantification of the vasculature of the CAM. We compared four widely used measurement techniques to identify the most appropriate one for the quantification of the vascular network of the CAM. The comparison of the different quantification methods suggested that the CAM assay application on the IKOSA platform is the most suitable image analysis application for the vasculature of the CAM. The new CAM application on the IKOSA platform turned out to be a reliable and feasible tool for practical use in angiogenesis research. This novel image analysis software enables a deeper exploration of various aspects of angiogenesis and might support future research on new anti-angiogenic strategies for cancer treatment.

Validation of a Drug Transporter Probe Cocktail Using the Prototypical Inhibitors Rifampin, Probenecid, Verapamil, and Cimetidine.

BACKGROUND AND OBJECTIVE: A novel cocktail containing four substrates of key drug transporters was previously optimized to eliminate mutual drug-drug interactions between the probes digoxin (P-glycoprotein substrate), furosemide (organic anion transporter 1/3), metformin (organic cation transporter 2, multidrug and toxin extrusion protein 1/2-K), and rosuvastatin (organic anion transporting polypeptide 1B1/3, breast cancer resistance protein). This clinical trial investigated the effects of four commonly employed drug transporter inhibitors on cocktail drug pharmacokinetics. METHODS: In a randomized open-label crossover trial in 45 healthy male subjects, treatment groups received the cocktail with or without single oral doses of rifampin, verapamil, cimetidine or probenecid. Concentrations of the probe drugs in serial plasma samples and urine fractions were measured by validated liquid chromatography-tandem mass spectrometry assays to assess systemic exposure. RESULTS: The results were generally in accordance with known in vitro and/or clinical drug-drug interaction data. Single-dose rifampin increased rosuvastatin area under the plasma concentration-time curve up to the last quantifiable concentration (AUC0-tz) by 248% and maximum plasma concentration (Cmax) by 1025%. Probenecid increased furosemide AUC0-tz by 172% and Cmax by 23%. Cimetidine reduced metformin renal clearance by 26%. The effect of single-dose verapamil on digoxin systemic exposure was less than expected from multiple-dose studies (AUC0-tz unaltered, Cmax + 22%). CONCLUSIONS: Taking all the interaction results together, the transporter cocktail is considered to be validated as a sensitive and specific tool for evaluating transporter-mediated drug-drug interactions in drug development. CLINICAL TRIAL REGISTRATION: EudraCT number 2017-001549-29.

Clinical Pharmacokinetics and Pharmacodynamics of Nintedanib.

Nintedanib is an oral, small-molecule tyrosine kinase inhibitor approved for the treatment of idiopathic pulmonary fibrosis and patients with advanced non-small cell cancer of adenocarcinoma tumour histology. Nintedanib competitively binds to the kinase domains of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). Studies in healthy volunteers and in patients with advanced cancer have shown that nintedanib has time-independent pharmacokinetic characteristics. Maximum plasma concentrations of nintedanib are reached approximately 2-4 h after oral administration and thereafter decline at least bi-exponentially. Over the investigated dose range of 50-450 mg once daily and 150-300 mg twice daily, nintedanib exposure increases are dose proportional. Nintedanib is metabolised via hydrolytic ester cleavage, resulting in the formation of the free acid moiety that is subsequently glucuronidated and excreted in the faeces. Less than 1% of drug-related radioactivity is eliminated in urine. The terminal elimination half-life of nintedanib is about 10-15 h. Accumulation after repeated twice-daily dosing is negligible. Sex and renal function have no influence on nintedanib pharmacokinetics, while effects of ethnicity, low body weight, older age and smoking are within the inter-patient variability range of nintedanib exposure and no dose adjustments are required. Administration of nintedanib in patients with moderate or severe hepatic impairment is not recommended, and patients with mild hepatic impairment should be monitored closely and the dose adjusted accordingly. Nintedanib has a low potential for drug-drug interactions, especially with drugs metabolised by cytochrome P450 enzymes. Concomitant treatment with potent inhibitors or inducers of the P-glycoprotein transporter can affect the pharmacokinetics of nintedanib. At an investigated dose of 200 mg twice daily, nintedanib does not have proarrhythmic potential.

The impact of poly-A microsatellite heterologies in meiotic recombination.

Meiotic recombination has strong, but poorly understood effects on short tandem repeat (STR) instability. Here, we screened thousands of single recombinant products with sperm typing to characterize the role of polymorphic poly-A repeats at a human recombination hotspot in terms of hotspot activity and STR evolution. We show that the length asymmetry between heterozygous poly-A's strongly influences the recombination outcome: a heterology of 10 A's (9A/19A) reduces the number of crossovers and elevates the frequency of non-crossovers, complex recombination products, and long conversion tracts. Moreover, the length of the heterology also influences the STR transmission during meiotic repair with a strong and significant insertion bias for the short heterology (6A/7A) and a deletion bias for the long heterology (9A/19A). In spite of this opposing insertion-/deletion-biased gene conversion, we find that poly-A's are enriched at human recombination hotspots that could have important consequences in hotspot activation.

The Role of Endometrial Volume and Endometrial and Subendometrial Vascularization Parameters in a Frozen Embryo Transfer Cycle.

The role of three-dimensional power Doppler ultrasonography of the endometrium in assisted reproduction is still far from clear. In this retrospective cohort study, transvaginal three-dimensional power Doppler examinations were performed 30 min before frozen-thawed embryo transfer. After pregnancy tests, two cohorts were established: P (pregnant, n = 31) and NP (nonpregnant, n = 31). The study only included nullipara with no uterine abnormalities who were undergoing infertility treatment at the Department of Gynecology, Obstetrics and Gynecological Endocrinology, Kepler University Hospital, Linz, Austria. The main outcome measures were the vascularization flow index (VFI), flow index (FI), and vascularization index (VI) in the endometrium/subendometrium, assessed using Virtual Organ Computer-aided AnaLysis (VOCAL™), and the endometrial volume. A total of 62 patients were enrolled in the study, forming two cohorts (pregnant, P; nonpregnant, NP). There were no significant differences between the two cohorts with regard to demographic data, numbers of embryos transferred, or embryo grading, but there was a significant difference in endometrial volume (cohort P, 3.17 ± 0.84 mL; cohort NP, 2.36 ± 0.9 mL; P = 0.001) and the pregnancy rate rises with larger volume. No differences were observed in the vascularization parameters FI, VFI, and VI in the endometrium and subendometrium. In the cohort of pregnant patients, there were 26 (41.9%) live births, with 21 term deliveries (80.8%). The endometrial volume was larger in the cohort of pregnant patients. Measurements were performed 30 min before embryo transfer, and no differences were observed in vascularization parameters in the subendometrium and endometrium.

Different Involvement of OAT in Renal Disposition of Oral Anticoagulants Rivaroxaban, Dabigatran, and Apixaban.

This study aimed to investigate the interactions of 3 anticoagulants, rivaroxaban, apixaban, and dabigatran, with 5 human solute carrier transporters, hOAT1, hOAT3, hOCT2, hOATP1B1, and hOATP1B3. Apixaban inhibited hOAT3, hOATP1B1, and hOATP1B3, and rivaroxaban inhibited hOAT3 and hOATP1B3, with IC50 values of >20 and >5 µM, respectively. The effect of dabigatran was negligible or very weak, so significant drug interactions at therapeutic doses are unlikely. Specific uptake of rivaroxaban was observed only in human and mouse OAT3-expressing cells. The Km for mouse Oat3 (mOat3) was 1.01 ± 0.70 µM. A defect in mOat3 reduced the kidney-to-plasma concentration ratio of rivaroxaban by 38% in mice. Probenecid treatment also reduced the kidney-to-plasma concentration ratio of rivaroxaban in rats by 73%. Neither mOat3 defect nor probenecid administration in rats reduced the renal clearance of rivaroxaban. The uptake of rivaroxaban by monkey kidney slices was temperature dependent and inhibited by probenecid but not by tetraethylammonium. Taken together, organic anion transporters, mainly OAT3, may mediate basolateral uptake of rivaroxaban in kidneys. hOAT3 could be an additional factor that differentiates the potential drug-drug interactions of the 3 anticoagulants in the urinary excretion process in clinical settings.

Clinical Pharmacokinetics and Pharmacodynamics of Afatinib.

Afatinib is an oral, irreversible ErbB family blocker that covalently binds to the kinase domains of epidermal growth factor receptor (EGFR), human EGFRs (HER) 2, and HER4, resulting in irreversible inhibition of tyrosine kinase autophosphorylation. Studies in healthy volunteers and patients with advanced solid tumours have shown that once-daily afatinib has time-independent pharmacokinetic characteristics. Maximum plasma concentrations of afatinib are reached approximately 2-5 h after oral administration and thereafter decline, at least bi-exponentially. Food reduces total exposure to afatinib. Over the clinical dose range of 20-50 mg, afatinib exposure increases slightly more than dose proportional. Afatinib metabolism is minimal, with unchanged drug predominantly excreted in the faeces and approximately 5 % in urine. Apart from the parent drug afatinib, the major circulation species in human plasma are the covalently bound adducts to plasma protein. The effective elimination half-life is approximately 37 h, consistent with an accumulation of drug exposure by 2.5- to 3.4-fold based on area under the plasma concentration-time curve (AUC) after multiple dosing. The pharmacokinetic profile of afatinib is consistent across a range of patient populations. Age, ethnicity, smoking status and hepatic function had no influence on afatinib pharmacokinetics, while females and patients with low body weight had increased exposure to afatinib. Renal function is correlated with afatinib exposure, but, as for sex and body weight, the effect size for patients with severe renal impairment (approximately 50 % increase in AUC) is only mildly relative to the extent of unexplained interpatient variability in afatinib exposure. Afatinib has a low potential as a victim or perpetrator of drug-drug interactions, especially with cytochrome P450-modulating agents. However, concomitant treatment with potent inhibitors or inducers of the P-glycoprotein transporter can affect the pharmacokinetics of afatinib. At a dose of 50 mg, afatinib does not have proarrhythmic potential.

Chromosomal Aneuploidies and Early Embryonic Developmental Arrest.

BACKGROUND: Selecting the best embryo for transfer, with the highest chance of achieving a vital pregnancy, is a major goal in current in vitro fertilization (IVF) technology. The high rate of embryonic developmental arrest during IVF treatment is one of the limitations in achieving this goal. Chromosomal abnormalities are possibly linked with chromosomal arrest and selection against abnormal fertilization products. The objective of this study was to evaluate the frequency and type of chromosomal abnormalities in preimplantation embryos with developmental arrest. MATERIALS AND METHODS: This cohort study included blastomeres of embryos with early developmental arrest that were biopsied and analyzed by fluorescence in-situ hybridization (FISH) with probes for chromosomes 13, 16, 18, 21 and 22. Forty-five couples undergoing IVF treatment were included, and 119 arrested embryos were biopsied. All probes were obtained from the Kinderwunsch Zentrum, Linz, Austria, between August 2009 and August 2011. RESULTS: Of these embryos, 31.6% were normal for all chromosomes tested, and 68.4% were abnormal. Eleven embryos were uniformly aneuploid, 20 were polyploid, 3 were haploid, 11 displayed mosaicism and 22 embryos exhibited chaotic chromosomal complement. CONCLUSION: Nearly 70% of arrested embryos exhibit chromosomal errors, making chromosomal abnormalities a major cause of embryonic arrest and may be a further explanation for the high developmental failure rates during culture of the embryos in the IVF setting.

The Use of Transporter Probe Drug Cocktails for the Assessment of Transporter-Based Drug-Drug Interactions in a Clinical Setting-Proposal of a Four Component Transporter Cocktail.

Probe drug cocktails are used clinically to assess the potential for drug-drug interactions (DDIs), and in particular, DDIs resulting from coadministration of substrates and inhibitors of cytochrome P450 enzymes. However, a probe drug cocktail has not been identified to assess DDIs involving inhibition of drug transporters. We propose a cocktail consisting of the following substrates to explore the potential for DDIs caused by inhibition of key transporters: digoxin (P-glycoprotein, P-gp), rosuvastatin (breast cancer resistance protein, BCRP; organic anion transporting polypeptides, OATP), metformin (organic cation transporter, OCT; multidrug and toxin extrusion transporters, MATE), and furosemide (organic anion transporter, OAT). Furosemide was evaluated in vitro, and is a substrate of OAT1 and OAT3, with Km values of 38.9 and 21.5 µM, respectively. Furosemide was also identified as a substrate of BCRP, OATP1B1, and OATP1B3. Furosemide inhibited BCRP (50% inhibition of drug transport: 170 µM), but did not inhibit OATP1B1, OATP1B3, OCT2, MATE1, and MATE2-K at concentrations below 300 µM, and P-gp at concentrations below 2000 µM. Conservative approaches for the estimation of the likelihood of in vivo DDIs indicate a remote chance of in vivo transporter inhibition by these probe drugs when administered at low single oral doses. This four component probe drug cocktail is therefore proposed for clinical evaluation.

Impact of endogenous esterase activity on in vitro p-glycoprotein profiling of dabigatran etexilate in Caco-2 monolayers.

Dabigatran etexilate, a double prodrug of dabigatran, is a reversible, competitive, direct thrombin inhibitor that has been approved for use in many countries. A recent guideline from the European Medicines Agency on drug-drug interactions proposed dabigatran etexilate as a sensitive in vivo and in vitro probe substrate for intestinal P-glycoprotein (P-gp) inhibition. We therefore performed a series of in vitro studies to determine the best experimental conditions for evaluation of P-gp involvement on the transport process of dabigatran etexilate across colorectal adenocarcinoma Caco-2 cell monolayers. Experiments using expressed carboxylesterase 1 (CES1) and CES2 bactosomes revealed that dabigatran etexilate was hydrolyzed into BIBR 1087 by CES1 expressed in our Caco-2 cells. The impact of CES1-mediated BIBR 1087 formation during transcellular transport experiments was assessed by comparing several combinations of three experimental approaches: radioactivity detection using [(14)C]dabigatran etexilate as substrate, liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification of dabigatran etexilate, and in the presence and absence of a CES inhibitor bis(p-nitrophenyl) phosphate (BNPP). The experimental approach that was based on the use of nonlabeled dabigatran etexilate together with LC-MS/MS quantification and the addition of BNPP was selected as the most favorable condition in which to correctly evaluate the permeability coefficient (Papp) of dabigatran etexilate and its transcellular transport by P-gp. The in vitro Caco-2 study at the selected condition revealed that dabigatran etexilate is a P-gp substrate with an efflux ratio of 13.8 and an intrinsic Papp, which is the Papp under the condition of complete blockage of P-gp by P-gp inhibitor, of 29 × 10(-6) cm/s.

Evaluation and prediction of potential drug-drug interactions of linagliptin using in vitro cell culture methods.

Linagliptin is a highly potent dipeptidyl peptidase-4 (DPP-4) inhibitor approved for the treatment of type 2 diabetes. Unlike other DPP-4 inhibitors, linagliptin is cleared primarily via the bile and gut. We used a panel of stably and transiently transfected cell lines to elucidate the carrier-mediated transport processes that are involved in linagliptin disposition in vivo and to assess the potential for drug-drug interactions (DDIs). Our results demonstrate that linagliptin is a substrate of organic cation transporter 2 (OCT2) and P-glycoprotein (P-gp) but not of organic anion-transporting polypeptide 1B1 and 1B3; organic anion transporter 1, 3, and 4; OCT1; or organic cation/carnitine transporter 1 and 2, suggesting that OCT2 and P-gp play a role in the disposition of linagliptin in vivo. Linagliptin inhibits transcellular transport of digoxin by P-gp with an apparent IC(50) of 66.1 µM, but it did not inhibit activity of multidrug resistance-associated protein 2 and breast cancer resistance protein as represented by transport of probe substrate into membrane vesicles from respective transporter-expressing cells. In addition, the inhibitory effect of linagliptin on major solute carrier transporter isoforms was investigated. Linagliptin showed inhibitory potency against only OCT1 and OCT2 out of all major solute carrier transporter isoforms examined, and those inhibition potencies, evaluated using three different in vitro probe substrates, were substrate-specific. Considering the low therapeutic plasma concentration of linagliptin, our data clearly suggest a very low risk for transporter-mediated DDIs with comedications in clinical practice.

Absolute quantification and differential expression of drug transporters, cytochrome P450 enzymes, and UDP-glucuronosyltransferases in cultured primary human hepatocytes.

The levels of metabolizing enzymes and transporters expressed in hepatocytes are decisive factors for hepatobiliary disposition of most drugs. Induction via nuclear receptor activation can significantly alter those levels, with the coregulation of multiple enzymes and transporters occurring to different extents. Here, we report the use of a targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method for concurrent quantification of multiple cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and transporter proteins in cultured primary human hepatocytes. The effects of culture format (i.e., sandwich culture versus conventional culture) and of dexamethasone (DEX) media concentrations on mRNA, protein, and activity levels were determined for three donors, and protein expression was compared with that in liver. In general, P450 and UGT expression was lower in hepatocyte cultures than that in liver, and CYP2C9 was found to be the most abundant P450 isoform expressed in cultured hepatocytes. The sandwich culture format and 0.1 µM DEX in media retained the protein expression in the hepatocytes closest to the levels found in liver. However, higher in vitro expression was observed for drug transporters, especially for multidrug resistance protein 1 and breast cancer resistance protein. Direct protein quantification was applied successfully to study in vitro induction in sandwich cultured primary hepatocytes in a 24-well format using the prototypical inducers rifampicin, omeprazole, and phenobarbital. We conclude that targeted absolute LC-MS/MS quantification of drug-metabolizing enzymes and transporters can broaden the scope and significantly increase the impact of in vitro drug metabolism studies, such as induction, as an important supplement or future alternative to mRNA and activity data.

Simultaneous absolute protein quantification of transporters, cytochromes P450, and UDP-glucuronosyltransferases as a novel approach for the characterization of individual human liver: comparison with mRNA levels and activities.

The purpose of the present study was to determine the absolute protein expression levels of multiple drug-metabolizing enzymes and transporters in 17 human liver biopsies, and to compare them with the mRNA expression levels and functional activities to evaluate the suitability of the three measures as parameters of hepatic metabolism. Absolute protein expression levels of 13 cytochrome P450 (P450) enzymes, NADPH-P450 reductase (P450R) and 6 UDP-glucuronosyltransferase (UGT) enzymes in microsomal fraction, and 22 transporters in plasma membrane fraction were determined using liquid chromatography/tandem mass spectrometry. CYP2C9, CYP2E1, CYP3A4, CYP2A6, UGT1A6, UGT2B7, UGT2B15, and P450R were abundantly expressed (more than 50 pmol/mg protein) in human liver microsomes. The protein expression levels of CYP3A4, CYP2B6, and CYP2C8 were each highly correlated with the corresponding enzyme activity and mRNA expression levels, whereas for other P450s, the protein expression levels were better correlated with the enzyme activities than the mRNA expression levels were. Among transporters, the protein expression level of organic anion-transporting polypeptide 1B1 was relatively highly correlated with the mRNA expression level. However, other transporters showed almost no correlation. These findings indicate that protein expression levels determined by the present simultaneous quantification method are a useful parameter to assess differences of hepatic function between individuals.

Afatinib pharmacokinetics and metabolism after oral administration to healthy male volunteers.

PURPOSE: To investigate the pharmacokinetics, metabolism and tolerability of afatinib (BIBW 2992), an oral irreversible ErbB family blocker, in healthy male volunteers. METHODS: In this open-label, single-center study, 8 healthy male volunteers received a single oral dose of 15 mg [(14)C]-radiolabeled afatinib (equivalent to 22.2 mg of the dimaleinate salt) as a solution. Blood, urine and fecal samples were collected for at least 96 hours (h) after dosing. Plasma and urine concentrations of afatinib were analyzed using high-performance liquid chromatography-tandem mass spectrometry. [(14)C]-radioactivity levels in plasma, whole blood, urine and feces were measured by liquid scintillation counting methods. Metabolite patterns were assessed by high-performance liquid chromatography. RESULTS: [(14)C]-radioactivity was mainly excreted via feces (85.4%). Overall recovery of [(14)C]-radioactivity was 89.5%, indicative of a complete mass balance. Afatinib was slowly absorbed, with maximum plasma concentrations achieved at a median of 6 h after dosing, declining thereafter in a biexponential manner. The geometric mean terminal half-life of afatinib was 33.9 h in plasma and longer for [(14)C]-radioactivity in plasma and whole blood. Apparent total body clearance for afatinib was high (geometric mean 1,530 mL/min). The high volume of distribution (4,500 L) in plasma may indicate a high tissue distribution. Afatinib was metabolized to only a minor extent, with the main metabolite afatinib covalently bound to plasma proteins. Oxidative metabolism mediated via cytochrome P-450 was of negligible importance for the elimination of afatinib. Afatinib was well tolerated. CONCLUSIONS: Afatinib displayed a complete mass balance with the main route of excretion via feces. Afatinib undergoes minimal metabolism.

The impact of pharmacogenetics of metabolic enzymes and transporters on the pharmacokinetics of telmisartan in healthy volunteers.

OBJECTIVE: Telmisartan is mainly taken up into the liver by organic anion transporting polypeptide (OATP) 1B3, conjugated with glucuronate, and excreted into the bile. We investigated the relationship between genotypes of metabolizing enzymes and transporters and pharmacokinetics of telmisartan in clinical study. We also checked which enzymes are responsible for telmisartan glucuronidation. MATERIALS AND METHODS: We collected blood samples from 57 healthy volunteers who had participated in a clinical trial of telmisartan and examined the relationship between 14 mutations in six transporters/metabolic enzymes and pharmacokinetics of telmisartan. We also performed an in-vitro glucuronidation assay with recombinant uridine 5'-diphospho-glucuronosyltransferases isoforms and human liver microsomes. RESULTS: In the clinical study, area under the plasma concentration-time curve value from time zero to infinity, of telmisartan in heterozygotes of SLCO1B3 (encoding protein: OATP1B3) rs11045585 tended to be larger than that in homozygotes of wild-type alleles. Unexpectedly, 19 heterozygotes of UGT1A1*28, whose function was decreased, significantly increased its oral clearance compared with homozygotes of UGT1A1*1 alleles (1090±690 vs. 620±430 ml/min/body). Metabolic clearance of telmisartan in human liver microsomes obtained from individuals with UGT1A1*28/*28 was higher compared with that of UGT1A1*1/*1 (168±33 vs. 93.3±27.3 µl/min/mg protein). Although telmisartan was metabolized by multiple UGT isoforms, in-vitro experiments revealed that UGT1A3 was estimated to be predominantly involved in telmisartan glucuronidation in human hepatocytes. CONCLUSION: UGT1A1*28 was thought to enhance the protein expression of UGT1A3 as reported most recently (Riedmaier et al. Clin Pharmacol Ther 2010; 87:65-73) and thereby increase glucuronidation activity of telmisartan and decrease the plasma concentration of telmisartan.

Pharmacokinetics and metabolism of BIBF 1120 after oral dosing to healthy male volunteers.

The pharmacokinetics and metabolism of BIBF 1120, an oral triple angiokinase inhibitor targeting vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), were studied in healthy male volunteers (n = 8) who had received a single oral dose of 100 mg [(14)C]-radiolabelled BIBF 1120 administered as solution. BIBF 1120 was well-tolerated and rapidly absorbed; median time to reach maximum plasma concentrations was 1.3 h and gMean terminal half-life was 13.7 h. A relatively high apparent total body clearance and volume of distribution possibly indicated a high tissue distribution. Plasma concentrations of BIBF 1120 plus carboxylate metabolite BIBF 1202 were lower than the total [(14)C]-radioactivity in plasma, indicating presence of additional metabolites. Total recovery in excreta was 94.7% 1 week post-dose; mass balance was considered complete after 96 h. BIBF 1120 and metabolites were mainly excreted via faeces. The major metabolic pathway for BIBF 1120 was methyl ester cleavage to BIBF 1202. Subsequently, the free carboxyl group of BIBF 1202 was glucuronidated to 1-O-acylglucuronide. Pathways of minor importance were oxidative N-demethylation to yield BIBF 1053, and oxidation of the piperazine moiety and conjugation. Glucuronidation of the parent drug and formylation of the secondary aliphatic amine of the piperazine ring played a minor role.

Age-related distribution of basal serum AMH level in women of reproductive age and a presumably healthy cohort.

In this study, day 1 to 4 serum anti-Müllerian hormone (AMH) level was analyzed in 2,741 patients attending our department for reproductive medicine or reproductive surgery, including a subgroup of 1,105 women who attended an assisted reproductive technology program because of a male factor as a presumably healthy subgroup. Day 1 to 4 serum AMH levels showed an age-dependent distribution and there is a wide range of AMH in each year of age analyzed, showing that even young women are at a risk of reduced ovarian reserve.