Identification of novel metabolites of abiraterone in human serum and their metabolic pathways.

Two novel abiraterone (Abi, 3ß-OH-Abi) metabolites in human serum were identified as 3α-OH-Abi and Δ5-Abi (D5A). Both metabolites were confirmed by their retention times on LC/MS and their product-ion mass spectra on LC-MS/MS compared to those of authentic compounds, which were chemically synthesized. The plausible metabolic pathways of these two metabolites are as follows: Abi is first oxidized to D5A by 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and then irreversibly converted to Δ4-Abi (D4A) by ∆5-∆4 isomerase. Presumably, D5A detection is difficult because of its rapid conversion to D4A and its low concentration in serum samples. In contrast, the low concentration 3α-OH-Abi was generated by reducing the remaining D5A using 3α-hydroxysteroid dehydrogenase (3α-HSD).

A Phase II Trial of Abiraterone With Dutasteride for Second-Generation Antiandrogen- and Chemotherapy-Naïve Patients With Castration-Resistant Prostate Cancer.

The development of a novel therapy to overcome primary and acquired resistance to abiraterone is an unmet need. This study aimed to evaluate the efficacy and safety of adding 5α-reductase inhibitor dutasteride to abiraterone, explore proof of concept, and identify candidates suitable for combination therapy. This phase II, single-arm, and open-label study enrolled second-generation antiandrogen- and chemotherapy-naïve patients with castration-resistant prostate cancer. Patients received abiraterone and prednisolone for 4 weeks, followed by adding dutasteride. The primary end point was a 50% prostate-specific antigen response rate. Serum concentrations of abiraterone and its metabolites as well as HSD3B1 and SRD5A2 genotypes were measured. The association between drug metabolism and genotypes and their impact on the efficacy of combination therapy were assessed. Among 21 patients, 18 (85.7%) achieved ≥50% PSA reduction. Median time to treatment failure was not reached during the median follow-up of 15.4 months. No patients experienced grade ≥3 adverse events. Although dutasteride reduced serum 3-keto-5α-abiraterone concentrations, higher serum 3-keto-5α-abiraterone concentrations on combination therapy were associated with a shorter time to treatment failure. HSD3B1 and SRD5A2 genotypes were associated with serum Δ4-abiraterone and 3-keto-5α-abiraterone concentrations before adding dutasteride, respectively. Time to treatment failure was longer in patients with homozygous wild-type HSD3B1, but comparable between those with the SRD5A2 genotype. The promising outcomes of this study warrant further investigation of combination therapy in a randomized trial. Stratification by HSD3B1 and SRD5A2 genetic profiles might identify patients suitable for combination therapy.

Progress in Chiral Stationary Phases Based on Proteins and Glycoproteins.

A lot of chiral stationary phases (CSPs) have been introduced for the purpose of analytical and preparative separations of enantiomers. CSPs based on proteins and glycoproteins have unique properties among those CSPs. This review article deals with the preparation of CSPs based on proteins and glycoproteins, their chiral recognition properties and mechanisms, focusing on the CSPs investigated in our group. The dealt proteins and glycoproteins are including bovine serum albumin, human serum albumin, lysozyme, pepsin, human α1-acid glycoprotein (AGP), chicken ovomucoid and chicken ovoglycoprotein (named chicken AGP).

Novel bone microenvironment model of castration-resistant prostate cancer with chitosan fiber matrix and osteoblasts.

The interaction between prostate cancer cells and osteoblasts is essential for the development of bone metastasis. Previously, novel androgen receptor axis-targeted agents (ARATs) were approved for metastatic castration-naïve and non-metastatic castration-resistant prostate cancer (CRPC); both of which are pivotal for investigating the association between the bone microenvironment and tumors. The present study established a novel in vitro 3D microenvironment model that simulated the bone microenvironment of CRPC, and evaluated the drug susceptibility of ARATs and the efficacy of the combination of abiraterone and dutasteride. Green fluorescent protein-transferred C4-2 cells (a CRPC cell line) and red fluorescent protein-transferred human osteoblasts differentiated from human mesenchymal stem cells were co-cultured in chitosan nanofiber matrix-coated culture plates to simulate the 3D scaffold of the bone microenvironment. The growth of C4-2 was quantified using live-cell imaging and the Cell3 iMager duos analysis system. The growth of C4-2 colonies were quantified for a maximum of 30 days. The expression of TGF-ß increased and promoted EMT in C4-2 cells co-cultured with osteoblasts, indicating resistance to ARATs. The IC50 of each drug and the combination effect of abiraterone and dutasteride were evaluated using this model. Combination treatment with abiraterone and dutasteride synergistically inhibited the growth of C2-4 colonies compared with individual investigational agents. This could be attributed to the reduction of 3-keto-5α-abiraterone, an androgen receptor agonist. The bone microenvironment model of the present study is unique and useful for evaluating new drug susceptibility testing in prostate cancer cells. This model may help to reveal the unknown mechanisms underlying micro- to clinical bone metastasis in prostate cancer.

Molecularly imprinted polymers for arbutin and rutin by modified precipitation polymerization and their application for selective extraction of rutin in nutritional supplements.

Molecularly imprinted polymers (MIPs) for glycosides, arbutin (ARB) and rutin (RUT), were prepared using methacrylamide (MAM) and 4-vinylpyridine (4-VPY) as functional monomers and divinylbenzene as a crosslinker by modified precipitation polymerization. The template molecule, ARB or RUT, was first dissolved in methanol, followed by precipitation polymerization using a mixture of acetonitrile and toluene as a porogenic solvent. The molar ratios of the template molecule, MAM and 4-VPY were optimized to achieve a high molecular recognition ability for ARB and RUT. The retention and molecular recognition properties of these MIPs were evaluated in HILIC or normal-phase mode. With an increase in the acetonitrile content in the mobile phase, the retention factor of ARB or RUT was increased. Furthermore, the MIPs for ARB and RUT showed the highest imprinting factors of 3.65 and 66.5 for the template molecules, respectively. Hydrogen bonding interactions such as N⋯H-O, C=O⋯H-O and NH⋯O-H between 4-VPY or MAM and hydroxy groups of d-glucose or d-rutinose could function in the recognition of a glycone. Furthermore, hydrogen bonding interactions between functional monomers and the hydroxy group(s) of hydroquinone or quercetin could function in the recognition of an aglycone. These results suggest that the MIPs could recognize both a glycone and aglycone via hydrogen bonding interactions. Furthermore, MIPs for RUT were successfully applied to extract RUT in nutritional supplements.

Synthesis of Molecularly Imprinted Polymers by Two-Step Swelling and Polymerization.

Synthesis of a molecularly imprinted polymer (MIP) by two-step swelling and polymerization is described. Monodisperse, spherical MIP particles, whose diameters are ca. 5-9µm, are prepared using a polystyrene particle as a shape template and dibutyl phthalate as an activating solvent. The obtained MIPs are suitable for separation media in liquid chromatography or solid-phase extraction media. Procedures for synthesis of MIPs and restricted access media (RAM)-MIP, packing of MIPs and RAM-MIPs, and application of MIPs and RAM-MIPs for selective separation and extraction of a target compound(s) are described.

Retention and molecular-recognition mechanisms of molecularly imprinted polymers for warfarin derivatives and their application for the determination of warfarin in human serum.

Monodisperse molecularly imprinted polymers (MIPs) for warfarin (WF), 4'-chlorowarfarin (CWF), 4'-bromowarfarin (BWF), 4'-nitrowarfarin (NWF) and 4'-methylwarfarin (MWF) (MIPWF, MIPCWF, MIPBWF, MIPNWF and MIPMWF, respectively) were prepared using 4-vinylpyridine (4-VPY) and ethylene glycol dimethacrylate as a functional monomer and crosslinker, respectively, by multi-step swelling and polymerization. The retention and molecular-recognition properties of those MIPs were evaluated in HILIC, and reversed- and normal-phase modes. According to 1H NMR studies, one-to-three complex formation of one WF or CWF molecule with three 4-VPY molecules occurred. Via computational approaches, the intermolecular interaction modes and energies between WF derivatives and 4-VPYs were evaluated by semi-empirical quantum chemistry methods and density functional theory calculations. Three major possible hydrogen bonding interaction modes were identified: the interactions between the 4-hydroxy group, α-proton (methylene C-H) and α-proton (methyl C-H) of the WF derivative and the nitrogen atoms of 4-VPYs. In HILIC and normal-phase modes, the interaction energies showed satisfactory correlations with the retention factors of the WF derivatives. In reversed-phase mode, the retention factors of the WF derivatives were described by the hydrophobicity and the acidity of the 4-hydroxy groups of the WF derivatives. These results demonstrate that three hydrogen bonding interactions in HILIC and normal-phase modes, and hydrogen bonding or ionic interactions and hydrophobic interactions in reversed-phase mode play important roles in the retention and molecular-recognition of the WF derivatives on MIPs. Furthermore, MIPBWF was successfully applied to the determination of WF in human serum by column-switching LC with high accuracy, precision and selectivity and without template-leakage problems.

Determination of Abiraterone and Its Metabolites in Human Serum by LC-ESI-TOF/MS Using Solid-phase Extraction.

We developed and validated a liquid chromatography-electrospray ionization-time of flight/mass spectrometry method for the determination of abiraterone (Abi) and its metabolites (Δ4-Abi, 3-keto-5α-Abi, 3α-OH-5α-Abi and 3ß-OH-5α-Abi) in human serum using Abi-d4 as the internal standard. As a pretreatment procedure of serum samples, solid-phase extraction based on a silica-gel cartridge was used. The relative recovery of Abi and its metabolites was over the ranges of 84.5 - 109.2% at a concentration of 6.0 ng mL-1 for Abi and 0.6 ng mL-1 for its metabolites. The method was free from matrix effects. The calibration curve of Abi was linear over the range of 2.0 - 400 ng mL-1 and those of its metabolites over the ranges 0.2 - 40 ng mL-1. The results of the intra- and inter-day accuracy and precision data were within the FDA acceptance criteria. The optimized method was applied for the determination of Abi and its metabolites in human serum after oral administration of Abi acetate.

Enantioseparation of warfarin derivatives on molecularly imprinted polymers for (S)- and (R)-chlorowarfarin.

Monodisperse molecularly imprinted polymers (MIPs) for warfarin (WF), 4'-chlorowarfarin (CWF), (S)-CWF and (R)-CWF (MIPWF, MIPCWF, MIP(S)-CWF and MIP(R)-CWF, respectively) were prepared using 4-vinylpyridine (4-VPY) and ethylene glycol dimethacrylate (EDMA) as a functional monomer and a crosslinker, respectively, by multi-step swelling and polymerization. The molar ratio of a template molecule, 4-VPY to EDMA was 6:18:25 or 4:18:25. The retention and molecular recognition properties of MIPWF and MIPCWF were evaluated using a mixture of sodium phosphate buffer or ammonium formate and acetonitrile in reversed-phase LC. WF and CWF on these MIPs gave the maximal retentions at mobile phase pH 7, and those retentions were decreased with an increase of acetonitrile content. The retention and imprinting factors were in the order of WF < CWF < 4'-bromowarfarin (BWF) on MIPWF and MIPCWF in neutral mobile phases. On the other hands, in acidic mobile phases the retention factors were in the same order with those in neutral mobile phases, while the imprinting factors of WF and CWF were higher on the respective MIPs. These results suggest that ionic or hydrogen bonding interactions, hydrophobic interactions and π-π interactions could work for the retention and molecular recognition of WF, CWF and BWF on these MIPs in a reversed-phase mode. Furthermore, MIP(S)-CWF and MIP(R)-CWF could separate WF, CWF and BWF enantiomers in acidic mobile phases.

Retention and molecular-recognition mechanisms of molecularly imprinted polymers for promazine derivatives.

Monodisperse molecularly imprinted polymers (MIPs) for promazine derivatives [promazine (PZ), methylpromazine (MPZ), chlorpromazine (CPZ) and bromopromazine (BPZ)], MIPPZ, MIPMPZ, MIPCPZ and MIPBPZ, were prepared using methacrylic acid (MAA) as a functional monomer and ethylene glycol dimethacrylate as a crosslinker by multi-step swelling and polymerization. The retention and molecular-recognition properties of the obtained MIPs were evaluated using LC in hydrophilic interaction chromatography (HILIC) and reversed-phase modes. In computational approaches, intermolecular interaction modes and energies between PZ derivatives and MAAs were evaluated at the HF/6-311G(d,p) level. The interaction energies of PZ, MPZ, CPZ and BPZ with 4 equivalents of MAAs were calculated. The results indicated that the interaction of the aliphatic amine moiety of a PZ derivative with MAA gave almost similar interaction energies at the HF/6-311G(d,p) level, and that the interaction of the sulfur atom of a phenothiazine scaffold with MAA was also the case. The third interaction of the aromatic amine of a PZ derivative with MAA was in the order of MPZ > PZ > CPZ > BPZ presumably due to the change of basicity by the electron-donating or electron-withdrawing effect of a subsituent. Furthermore, the fourth attractive modes of CPZ and BPZ were suggested to be the interaction of their halogen atoms with MAA through both halogen bonding and hydrogen bonding, while PZ and MPZ were suggested to have the weak C-H ⋅⋅⋅ π interaction with MAA. In HILIC mode, the interaction energies at the HF method had good correlation with the retention factor of a PZ derivative on each MIP, indicating that in addition to the shape recognition, the attractive electrostatic interactions would be more responsible for its retention rather than the dispersion energies. Furthermore, in addition to the shape recognition, ionic and hydrophobic interactions, and halogen bonding and hydrogen bonding (the last interaction seems to be weak) seem to work for the retention and molecular-recognition of PZ derivatives on the MIPs in reversed-phase mode.

Evaluation of molecularly imprinted polymers for chlorpromazine and bromopromazine prepared by multi-step swelling and polymerization method-The application for the determination of chlorpromazine and its metabolites in rat plasma by column-switching LC.

Monodisperse molecularly imprinted polymers (MIPs) for chlorpromazine (CPZ) and bromopromazine (BPZ), MIPCPZ and MIPBPZ, were prepared using methacrylic acid as a functional monomer and ethylene glycol dimethacrylate as a crosslinker by multi-step swelling and polymerization. The retention and molecular-recognition properties of MIPCPZ and MIPBPZ were evaluated using a mixture of potassium phosphate buffer and acetonitrile or a mixture of water and acetonitrile including ammonium formate as a mobile phase in reversed-phase LC. On MIPBPZ, CPZ, BPZ and imipramine (IMP) gave the maximal retention factors at a mobile-phase pH 8, while the maximal imprinting factors were obtained at a mobile-phase pH 7. Each MIP recognized a template molecule the most, while CPZ metabolites, desmethyl CPZ (DM-CPZ), CPZ sulfoxide (CPZ-SO) and 7-hydroxy CPZ (7-OH-CPZ), were moderately recognized on MIPCPZ and MIPBPZ. Furthermore, both MIPs gave the similar retention and molecular-recognition for CPZ and its metabolites. For avoiding the template-leakage problems, MIPBPZ was used as the pretreatment column for the determination of CPZ and its metabolites in rat plasma in column-switching LC with UV detection. In addition to DM-CPZ and CPZ-SO, didesmethyl CPZ (DDM-CPZ) and CPZ N-oxide (CPZ-NO) were speculated as the metabolite in rat plasma after administration of CPZ using LC-ESI-TOF-MS, while 7-OH-CPZ was not detected. The column-switching LC method was validated and applied for the determination of CPZ and its metabolites, DM-CPZ, DDM-CPZ, CPZ-SO and CPZ-NO, in rat plasma after intravenous and oral administration of CPZ using IMP as an internal standard.

Preparation and Evaluation of Molecularly Imprinted Polymers for Promazine and Chlorpromazine by Multi-step Swelling and Polymerization: the Application for the Determination of Promazine in Rat Serum by Column-switching LC.

Molecularly imprinted polymers (MIPs) for promazine (PZ) and chlorpromazine (CPZ), MIPPZ and MIPCPZ, were prepared by multi-step swelling and polymerization using methacrylic acid as a functional monomer and ethylene glycol dimethacrylate as a crosslinker. The retention and molecular-recognition properties of MIPPZ and MIPCPZ were evaluated using a mixture of potassium phosphate buffer and acetonitrile, or a mixture of ammonium formate and acetonitrile as the mobile phase in LC. PZ and CPZ gave the maximal retentions on MIPPZ and MIPCPZ at an apparent pH 8.2 using a mixture of potassium phosphate buffer and acetonitrile as the mobile phase. The retentions of PZ and CPZ decreased with an increase of acetonitrile contents from 70 to 90 vol% using a mixture of ammonium formate and acetonitrile as the mobile phase. The template molecules (PZ and CPZ, respectively) were recognized the most on the respective MIPs, and the imprinting factor of PZ was higher on MIPCPZ than on MIPPZ. These results indicate that in addition to shape recognition, ionic and hydrophobic interactions seem to work for the retention and molecular-recognition of PZ and CPZ on the MIPs. MIPCPZ was successfully utilized for the selective extraction of PZ in rat-serum samples in column-switching LC with fluorescence detection.

Pharmacogenetics-based area-under-curve model can predict efficacy and adverse events from axitinib in individual patients with advanced renal cell carcinoma.

We investigated the relationship between axitinib pharmacogenetics and clinical efficacy/adverse events in advanced renal cell carcinoma (RCC) and established a model to predict clinical efficacy and adverse events using pharmacokinetic and gene polymorphisms related to drug metabolism and efflux in a phase II trial. We prospectively evaluated the area under the plasma concentration-time curve (AUC) of axitinib, objective response rate, and adverse events in 44 consecutive advanced RCC patients treated with axitinib. To establish a model for predicting clinical efficacy and adverse events, polymorphisms in genes including ABC transporters (ABCB1 and ABCG2), UGT1A, and OR2B11 were analyzed by whole-exome sequencing, Sanger sequencing, and DNA microarray. To validate this prediction model, calculated AUC by 6 gene polymorphisms was compared with actual AUC in 16 additional consecutive patients prospectively. Actual AUC significantly correlated with the objective response rate (P = 0.0002) and adverse events (hand-foot syndrome, P = 0.0055; and hypothyroidism, P = 0.0381). Calculated AUC significantly correlated with actual AUC (P < 0.0001), and correctly predicted objective response rate (P = 0.0044) as well as adverse events (P = 0.0191 and 0.0082, respectively). In the validation study, calculated AUC prior to axitinib treatment precisely predicted actual AUC after axitinib treatment (P = 0.0066). Our pharmacogenetics-based AUC prediction model may determine the optimal initial dose of axitinib, and thus facilitate better treatment of patients with advanced RCC.

Preparation of molecularly imprinted polymers for warfarin and coumachlor by multi-step swelling and polymerization method and their imprinting effects.

Monodisperse molecularly imprinted polymers (MIPs) for warfarin (WF) and coumachlor (CC), MIPWF and MIPCC, respectively, were prepared using 4-vinylpyridine (4-VPY) as a functional monomer and ethylene glycole dimethacrylate (EDMA) as a crosslinker by multi-step swelling and polymerization. Six kinds of MIPWF, MIPWF1 - MIPWF6, were prepared varying the concentrations of WF and 4-VPY, while maintaining the EDMA concentration constant, and their retention and molecular recognition properties were evaluated using a mixture of sodium phosphate buffer and acetonitrile as a mobile phase in LC. In addition to shape recognition, hydrogen bonding, ionic and hydrophobic interactions could affect the retention and molecular recognition of WF on MIPWF, and ionic interactions seem to govern the retention and molecular recognition of WF above mobile phase pH 6 associated with higher molar ratio of 4-VPY to EDMA. Furthermore, MIPCC was prepared under the same conditions with MIPWF6, which gave the highest imprinting factor for WF. WF could be recognized more strongly on MIPCC than MIPWF6, and the imprinting factors of WF on MIPWF6 and MIPCC, respectively, are 2.68 and 5.03 using 20mM sodium phosphate buffer - acetonitrile (30/70, v/v)(final pH 6.1) as the mobile phase. This result indicates that the use of CC as a template molecule instead of WF could be useful for getting a higher imprinting factor for WF and for avoiding the leakage problem in the assay of WF in LC.

Molecularly imprinted polymer for glutathione by modified precipitation polymerization and its application to determination of glutathione in supplements.

Molecularly imprinted polymers (MIP) particles for glutathione (GSH) with a narrow particle size distribution were prepared by modified precipitation polymerization using methacrylic acid as a functional monomer, divinylbenzene as a crosslinker and water as a co-solvent. The particle diameters of the MIP and non-imprinted polymer (NIP) prepared under the optimum conditions were 3.81±0.95 (average±standard deviation) and 3.39±1.22µm, respectively. The retention and molecular-recognition properties of the prepared MIP were evaluated using a mixture of acetonitrile and water as a mobile phase in hydrophilic interaction chromatography. With an increase of acetonitrile content, the retention factor of GSH was increased on the MIP. In addition to shape recognition, hydrophilic interactions seem to work for the recognition of GSH on the MIP. The MIP had a specific molecular-recognition ability for GSH, while glutathione disulfide, l-Glu, l-Cys, Gly-Gly and l-Cys-Gly could not be retained or recognized on the MIP. The effect of column temperature revealed that the separation of GSH on the MIP was entropically driven. Binding experiments and Scatchard analyses revealed that one binding sites were formed on both the MIP and NIP, while the MIP gave higher affinity and capacity for GSH than the NIP. Furthermore, the MIP was successfully applied for determination of GSH in the supplements.