Multinuclear NMR Study on the Formation and Polyol-Induced Deformation Mechanisms of Wormlike Micelles Composed of Cetyltrimethylammonium Bromide and 3-Fluorophenylboronic Acid.

We had previously confirmed a glucose-responsive decrease in the viscosity of cetyltrimethylammonium bromide (CTAB) and phenylboronic acid (PBA) wormlike micelle (WLM) systems. However, the mechanisms of the formation of WLMs and the decrease in viscosity with glucose addition have not been determined. In this study, we elucidated the mechanisms using 3-fluorophenylboronic acid (3FPBA) based on 11B NMR and 19F NMR analyses. The system in 60 mM CTAB/60 mM 3FPBA at pH 7.4 demonstrated high viscoelasticity, and the formation of WLMs in the system was confirmed by rheological characteristics. The 11B NMR spectrum at pH 7.4 revealed that 3FPBA existed in a neutral form with sp2-hybridized boron; however, the 11B signal disappeared in the presence of CTAB. In contrast, 19F NMR studies indicated that the quaternary ammonium ion of CTAB interacts with the phenyl group of 3FPBA in the sp2 form via cation-π interactions. PBA derivatives react with various polyols; thus, we investigated the change in the viscous system after the addition of sugar and sugar alcohols. The viscosity of the WLMs decreased with increased polyol concentration, especially those of fructose and mannitol, in which the decrease was apparent at 40-160 mM polyols. The 19F NMR spectra revealed that polyol addition induced decrease in the sp2 form of 3FPBA and increase in the sp3 form of 3FPBA. Based on the results, we propose the following mechanism of the polyol response: (1) The WLMs are stabilized by CTAB and 3FPBA in the sp2 form using cation-π interactions as the driving force. (2) When polyol is added to the system, the sp2 form of 3FPBA decreases and its sp3 form increases. (3) This change means that the structural component of WLMs decreases, which induces the disruption of WLMs, and the viscosity decreases. The formation and deformation mechanisms of the WLMs determined in this study are notable because 3FPBA interacts as a neutral compound, whereas CTAB often interacts with anionic aromatic compounds to form WLMs. Without 19F NMR measurements, these mechanisms would not have been discovered.

Glucose Responsive Rheological Change and Drug Release from a Novel Worm-like Micelle Gel Formed in Cetyltrimethylammonium Bromide/Phenylboronic Acid/Water System.

A novel glucose (Glc)-responsive gel formed by worm-like micelles (WLMs) has the potential to provide a self-regulating insulin delivery system. We have prepared a WLM gel system using 75 mM cetyltrimethylammonium bromide, 75 mM phenylboronic acid, and water. At pH 9.4, this gel-like system was highly viscous and supported its own weight, and dynamic viscoelasticity measurement indicated that it contained long and entangled WLMs. The visual observation of gels prepared to include >6 mM Glc revealed that these adopted a sol-like appearance, whereas those prepared to include a control compound (2-10 mM diethylene glycol) retained their gel-like appearance. The storage modulus ( G') of this system decreased as the Glc concentration increased (2-10 mM), indicating a gradual shortening of the WLMs. In vitro release was evaluated using a test compound (fluorescein isothiocyanate dextran) in a microsized flow system. By 120 min, the release of this compound from the WLM gel was around 27-fold greater in the presence of 100 mM Glc than without Glc or with 100 mM diethylene glycol. This demonstrated the successful preparation of a WLM gel that showed an altered drug release rate, depending on Glc concentration.

In vitro metabolism of alectinib, a novel potent ALK inhibitor, in human: contribution of CYP3A enzymes.

1. The in vitro metabolism of alectinib, a potent and highly selective oral anaplastic lymphoma kinase inhibitor, was investigated. 2. The main metabolite (M4) in primary human hepatocytes was identified, which is produced by deethylation at the morpholine ring. Three minor metabolites (M6, M1a, and M1b) were also identified, and a minor peak of hydroxylated alectinib (M5) was detected as a possible precursor of M4, M1a, and M1b. 3. M4, an important active major metabolite, was produced and further metabolized to M6 by CYP3A, indicating that CYP3A enzymes were the principal contributors to this route. M5 is possibly produced by CYP3A and other isoforms as the primary step in metabolism, followed by oxidation to M4 mainly by CYP3A. Alternatively, M5 could be oxidized to M1a and M1b via an NAD-dependent process. None of the non-CYP3A-mediated metabolism appeared to be major. 4. In conclusion, this study suggests that involvement of multiple enzymes in the metabolism of alectinib reduces its potential for drug-drug interactions.

Metabolites of alectinib in human: their identification and pharmacological activity.

Two metabolites (M4 and M1b) in plasma and four metabolites (M4, M6, M1a and M1b) in faeces were detected through the human ADME study following a single oral administration of [14C]alectinib, a small-molecule anaplastic lymphoma kinase inhibitor, to healthy subjects. In the present study, M1a and M1b, which chemical structures had not been identified prior to the human ADME study, were identified as isomers of a carboxylate metabolite oxidatively cleaved at the morpholine ring. In faeces, M4 and M1b were the main metabolites, which shows that the biotransformation to M4 and M1b represents two main metabolic pathways for alectinib. In plasma, M4 was a major metabolite and M1b was a minor metabolite. The contribution to in vivo pharmacological activity of these circulating metabolites was assessed from their in vitro pharmacological activity and plasma protein binding. M4 had a similar cancer cell growth inhibitory activity and plasma protein binding to that of alectinib, suggesting its contribution to the antitumor activity of alectinib, whereas the pharmacological activity of M1b was insignificant.

Absorption, distribution, metabolism and excretion (ADME) of the ALK inhibitor alectinib: results from an absolute bioavailability and mass balance study in healthy subjects.

1. Alectinib is a highly selective, central nervous system-active small molecule anaplastic lymphoma kinase inhibitor. 2. The absolute bioavailability, metabolism, excretion and pharmacokinetics of alectinib were studied in a two-period single-sequence crossover study. A 50 µg radiolabelled intravenous microdose of alectinib was co-administered with a single 600 mg oral dose of alectinib in the first period, and a single 600 mg/67 µCi oral dose of radiolabelled alectinib was administered in the second period to six healthy male subjects. 3. The absolute bioavailability of alectinib was moderate at 36.9%. Geometric mean clearance was 34.5 L/h, volume of distribution was 475 L and the hepatic extraction ratio was low (0.14). 4. Near-complete recovery of administered radioactivity was achieved within 168 h post-dose (98.2%) with excretion predominantly in faeces (97.8%) and negligible excretion in urine (0.456%). Alectinib and its major active metabolite, M4, were the main components in plasma, accounting for 76% of total plasma radioactivity. In faeces, 84% of dose was excreted as unchanged alectinib with metabolites M4, M1a/b and M6 contributing to 5.8%, 7.2% and 0.2% of dose, respectively. 5. This novel study design characterised the full absorption, distribution, metabolism and excretion properties in each subject, providing insight into alectinib absorption and disposition in humans.

In vitro profiling of the metabolism and drug-drug interaction of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, using human liver microsomes, human hepatocytes, and recombinant human CYP.

Abstract 1. The metabolism and drug-drug interaction (DDI) risk of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, were evaluated by in vitro studies using human liver microsomes, human hepatocytes, and recombinant human CYPs. 2. The main metabolite of tofogliflozin was the carboxylated derivative (M1) in human hepatocytes, which was the same as in vivo. The metabolic pathway of tofogliflozin to M1 was considered to be as follows: first, tofogliflozin was catalyzed to the primary hydroxylated derivative (M4) by CYP2C18, CYP4A11 and CYP4F3B, then M4 was oxidized to M1. 3. Tofogliflozin had no induction potential on CYP1A2 and CYP3A4. Neither tofogliflozin nor M1 had inhibition potential on CYPs, with the exception of a weak CYP2C19 inhibition by M1. 4. Not only are multiple metabolic enzymes involved in the tofogliflozin metabolism, but the drug is also excreted into urine after oral administration, indicating that tofogliflozin is eliminated through multiple pathways. Thus, the exposure of tofogliflozin would not be significantly altered by DDI caused by any co-administered drugs. Also, tofogliflozin seems not to cause significant DDI of co-administered drugs because tofogliflozin has no CYP induction or inhibition potency, and the main metabolite M1 has no clinically relevant CYP inhibition potency.

Metabolism and mass balance of SGLT2 inhibitor tofogliflozin following oral administration to humans.

1. Tofogliflozin is a novel and selective SGLT2 inhibitor increasing glucosuria by inhibition of glucose re-absorption in the kidney for the treatment of type 2 diabetes mellitus. 2. In this study, the metabolism and the mass balance of tofogliflozin was evaluated following administration of a single oral dose of 20 mg [(14)C]-tofogliflozin to six healthy subjects. 3. Tofogliflozin underwent mainly oxidative metabolism in the ethylphenyl moiety, but also minor glucuronide conjugates of metabolites and the parent drug were formed. 4. In plasma, the parent drug and its major phenyl acetic acid metabolite M1 accounted for 42% and 52% of the total drug-related material, respectively. The hydroxyl metabolites and their successor ketone metabolite showed an exposure well below 5%, along with an acyl glucuronide of M1. 5. Tofogliflozin was completely absorbed with subsequent predominate metabolic clearance and a small contribution of direct urinary elimination. Approximately, 76% of the dose was excreted in urine and 20% in faeces within 72 h. The high absorption of tofogliflozin was exemplified by the small trace of parent drug in faeces. The phenyl acetic acid metabolite M1 was the major component excreted in urine and faeces accounting for more than half of the dose. Tofogliflozin demonstrated a high metabolic turnover.

A novel double-tracer technique to characterize absorption, distribution, metabolism and excretion (ADME) of [14C]tofogliflozin after oral administration and concomitant intravenous microdose administration of [13C]tofogliflozin in humans.

BACKGROUND: Human mass balance studies and the assessment of absolute oral bioavailability (F) are usually assessed in separate studies. Intravenous microdose administration of an isotope tracer concomitant to an unlabeled oral dose is an emerging technique to assess F. We report a novel double-tracer approach implemented for tofogliflozin combining oral administration of a radiolabel tracer with concomitant intravenous administration of a stable isotope tracer. Tofogliflozin is a potent and selective sodium/glucose cotransporter 2 inhibitor for the treatment of type 2 diabetes mellitus currently in clinical development. OBJECTIVES: The objectives of the present study were to assess the systemic exposure of major circulating metabolites, excretion balance, F and contribution of renal clearance (CLR) to total clearance (CL) of tofogliflozin in healthy subjects within one study applying a novel double-tracer technique. METHODS: Six healthy male subjects received 20 mg [(12)C/(14)C]tofogliflozin (3.73 MBq) orally and a concomitant microdose of 0.1 mg [(13)C]tofogliflozin intravenously. Pharmacokinetics of tofogliflozin were determined for the oral and intravenous route; the pharmacokinetics of the metabolites M1 and M5 were determined for the oral route. Quantification of [(12)C]tofogliflozin in plasma and urine and [(13)C]tofogliflozin in plasma was performed by selective LC-MS/MS methods. For the pre-selected metabolites of tofogliflozin, M1 and M5, a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied to plasma and urine samples. Total radioactivity was assessed in plasma, urine and feces. Pharmacokinetic analysis was conducted by non-compartmental methods. RESULTS: The pharmacokinetics of tofogliflozin in healthy subjects were characterized by an F of 97.5 ± 12.3 %, CL of 10.0 ± 1.3 l/h and volume of distribution at steady-state (V(ss)) of 50.6 ± 6.7 l. The main route of elimination of total drug-related material was by excretion into urine (77.0 ± 4.1 % of the dose). The observed CL(R) of 25.7 ± 5.0 ml/min was higher than the product of the estimated glomerular filtration rate (eGFR) and fraction unbound in plasma (f(u)) (eGFR × f(u) 15 ml/min), indicating the presence of net active tubular secretion in the renal elimination of tofogliflozin. However, CLR contributed only 15.5 % to the CL of tofogliflozin, suggesting that reductions in CLR by renal impairment won't significantly affect systemic exposure to tofogliflozin. Tofogliflozin and its metabolite M1 were the only major circulating entities accounting for 46 ± 8.6 and 50 ± 8.2 %, respectively, of total circulating drug-related material, while the metabolite M5 was a minor circulating metabolite accounting for 3.0 ± 0.3 % of total circulating drug-related material. Both the M1 and M5 metabolites were excreted into urine and the major metabolite M1 did not exhibit active tubular secretion. CONCLUSIONS: These results demonstrate the utility of the double-tracer approach to provide essential pharmacokinetic data and excretion data for drug-related material in one study at the same dosing occasion. The data obtained allowed the characterization of absorption, distribution, metabolism and excretion of tofogliflozin. Tofogliflozin exhibited highly favorable pharmacokinetic properties as demonstrated by its high F, low CL and a low V(ss. The presence of only one major circulating metabolite of tofogliflozin was unambiguously demonstrated. As a drug targeting the kidney, luminal exposure of the kidney is achieved by renal filtration and active tubular secretion.

Perspectives on non-clinical safety evaluation of drug metabolites through the JSOT workshop.

The prompt and appropriate safety assessment of drug metabolite(s) was mentioned in regulatory guidances such as an International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance, entitled "Guidance on Non-clinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals" (ICH M3(R2)) implemented in January 1 of 2011 in Japan, and has become a significant issue in the drug development. Upon release of ICH M3(R2) Step 4, a survey was conducted between March and April 2010 on the safety assessment of drug metabolites in 63 member companies of the Japan Pharmaceutical Manufacturers Association (JPMA). The Pharmacokinetics Team in the Non-Clinical Evaluation Expert Committee in JPMA conducted a questionnaire survey and compiled the results to comprehend how safety of drug metabolites are currently assessed at research-based pharmaceutical companies in Japan. The assessment of "Metabolites in Safety Testing" (MIST) can be divided into three stages based on the research purpose as follows: MIST 1 is a stage of estimating human drug metabolites and predicting their potential risks, MIST 2 is a stage of deciding the necessity for non-clinical safety studies, and MIST 3 is a stage of conducting non-clinical safety studies. In this paper, we propose typical approaches on safety assessment of metabolites that meet the purpose of each stage, considering the current level of scientific technology. Our proposals are based on the results from our survey and a symposium about the safety assessment of drug metabolites at the 37th annual meeting of the Japanese Society of Toxicology held in June 2010.

Differentiation of organ availability by sequential and simultaneous analyses: intestinal conjugative metabolism impacts on intestinal availability in humans.

The impact of intestinal conjugative metabolism on oral bioavailability was assessed by sequential and simultaneous analyses of the reported data in humans. The data were retrieved from reports on drugs that are metabolized by sulfate conjugation, and the organ availabilities affecting oral bioavailability were differentiated. Sequential analysis gave the following results. The intestinal availability (Fg) of salbutamol was 0.700, whereas hepatic availability (Fh) and bioavailability (F) were 0.893 and 0.493, respectively. Fg of (+)-terbutaline, (-)-terbutaline, and (+/-)-terbutaline was 0.128, 0.254, and 0.250, respectively. In contrast, Fh of (+)-terbutaline, (-)-terbutaline, and (+/-)-terbutaline was 0.979, 0.971, and 0.946, respectively. Fg and Fh of ethynylestradiol were 0.536 and 0.780, respectively. Simultaneous analysis also gave similar results, although the sequential analysis overestimated the intestinal availability. These results indicate that intestinal sulfation metabolism has more impact on intestinal availability than on hepatic availability, resulting in low bioavailability in humans.