A high-performance liquid chromatography assay with a triazole-bonded column for evaluation of d-amino acid oxidase activity.

Elution profiles of kynurenic acid (KYNA) and 7-chlorokynurenic acid (Cl-KYNA) were examined by high-performance liquid chromatography (HPLC) using a triazole-bonded stationary phase column (Cosmosil® HILIC) under isocratic elution of a mobile phase consisting of CH3 CN-aqueous 10 mm ammonium formate between pH 3.0 and 6.0. The capacity factors of KYNA and Cl-KYNA varied with both the CH3 CN content and the pH of the mobile phase. The elution order of KYNA and Cl-KYNA was reversed between the CH3 CN- and H2 O-rich mobile phases, suggesting that hydrophilic interactions and anion-exchange interactions caused retention of KYNA and Cl-KYNA in the CH3 CN- and H2 O-rich mobile phases, respectively. The present HPLC method using a triazole-bonded column and fluorescence detection (excitation 250 nm, emission 398 nm) was applied to monitor in vitro production of KYNA from d-kynurenine (d-KYN) by d-amino acid oxidase (DAO) using Cl-KYNA as an internal standard. A single KYNA peak was clearly observed after enzymatic reaction of d-KYN with DAO. Production of KYNA from d-KYN was suppressed by the addition of commercial DAO inhibitors. The present HPLC method can be used to evaluate DAO activity and DAO inhibitory effects in candidate drugs for the treatment of schizophrenia.

Enantiomeric Separation of Monosubstituted Tryptophan Derivatives and Metabolites by HPLC with a Cinchona Alkaloid-Based Zwitterionic Chiral Stationary Phase and Its Application to the Evaluation of the Optical Purity of Synthesized 6-Chloro-l-Tryptophan.

6-Chlorotryptophan possesses unique bioactivity and can be used as a precursor for several bioactive compounds in medicinal chemistry. It was enantioselectively synthesized by condensing 6-chloroindole with racemic N-acetylserine, followed by enzymatic hydrolysis with l-aminoacylase (EC 3.5.1.14). The optical purity was examined by conducting high-performance liquid chromatography with a Cinchona alkaloid-based zwitterionic chiral stationary phase (CSP) [CHIRALPAK(®) ZWIX(+)], which bears a chiral trans-2-aminocyclohexanesulfonic acid moiety tagged at C-9 of the Cinchona alka-loid. The zwitterionic CSP enabled efficient enantiomeric separations of monosubstituted tryptophan derivatives 1-methyltryptophan, 5-methyltryptophan, 6-methyltryptophan, 5-methoxytryptophan, and 6-chlorotryptophan with a methanol/H2O (98/2) mobile phase containing formic acid (FA) and diethylamine (DEA) additives. The mobile phase contains 25-75 mM FA and 20-50 mM DEA, enabling good separation of the enantiomers of each tryptophan derivative (α > 1.25). Thus, the optical purity of the synthesized 6-chloro-l-tryptophan was easily determined (greater than 99.0%) using HPLC with the zwitterionic CSP.

Evaluation of human D-amino acid oxidase inhibition by anti-psychotic drugs in vitro.

It is of importance to determine whether antipsychotic drugs currently prescribed for schizophrenia exert D-amino acid oxidase (DAO)-inhibitory effects. We first investigated whether human (h)DAO can metabolize D-kynurenine (D-KYN) to produce the fluorescent compound kynurenic acid (KYNA) by using high-performance liquid chromatography with mass spectrometry, and fluorescence spectrometry. After confirmation of KYNA production from D-KYN by hDAO, 8 first- and second-generation antipsychotic drugs, and 6 drugs often prescribed concomitantly, were assayed for hDAO-inhibitory effects by using in vitro fluorometric methods with D-KYN as the substrate. DAO inhibitors 3-methylpyrazole-5-carboxylic acid and 4H-thieno[3,2-b]pyrrole-5-carboxylic acid inhibited KYNA production in a dose-dependent manner. Similarly, the second-generation antipsychotics blonanserin and risperidone were found to possess relatively strong hDAO-inhibitory effects in vitro (5.29 ± 0.47 µM and 4.70 ± 0.17 µM, respectively). With regard to blonanserin and risperidone, DAO-inhibitory effects should be taken into consideration in the context of their in vivo pharmacotherapeutic efficacy.

Enantiomeric separation of tolperisone and eperisone by reversed-phase HPLC with cellulose tris(3-chloro-4-methylphenylcarbamate)-coated chiral column.

Enantiomeric separations of centrally acting muscle relaxants, that is, tolperisone (TOL) and eperisone (EP), that are marketed as racemates were investigated by reversed-phase high-performance liquid chromatography (HPLC) on a polysaccharide-based chiral column. Both TOL and EP are basic drugs because they contain a tertiary amino group and have similar chemical structures with the exception of the p-methylphenyl and p-ethylphenyl groups in TOL and EP, respectively. A reversed-phase chiral column, that is, a Chiralcel OZ-RH column, which bears cellulose tris(3-chloro-4-methylphenylcarbamate) as the chiral moiety, was effective for the enantiomeric separation of TOL and EP enantiomers. The separation factor and resolution values obtained for TOL were 1.22 and 1.66, respectively, and those for EP were 1.21 and 2.24, respectively, using a 20 mm ammonium acetate in H2 O (pH 8.0 and 7.0, respectively)-CH3 CN (70:30) mobile phase. Using the proposed HPLC conditions, it was found that (R)-TOL eluted faster than (S)-TOL, as revealed by the optical rotation and circular dichroism spectroscopy. In contrast, EP was easily racemized under the experimental conditions, and hence, the elution order was not determined.

Fluorimetric assay for D-amino acid oxidase activity in rat brain homogenate by using D-kynurenine as a substrate.

An easy fluorimetric assay for measuring D-amino acid oxidase (DAAO) activity by using one of the D-amino acids--D-kynurenine (D-KYN)--as a substrate was applied to assess DAAO activity in the cerebrum, cerebellum, and brainstem of Sprague-Dawley (SD) male rats. In this assay, DAAO produces kynurenic acid (KYNA) from D-KYN, and the fluorescence originating from KYNA can then be used to evaluate DAAO activity. Here, pellet fractions obtained by centrifugation of brain homogenates were allowed to react enzymatically with D-KYN. The addition of specific DAAO inhibitors, such as 3-methylpyrazole-5-carboxylic acid and 4H-thieno [3, 2-b] pyrrole-5-carboxylic acid (Compound 8), significantly attenuated the fluorescence intensity of KYNA, suggesting that DAAO present in the rat brain homogenates was responsible for the production of KYNA. In contrast, an inhibitor of aminotransferase (AT), aminooxyacetic acid, did not decrease KYNA production from D-KYN, meaning that AT could not metabolize D-KYN to KYNA under the present conditions. Moreover, the DAAO activity measured by the proposed assay correlated well with DAAO mRNA expression (r = 0.9982) determined by real-time polymerase chain reaction. Taken together, these findings show that the proposed fluorimetric assay can be used to evaluate DAAO activity in rat brain.

Inhibition of D-amino acid oxidase activity by antipsychotic drugs evaluated by a fluorometric assay using D-kynurenine as substrate.

A facile fluorometric assay using D-kynurenine as a substrate was utilized for evaluating the inhibition of D-amino acid oxidase (DAAO), which is one of the products of a susceptibility gene for schizophrenia, by commercial antipsychotic drugs, namely, chlorpromazine (CPZ), carbamazepine, sulpiride, quetiapine, and imipramine. CPZ inhibited DAAO (65.8 ± 13.2 µM, n = 3) as reported previously, and other drugs also inhibited DAAO activity. Among these, quetiapine had the smallest IC(50) value (19.5 ± 2.60 µM, n = 3). The proposed assay can be useful for the evaluation or screening of DAAO-inhibitory drugs.

Physicochemical and pharmacological characterization of novel vasoactive intestinal peptide derivatives with improved stability.

Previously, [R(15,20,21), L(17)]-VIP-GRR (IK312532), a long-acting VIP derivative, was proposed as potential drug candidate for the treatment of asthma/COPD. The present work is aimed to elucidate solution-state stability of IK312532 and to develop further stabilized derivative with equipotent or higher biological functions. A stability study on IK312532 was carried out in solution state, and degradation mechanism was deduced by UPLC-MS and amino acid analyses. Three novel VIP derivatives were designed and chemically synthesized on the basis of stability data, being subjected to physicochemical and pharmacological characterization. Solution-state stability studies revealed the gradual degradation of IK312532, following pseudo-first-order kinetics. Chemical modification of IK312532, mainly position at 24, resulted in marked improvement of stability, although the chemical modification had no influence on the secondary structure, receptor binding, and activation of adenylate cyclase in rat lung cells. Novel derivatives also exhibited more potent neurite outgrowth in rat pheochromocytoma PC12 cells when compared to VIP and IK312532, possibly due to improved stability. Deamination of Asn at position 24 might be responsible for degradation of VIP derivative, and stability and chemical modification studies led us to the successful development of novel VIP derivatives with higher stability and biological functions.

Structural transition of glucagon in the concentrated solution observed by electrophoretic and spectroscopic techniques.

Glucagon, a polypeptide hormone consisting of 29 amino acid residues, tends to form gel-like fibrillar aggregates, and the glucagon fibril, as well as other pathologically related fibrils including prion, amylin, and beta-amyloid, have been found to be cytotoxic through the activation of apoptotic signaling pathways. To understand the aggregation properties of glucagon fibril, we have characterized and compared the physicochemical properties of glucagon, secretin, a member of the glucagon superfamily, and amylin using analytical techniques including capillary electrophoresis (CE), circular dichroism (CD), FT-IR, FT-Raman, transmission electron microscopy (TEM), and beta-sheet-imaging probe. Aging treatment of glucagon resulted in the formation of fibrillar aggregates in time- and concentration-dependent manner, and FT-IR and FT-Raman analyses showed the spectral shift of amide I band, suggesting the conformational changes from alpha-helix to beta-sheet structure. Interestingly, secretin, having high sequential and secondary structural homology with glucagon, did not generate the fibrillar aggregates at the conditions tested. In addition, we evaluated the association state of glucagon at various pHs raging from pH 2.0 to 3.5 using CE. Based on the CE data, the rate constants of glucagon aggregation were calculated to be 0.002 +/- 0.004/h and 0.080 +/- 0.011/h for aging at pH 2.0 and 3.5, respectively, suggesting the pH dependence of self-association. CE showed the potential to separate and detect the glucagon aggregates and intermediates during aging process.

Mishandling of the therapeutic peptide glucagon generates cytotoxic amyloidogenic fibrils.

PURPOSE: Some therapeutic peptides exhibit amyloidogenic properties that cause insolubility and cytotoxicity against neuronal cells in vitro. Here, we characterize the conformational change in monomeric therapeutic peptide to its fibrillar aggregate in order to prevent amyloidogenic formation during clinical application. METHODS: Therapeutic peptides including glucagon, porcine secretin, and salmon calcitonin were dissolved in acidic solution at concentrations ranging from 1 mg/ml to 80 mg/ml and then aged at 37 degrees C. Amyloidogenic properties were assessed by circular dichroism (CD), electron microscopy (EM), staining with beta-sheet-specific dyes, and size-exclusion chromatography (SEC). Cytotoxic characteristics were determined concomitantly. RESULTS: By aging at 2.5 mg/ml or higher for 24 h, monomeric glucagon was converted to fibrillar aggregates consisting of a beta-sheet-rich structure with multimeric states of glucagon. Although no aggregation was observed by aging at the clinical concentration of 1 mg/ml for 1 day, 30-day aging resulted in the generation of fibrillar aggregates. The addition of anti-glucagon serum significantly inhibited fibrillar conversion of monomeric glucagon. Glucagon fibrils induced significant cell death and activated an apoptotic enzyme, caspase-3, in PC12 cells and NIH-3T3 cells. Caspase inhibitors attenuated this toxicity in a dose-dependent manner, indicating the involvement of apoptotic signaling pathways in the fibrillar formation of glucagon. On the contrary to glucagon, salmon calcitonin exhibited aggregation at a much higher concentration of 40 mg/ml and secretin showed no aggregation at the concentration as high as 75 mg/ml. CONCLUSIONS: These results indicated that glucagon was self-associated by its beta-sheet-rich intermolecular structure during the aging process under concentrated conditions to induce fibrillar aggregates. Glucagon has the same amyloidogenic propensities as pathologically related peptides such as beta-amyloid (Abeta)1-42 and prion protein fragment (PrP)106-126 including conformational change to a beta-sheet-rich structure and cytotoxic effects by activating caspases. These findings suggest that inappropriate preparation and application of therapeutic glucagon may cause undesirable insoluble products and side effects such as amyloidosis in clinical application.