Determination of intracellular tenofovir-diphosphate and emtricitabine-triphosphate concentrations in dried blood spots for pre-exposure prophylaxis adherence.

OBJECTIVES: We measured the intracellular concentrations of tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) in dried blood spots (DBS) for pre-exposure prophylaxis (PrEP) adherence using sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: A total of 191 DBS were obtained from 85 participants who were receiving tenofovir disoproxil fumarate (TDF; 300 mg) and emtricitabine (FTC; 200 mg) as PrEP at the Sexual Health Clinic, National Center for Global Health and Medicine, Tokyo, Japan. DBS punch (3 mm) added to 25 µL of 50% methanol and 400 µL of internal standard solution was used for solid phase extraction. Chromatographic separation was achieved on an Atlantis Premier BEH C18 AX Column (50 mm × 2.1 mm i.d.; particle size 1.7 µm) using gradient elution (flow rate: 0.6 mL/min); injection volume: 7 µL and run time: 5.5 min. Calibration curves for the two drugs were linear in the range 0.05-12.5 ng/punch. RESULTS: We determined the intracellular TFV-DP and FTC-TP concentrations in 191 DBS obtained from 85 patients administered with TDF and FTC as PrEP. The analytical performance data (calibration curve and QC samples) for all the analytical runs met the acceptance criteria. Intracellular concentrations of TFV-DP and FTC-TP in the DBS remained stable for at least 24 h after oral administration. CONCLUSIONS: A multiplex LC-MS/MS method was successfully developed for DBS, which can be useful for monitoring the levels of TFV-DP and FTC-TP in individuals receiving PrEP.

Steady-state pharmacokinetics of plasma tenofovir alafenamide (TAF), tenofovir (TFV) and emtricitabine (FTC), and intracellular TFV-diphosphate and FTC-triphosphate in HIV-1 infected old Japanese patients treated with bictegravir/FTC/TAF.

Emtricitabine (FTC) plus tenofovir alafenamide (TAF) has demonstrated efficacy and safety for pre-exposure prophylaxis (PrEP) to prevent HIV-1 infection. We measured the plasma PK of FTC, tenofovir (TFV), and TAF in a steady-state pharmacokinetic (PK) study of bictegravir/FTC/TAF in HIV-1-infected patients. Furthermore, validated liquid chromatography-tandem mass spectrometry was used to measure intracellular TFV-diphosphate (DP) and FTC-triphosphate (TP), the active metabolites of TFV and FTC, respectively. Plasma and dried blood spot samples were collected from 10 male patients aged ≥ 50 years at various time intervals: 0 (trough), 1, 2, 3, 4, 6, 8, 12, and 24 h after drug administration. The mean ± standard deviation of plasma PK parameters were as follows: The maximum concentrations of TAF, TFV, and FTC were 104.0 ± 72.5, 27.9 ± 5.2, and 3,976.0 ± 683.6 ng/mL, respectively. Additionally, their terminal elimination half-lives were 0.6 ± 0.5, 31.6 ± 10.4, and 6.9 ± 1.4 h, respectively. These results were consistent with previously reported data. The intracellular levels of TFV-DP and FTC-TP varied widely among individuals; however, they remained stable over 24 h in each individual at approximately 1,000-1,500 and 2,000-3,000 fmol/punch, respectively, indicating that plasma concentrations did not affect the intracellular concentrations of their active metabolites. These results demonstrated that measuring intracellular TFV-DP and FTC-TP could be useful for monitoring adherence to PrEP in clients on this regimen.

Impact of ramucirumab pharmacokinetics in combination with docetaxel on the efficacy and survival in patients with advanced non-small cell lung cancer.

OBJECTIVES: Ramucirumab, an anti-vascular endothelial growth factor receptor-2 antibody, has been approved for the treatment of non-small cell lung cancer (NSCLC); however, its pharmacokinetic properties in clinical practice are unknown. We aimed to measure ramucirumab concentrations and conduct a retrospective pharmacokinetic analysis using real-world data. MATERIALS AND METHODS: Patients with stage III-IV and recurrent NSCLC who received ramucirumab plus docetaxel were evaluated in this study. After the first administration, the ramucirumab trough concentration (Ctrough) was measured using liquid chromatography-mass spectrometry. Patient characteristics, adverse events, tumor response, and survival time were retrospectively extracted from medical records from August 2, 2016 to July 16, 2021. RESULTS: A total of 131 patients were examined to assess serum ramucirumab concentrations. Ctrough ranged from below the lower limit of quantification (BLQ) to 48.8 µg/mL (BLQ ≤ 1st quartile (Q1) ≤ 7.34, 7.34 < 2nd quartile (Q2) ≤ 14.7, 14.7 < 3rd quartile (Q3) ≤ 21.9 and 21.9 < 4th quartile (Q4) ≤ 48.8 µg/mL). The overall response rate was significantly higher in Q2-4 than that in Q1 (p = 0.011). The median progression-free survival was marginally longer, and overall survival was significantly longer in Q2-4 (p = 0.009). The Glasgow prognostic score (GPS) in Q1 was significantly higher than in Q2-4 (p = 0.034) and associated with Ctrough (p = 0.002). CONCLUSION: Patients with higher ramucirumab exposure had a high ORR and prolonged survival time, whereas patients with lower ramucirumab exposure were characterized by a high GPS and poor prognosis. Cachexia may reduce the exposure level of ramucirumab in certain patients, reducing the clinical benefits of ramucirumab treatment.

Development of an analytical method to determine E7130 concentration in mouse plasma by micro-sampling using ultra-performance liquid chromatography-high resolution mass spectrometry.

E7130 is a novel microtubule inhibitor and a promising tumor microenvironment ameliorator. Since the amount of the administration in preclinical study is very small due to the high potency of E7130, this study aimed to establish a sensitive analytical method to measure E7130 concentration in mouse plasma samples obtained via microsampling. A sensitive and validated method was developed based on ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). Chromatographic separation was achieved using a Waters ACQUITY UPLC BEH C18 1.7 µm (2.1 × 50 mm) column. Mobile phase A comprised 0.1% formic acid and 10 mM ammonium formate in water, and mobile phase B was methanol. A gradient elution was applied at a flow rate of 0.5 mL/min. The calibration curve drawn was linear in the 0.2-100 ng/mL E7130 concentration range for mouse plasma microsamples (10 µL). Analytical results demonstrated good precision (<6.7%) and accuracy (88.5%-100.0%) in E7130 quantitation, indicating that UHPLC-HRMS is a useful method for pharmacokinetic analysis and a valuable approach for the quantitation of hardly fragmented compounds.

Comparative Study on the Efficacy and Exposure of Molecular Target Agents in Non-small Cell Lung Cancer PDX Models with Driver Genetic Alterations.

Patient-derived xenografts (PDX) can adequately reflect clinical drug efficacy. However, the methods for evaluating drug efficacy are not fully established. We selected five non-small cell lung cancer (NSCLC) PDXs with genetic alterations from established PDXs and the corresponding molecular targeted therapy was administered orally for 21 consecutive days. Genetic analysis, measurement of drug concentrations in blood and tumors using LC/MS-MS, and analysis of drug distribution in tumors using matrix-assisted laser desorption/ionization mass spectrometry were performed. Fifteen (20%) PDXs were established using samples collected from 76 patients with NSCLC with genetic alterations. The genetic alterations observed in original patients were largely maintained in PDXs. We compared the drug efficacy in original patients and PDX models; the efficacies against certain PDXs correlated with the clinical effects, while those against the others did not. We determined blood and intratumor concentrations in the PDX model, but both concentrations were low, and no evident correlation with the drug efficacy could be observed. The intratumoral spatial distribution of the drugs was both homogeneous and heterogeneous for each drug, and the distribution was independent of the expression of the target protein. The evaluation of drug efficacy in PDXs enabled partial reproduction of the therapeutic effect in original patients. A more detailed analysis of systemic and intratumoral pharmacokinetics may help clarify the mode of action of drugs. Further development of evaluation methods and indices to improve the prediction accuracy of clinical efficacy is warranted.

Autofluorescence-based high-throughput isolation of nonbleaching Cyanidioschyzon merolae strains under nitrogen-depletion.

Photosynthetic organisms maintain optimum levels of photosynthetic pigments in response to environmental changes to adapt to the conditions. The identification of cyanobacteria strains that alleviate bleaching has revealed genes that regulate levels of phycobilisome, the main light-harvesting complex. In contrast, the mechanisms of pigment degradation in algae remain unclear, as no nonbleaching strains have previously been isolated. To address this issue, this study attempted to isolate nonbleaching strains of the unicellular red alga Cyanidioschyzon merolae after exposure to nitrogen (N)-depletion based on autofluorescence information. After four weeks under N-depletion, 13 cells from 500,000 cells with almost identical pre- and post-depletion chlorophyll a (Chl a) and/or phycocyanin autofluorescence intensities were identified. These nonbleaching candidate strains were sorted via a cell sorter, isolated on solid medium, and their post-N-depletion Chl a and phycocyanin levels were analyzed. Chl a levels of these nonbleaching candidate strains were lower at 1-4 weeks of N-depletion similar to the control strains, however, their phycocyanin levels were unchanged. Thus, we successfully isolated nonbleaching C. merolae strains in which phycocyanin was not degraded under N-depletion, via autofluorescence spectroscopy and cell sorting. This versatile method will help to elucidate the mechanisms regulating pigments in microalgae.

Tissue 2-Hydroxyglutarate and Preoperative Seizures in Patients With Diffuse Gliomas.

BACKGROUND AND OBJECTIVES: Mutant isocitrate dehydrogenase (IDH) 1/2 gene products gain a new ability to produce D-2-hydroxyglutarate (D2HG). IDH1/2 mutations are thought to be associated with seizures owing to the structural similarity between D2HG and glutamate. However, the effects of D2HG on seizures in clinical settings are not fully understood. We sought to investigate the relationship between tissue 2-hydroxyglutarate (2HG) concentration and preoperative seizures using clinical samples. METHODS: We included 104 consecutive patients with diffuse glioma who underwent surgery from August 2008 to May 2016 and whose clinical presentation and IDH1/2 status were identified. The presence of preoperative seizures, tumor location, histopathologic diagnosis, IDH1/2 status, and 1p/19q codeletion were assessed from the patient charts. Tissue 2HG concentration was measured using liquid chromatography-tandem mass spectrometry. To evaluate 2HG distribution without artefactual tissue disruption, we applied matrix-assisted laser desorption/ionization high-resolution mass spectrometry imaging (MALDI-MSI) in 12 patients' surgically resected samples. We assessed the correlation of preoperative seizures with tissue 2HG concentration, IDH1/2 status, WHO grade, and 1p/19q codeletion. RESULTS: Tissue 2HG concentration was higher in IDH1/2 mutant tumors (IDH-Mut, n = 42) than in IDH1/2 wild-type tumors (IDH-WT, n = 62) (median 4,860 ng/mg vs 75 ng/mg) (p < 0.0001). MALDI-MSI could detect 2HG signals in IDH-Mut, but not in IDH-WT. Preoperative seizures were observed in 64.3% of patients with IDH-Mut and 21.0% patients with IDH-WT (p < 0.0001). The optimal cutoff value of tissue 2HG concentration for predicting preoperative seizures was 1,190 ng/mg, as calculated by the receiver operating characteristic curve. Increased preoperative seizure risk was only observed in tumors with 2HG concentration above the cutoff value among IDH-Mut (IDH-Mut with above the cutoff value: 71.4% vs IDH-Mut with below the cutoff value: 28.6%; p = 0.031). Multivariate analysis, including IDH1/2 mutation status, tissue 2HG concentration, WHO grade, and 1p/19q codeletion, revealed that only increased tissue 2HG concentration was associated with preoperative seizures (odds ratio 5.86, 95% confidence interval 1.02-48.5; p = 0.048). DISCUSSION: We showed that high tissue 2HG concentration was associated with preoperative seizures, suggesting that excess 2HG increases risk of preoperative seizures in IDH1/2 mutant tumors.

A procedure for method development and protein binding ratio as the indicator of sensitivity with anticancer agents on MALDI mass spectrometry imaging.

The concentration and distribution of a drug and its metabolites in tissues are key factors for elucidating both drug efficacy and toxicity in drug development. In this study we developed a pharamaco-imaging procedure for 12 agents and investigated the relationship between the properties of target compounds and the sensitivities of detection in matrix-assisted laser desorption/ionization-mass spectrometer imaging (MALDI-MSI). We prepared mock samples with mouse liver homogenates diluted with gelatin solution, limit of detection concentrations of each compound was confirmed. The correlation was evaluated between the intensities of mass signals obtained in MALDI-MSI with each test compound (the intensities of the test compounds) at a consistent concentration and the properties of each test compound. The liver homogenate diluted with gelatin solution showed easier handling and lower coefficients of variation than did liver homogenate only, and can be used as a good surrogate matrix. Based on the analysis of 12 agents, the protein binding ratios showed significant correlation to the detection sensitivities. We presented a procedure for standardization of pharmaco-imaging method development with an in-tissue method using MALDI-MS. Our results indicated the correlation between test compound's sensitivity and their protein binding ratios in plasma or serum.

Investigation of Potential Pharmacokinetic Interactions Between Teneligliptin and Metformin in Steady-state Conditions in Healthy Adults.

PURPOSE: We assessed the effects of coadministration of metformin and teneligliptin on their pharmacokinetics in steady-state conditions relative to the administration of either drug alone. METHODS: This was a Phase I, single-center, open-label, 2-way parallel-group study in healthy male and female subjects. Subjects in group 1 (n = 20) were administered 40 mg of teneligliptin once daily for 5 days, and 850 mg of metformin BID was added to ongoing teneligliptin for an additional 3 days. The subjects in group 2 (n = 20) were administered 850 mg of metformin BID for 3 days, and 40 mg of teneligliptin once daily was added to ongoing metformin for an additional 5 days. Pharmacokinetic outcomes were the AUC0-τ and Cmax of metformin and teneligliptin when administered alone or in combination. FINDINGS: Ten male and 10 female subjects participated in each group (mean ± SD age 39.2 ± 11.6 years [range, 19-63 years] in group 1, 47.6 ± 11.9 years [27-64] in group 2; mean ± SD BMI 23.36 ± 2.45 in group 1, 24.56 ± 2.54 in group 2). One female subject in each group was withdrawn because of an adverse event (AE) (vomiting). All 20 subjects in each group were included in the safety analyses, and 19 subjects in each group were included in the pharmacokinetic analyses. The geometric least square means ratio (teneligliptin plus metformin/teneligliptin alone) for Cmax and the AUC0-τ for teneligliptin were 0.907 (90% CI, 0.853-0.965) and 1.042 (90% CI, 0.997-1.089), respectively. The geometric least square means ratio (metformin plus teneligliptin/metformin alone) for the Cmax and AUC0-τ for metformin were 1.057 (90% CI, 0.974-1.148) and 1.209 (90% CI, 1.143-1.278). The 90% CIs were within the prespecified threshold for equivalence (0.80-1.25), except for the AUC0-τ for metformin, which was increased by teneligliptin by 20% relative to metformin alone. In group 1, nine subjects experienced 25 AEs during treatment with teneligliptin alone and 10 subjects experienced 15 AEs during treatment with teneligliptin plus metformin. In group 2, eight subjects experienced 11 AEs during treatment with metformin alone and 11 subjects experienced 18 AEs during treatment with metformin plus teneligliptin. Two AEs in each treatment group were rated as severe. Results of in vitro experiments suggest that teneligliptin-mediated inhibition of organic cation transporter-2 does not increase metformin exposure. IMPLICATIONS: Coadministration of teneligliptin and metformin was well tolerated by these healthy subjects during the 8-day treatment period. Coadministration with metformin did not affect the pharmacokinetics of teneligliptin. Although coadministration with teneligliptin increased exposure to metformin, this change is unlikely to be clinically relevant. European Clinical Trials Database identifier: 2007-001511-29.

Slow R-warfarin 7-hydroxylation mediated by P450 2C19 genetic variants in cynomolgus monkeys in vivo.

Cynomolgus monkeys are widely used as non-human primate species in preclinical studies, due to their close evolutionary relationship to humans. Monkey cytochrome P450 2C19 (formerly known as P450 2C75), highly homologous to human P450 2C19, has been identified to be R-warfarin 7-hydroxylase in cynomolgus monkeys. In the present study, the in vivo pharmacokinetics of stereoselective warfarin and metabolites at a dose of 1.0mg/kg were investigated after oral and intravenous administration of racemic warfarin to fasted male cynomolgus monkeys (n=11, from Indochina, 4-8 years of age, 3.5-7.4kg of body weight), which had been genotyped for P450 2C19 [c.298TT>AA; c.308C>T; and c.334ATC>CTT]. Kinetic parameters for S-warfarin were not different among the homozygous mutant, heterozygous mutant, and wild type groups; however, values of elimination half-lives, area under the curves, and total body clearance of R-warfarin in the homozygous mutant group showed one-order differences from those values in the wild type group after oral or intravenous administration. R-Warfarin 7-hydroxylations in vivo in homozygous mutant groups were slow compared to wild type or heterozygous mutant groups. These results demonstrate that inter-animal variations of R-warfarin clearance in cynomolgus monkeys are associated with P450 2C19 genetic variants [p.Phe100Asn, p.Ala103Val, and p.Ile112Leu]. Because some interindividual variability of P450 2C-dependent drug metabolism in cynomolgus monkeys, similarly in humans, is accounted for by polymorphic P450 2C19 variants, genotyping of drug metabolism enzymes should be considered before and after P450-dependent drug metabolism testing and evaluations in cynomolgus monkeys.

Clinical application of a custom AmpliSeq library and ion torrent PGM sequencing to comprehensive mutation screening for deafness genes.

BACKGROUND: Congenital hearing loss is one of the most common sensory disorders, with 50-70% of cases attributable to genetic causes. Although recent advances in the identification of deafness genes have resulted in more accurate molecular diagnosis, leading to the better determination of suitable clinical interventions, difficulties remain with regard to clinical applications due to the extreme genetic heterogeneity of deafness. AIM: Toward more effective genetic testing, we adopted Massively Parallel DNA Sequencing (MPS) of target genes using an Ion PGM™ system and an Ion AmpliSeq™ panel to diagnose common mutations responsible for deafness and discover rare causative gene mutations. Before its clinical application, we investigated the accuracy of MPS-based genetic testing. RESULTS: We compared the results of Invader assay-based genetic screening, the accuracy of which has already been verified in previous studies, with those of MPS-based genetic testing for a large population of Japanese deafness patients and revealed that over 99.98% of the results were the same for each genetic testing system. CONCLUSION: The Ion Personal Genome Machine system had sufficient uniformity and accuracy for application to the clinical diagnosis of common causative mutations and efficiently identified rare causative mutations and/or mutation candidates.

Effect of ketoconazole on the pharmacokinetics of the dipeptidyl peptidase-4 inhibitor teneligliptin: an open-label study in healthy white subjects in Germany.

OBJECTIVE: The aim of this study was to examine the effect of ketoconazole, a potent cytochrome P450 (CYP) 3A4 and P-glycoprotein (P-gp) inhibitor, on teneligliptin pharmacokinetics and to evaluate the safety of combined administration of teneligliptin with ketoconazole. METHODS: This open-label, fixed-sequence study was conducted in 16 healthy adult volunteers in Germany. On day 1, under fasting conditions, 20 mg of teneligliptin was administered to evaluate the pharmacokinetics of teneligliptin alone. For 3 days (days 8-10), 400 mg of ketoconazole was administered once daily. On day 11, teneligliptin 20 mg and ketoconazole 400 mg were concurrently administered, and for 2 days (days 12 and 13), ketoconazole was administered once daily. The pharmacokinetic parameters (Cmax, Tmax, AUC, terminal t½, apparent total plasma clearance, and Vd during the terminal phase) of teneligliptin on days 1 and 11 were calculated. The safety profile was evaluated based on adverse events and clinical findings. To investigate the role of human P-gp in membrane permeation of teneligliptin, an in vitro study was performed to measure the transcellular transport of teneligliptin across monolayers of human P-gp-expressing cells and control cells. RESULTS: For Cmax and AUC, the geometric least squares mean ratios (90% CIs) of teneligliptin with ketoconazole to teneligliptin alone were 1.37 (1.25-1.50) and 1.49 (1.39-1.60), respectively. There was no change in t½ of the terminal elimination phase. In addition, the tolerability of teneligliptin coadministered with ketoconazole was acceptable. The in vitro study revealed corrected efflux ratios for teneligliptin of 6.81 and 5.27 at teneligliptin concentrations of 1 and 10 µM, respectively. CONCLUSIONS: Because the exposure to teneligliptin in combined administration with ketoconazole, a potent CYP3A4 and P-gp inhibitor, was less than twice that of administration of teneligliptin alone, it is suggested that combined administration of teneligliptin with drugs and foods that inhibit CYP3A4 should not cause a marked increase in exposure. The results of our in vitro study suggest that teneligliptin is a substrate of P-gp. CLINICAL TRIAL REGISTRATION: EudraCT No. 2009-016652-51.

Metabolism and disposition of the dipeptidyl peptidase IV inhibitor teneligliptin in humans.

1. The absorption, metabolism and excretion of teneligliptin were investigated in healthy male subjects after a single oral dose of 20 mg [(14)C]teneligliptin. 2. Total plasma radioactivity reached the peak concentration at 1.33 h after administration and thereafter disappeared in a biphasic manner. By 216 h after administration, ≥90% of the administered radioactivity was excreted, and the cumulative excretion in the urine and faeces was 45.4% and 46.5%, respectively. 3. The most abundant metabolite in plasma was a thiazolidine-1-oxide derivative (designated as M1), which accounted for 14.7% of the plasma AUC (area under the plasma concentration versus time curve) of the total radioactivity. The major components excreted in urine were teneligliptin and M1, accounting for 14.8% and 17.7% of the dose, respectively, by 120 h, whereas in faeces, teneligliptin was the major component (26.1% of the dose), followed by M1 (4.0%). 4. CYP3A4 and FMO3 are the major enzymes responsible for the metabolism of teneligliptin in humans. 5. This study indicates the involvement of renal excretion and multiple metabolic pathways in the elimination of teneligliptin from the human body. Teneligliptin is unlikely to cause conspicuous drug interactions or changes in its pharmacokinetics patients with renal or hepatic impairment, due to a balance in the elimination pathways.

Simultaneous genomic identification and profiling of a single cell using semiconductor-based next generation sequencing.

Combining single-cell methods and next-generation sequencing should provide a powerful means to understand single-cell biology and obviate the effects of sample heterogeneity. Here we report a single-cell identification method and seamless cancer gene profiling using semiconductor-based massively parallel sequencing. A549 cells (adenocarcinomic human alveolar basal epithelial cell line) were used as a model. Single-cell capture was performed using laser capture microdissection (LCM) with an Arcturus® XT system, and a captured single cell and a bulk population of A549 cells (≈ 10(6) cells) were subjected to whole genome amplification (WGA). For cell identification, a multiplex PCR method (AmpliSeq™ SNP HID panel) was used to enrich 136 highly discriminatory SNPs with a genotype concordance probability of 10(31-35). For cancer gene profiling, we used mutation profiling that was performed in parallel using a hotspot panel for 50 cancer-related genes. Sequencing was performed using a semiconductor-based bench top sequencer. The distribution of sequence reads for both HID and Cancer panel amplicons was consistent across these samples. For the bulk population of cells, the percentages of sequence covered at coverage of more than 100 × were 99.04% for the HID panel and 98.83% for the Cancer panel, while for the single cell percentages of sequence covered at coverage of more than 100 × were 55.93% for the HID panel and 65.96% for the Cancer panel. Partial amplification failure or randomly distributed non-amplified regions across samples from single cells during the WGA procedures or random allele drop out probably caused these differences. However, comparative analyses showed that this method successfully discriminated a single A549 cancer cell from a bulk population of A549 cells. Thus, our approach provides a powerful means to overcome tumor sample heterogeneity when searching for somatic mutations.

Draft Genome Sequence of Pediococcus lolii NGRI 0510Q(T) Isolated from Ryegrass Silage.

Pediococcus lolii NGRI 0510Q(T) was isolated from ryegrass silage produced on Ishigaki Island, Okinawa Prefecture, Japan. Here we present a draft genome sequence for this strain, consisting of 103 contigs for a total of 2,047,078 bp, 2,154 predicted coding sequences, and a G+C content of 42.1%.

Discovery and preclinical profile of teneligliptin (3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl]thiazolidine): a highly potent, selective, long-lasting and orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes.

Dipeptidyl peptidase IV (DPP-4) inhibition is suitable mechanism for once daily oral dosing regimen because of its low risk of hypoglycemia. We explored linked bicyclic heteroarylpiperazines substituted at the γ-position of the proline structure in the course of the investigation of l-prolylthiazolidines. The efforts led to the discovery of a highly potent, selective, long-lasting and orally active DPP-4 inhibitor, 3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl]thiazolidine (8 g), which has a unique structure characterized by five consecutive rings. An X-ray co-crystal structure of 8 g in DPP-4 demonstrated that the key interaction between the phenyl ring on the pyrazole and the S(2) extensive subsite of DPP-4 not only boosted potency, but also increased selectivity. Compound 8 g, at 0.03 mg/kg or higher doses, significantly inhibited the increase of plasma glucose levels after an oral glucose load in Zucker fatty rats. Compound 8 g (teneligliptin) has been approved for the treatment of type 2 diabetes in Japan.

Fused bicyclic heteroarylpiperazine-substituted L-prolylthiazolidines as highly potent DPP-4 inhibitors lacking the electrophilic nitrile group.

Hypoglycemic agents with a mechanism of depeptidyl peptidase IV (DPP-4) inhibition are suitable for once daily oral dosing. It is difficult to strike a balance between inhibitory activity and duration of action in plasma for inhibitors bearing an electrophilic nitrile group. We explored fused bicyclic heteroarylpiperazine substituted at the γ-position of the proline structure in the investigation of L-prolylthiazolidines lacking the electrophilic nitrile. Among them, 2-trifluoroquinolyl compound 8g is the most potent, long-lasting DPP-4 inhibitor (IC(50) = 0.37 nmol/L) with high selectivity against other related peptidases. X-ray crystal structure determination of 8g indicates that CH-π interactions generated between the quinolyl ring and the guanidinyl group of Arg358 enhances the DPP-4 inhibitory activity and selectivity.

[(S)-gamma-(4-Aryl-1-piperazinyl)-l-prolyl]thiazolidines as a novel series of highly potent and long-lasting DPP-IV inhibitors.

In the search for an inhibitor of dipeptidyl peptidase IV (DPP-IV) highly potent both in vitro and in vivo, we synthesized a series of L-prolylthiazolidine-based DPP-IV inhibitors having 4-arylpiperazine or 4-arylpiperidine at the gamma-position of the proline structure. Of these compounds, the 4-(5-nitro-2-pyridyl)piperazine analog 21e showed a sub-nanomolar (IC(50)=0.92 nmol/L) DPP-IV inhibitory activity and a long-lasting in vivo DPP-IV inhibition profile.

Lead optimization of [(S)-gamma-(arylamino)prolyl]thiazolidine focused on gamma-substituent: Indoline compounds as potent DPP-IV inhibitors.

Dipeptidyl peptidase IV (DPP-IV) inhibitors are looked to as a potential new antidiabetic agent class. A series of [(S)-gamma-(arylamino)prolyl]thiazolidine compounds in which the electrophilic nitrile is removed are chemically stable DPP-IV inhibitors. To discover a structure for the gamma-substituent of the proline moiety more suitable for interacting with the S(2) pocket of DPP-IV, optimization focused on the gamma-substituent was carried out. The indoline compound 22e showed a DPP-IV-inhibitory activity 100-fold more potent than that of the prolylthiazolidine 10 and comparable to that of NVP-DPP728. It also displayed improved inhibitory selectivity for DPP-IV over DPP8 and DPP9 compared to compound 10. Indoline compounds such as 22e have a rigid conformation with double restriction of the aromatic moiety by proline and indoline structures to promote interaction with the binding site in the S(2) pocket of DPP-IV. The double restriction effect provides a potent inhibitory activity which compensates for the decrease in activity caused by removing the electrophilic nitrile.