[Fluorescence enhancement of flavoxate hydrochloride in alkali solution and its application in pharmaceutical analysis].

Fluorescence enhancement reaction of flavoxate hydrochloride (FX) in strong alkali solution was studied, the mechanism of the reaction was investigated, and a novel fluorimetric method for analysis of FX in drug sample was established. FX has no intrinsic fluorescence, but it can slowly produce fluorescence in strong alkali solution. Heating can promote the fluorescence enhancement reaction. In 3D fluorescence spectra of the decomposition product of FX, two fluorescence peaks, located respectively at excitation wavelengths λex/ emission wavelength λem =223/410 nm, and 302/410 nm, were observed. Using quinine sulfate as a reference, fluorescence quantum yield of the decomposition product was measured to be 0.50. The structural characteriza- tion and spectral analysis of the decomposition product reveal that ester bond hydrolysis reaction of FX is firstly occurred during heating process, forming 3-methylflavone-8-carboxylic acid (MFA), then a cleavage reaction of the γ-pyrone ring of MFA occurred, producing α, ß-unsaturated ketone. This product includes adjacent hydroxyl benzoic acid group in its molecule, which can form intramolecular hydrogen bond under alkaline condition, so that increase the conjugate degree and enhance the rigidity of the molecule, and thereby cause fluorescence enhancement. Based on this fluorescence enhancement reaction, a fluorimetric method was proposed for the determination of FX. A linear calibration curve covered the concentration range 0.020 3-0.487 µg · mL. The regression equation was I(F) = 23.9 + 5357.3 c, with correlation coefficient r = 0.999 7 (n = 8), detection limit D = 1.1 ng · mL(-1). The method was applied to the analysis of FX tablets, with a spiked recovery rate of 100.2%. The reliability of the method was verified by a UV-spectrophotometric method.

Spectrofluorimetric determination of 3-methylflavone-8-carboxylic acid, the main active metabolite of flavoxate hydrochloride in human urine.

A simple, sensitive and selective spectrofluorimetric method has been developed for the determination of 3-methylflavone-8-carboxylic acid as the main active metabolite of flavoxate hydrochloride in human urine. The proposed method was based on the measurement of the native fluorescence of the metabolite in methanol at an emission wavelength 390 nm, upon excitation at 338 nm. Moreover, the urinary excretion pattern has been calculated using the proposed method. Taking the advantage that 3-methylflavone-8-carboxylic acid is also the alkaline degradate, the proposed method was applied to in vitro determination of flavoxate hydrochloride in tablets dosage form via the measurement of its corresponding degradate. The method was validated in accordance with the ICH requirements and statistically compared to the official method with no significant difference in performance.

Molecularly imprinted polymer for selective extraction of 3-methylflavone-8-carboxylic acid from human urine followed by its determination using zwitterionic hydrophilic interaction liquid chromatography.

A novel water-compatible molecularly imprinted SPE combined with zwitterionic hydrophilic interaction liquid chromatography method for selective extraction and determination of 3-methylflavone-8-carboxylic acid (MFA), the main active metabolite of flavoxate in human urine, was developed and validated. The effects of progenic solvents, pH, cross linker and amount of monomer were studied to optimize the efficiency and selectivity. The molecularly imprinted polymer showed good specific adsorption capacity with an optimum of 200 µmol/g at pH 7.5 and selective extraction of MFA from human urine. The recovery of MFA from human urine was >98%. The lower limit of quantification was 1.20 µg/mL. The proposed method overcomes the matrix effects of endogenous substances generally encountered during direct analysis of urine sample.

Liquid chromatographic fingerprint of 3-methylflavone-8-carboxylic acid established for its synthesis control analysis.

A high-performance liquid chromatographic method was proposed for the separation of relative impurities in industrial 3-methylflavone-8-carboxylic acid (MFCA) and its intermediate methyl 3-propionylsalicylate (MPS). Benzoic acid (BA), MPS, 6-chloro-3-methylflavone-8-carboxylic acid (MFCA-Cl) and methyl 5-chloro-3-propionylsalicylate (MPS-Cl) in MFCA, and MPS-Cl in MPS were respectively quantified by an external standard method. As results showed, the linearity of standard curves was excellent with the relative coefficients of over 0.999 for all the detected components, and the intra- and inter-day precisions of impurities determination were satisfactory with the relative standard deviation of not more than 8.0%. Under the selected experimental condition, the chromatographic fingerprints of MFCA and MPS were drawn synthetically, and the transfer of impurities in the stepwise reactions of MFCA manufacture was elucidated. The fingerprints have great potential in instructing the production of MFCA for industrial use and in conducting the conversion of relative impurities.

High-performance liquid chromatographic determination of flavoxate hydrochloride and its hydrolysis product.

Liquid chromatographic method was presented for the determination of flavoxate hydrochloride (FX) and its hydrolysis product. The method was based on high-performance liquid chromatographic (HPLC) separation of FX from its hydrolysis product on CN column using a mobile phase consisting of acetonitrile-12 mM ammonium acetate (45:55, vol/vol, pH 4.0) with UV detection at 220 nm and flow rate of 1.5 mL min(-1). The proposed HPLC method for the determination of FX was utilized to investigate the kinetics of acidic hydrolytic process at different temperatures and to calculate its activation energy. In addition, the proposed HPLC method was used for pH-rate profile study of hydrolysis of FX in Britton-Robinson buffer solutions. The 3-methylflavone-8-carboxylic acid ethyl ester, as impurity of flavoxate hydrochloride, can be separated by the proposed HPLC method.

Development of a high-performance liquid chromatographic method for bioequivalence study of flavoxate tablets.

An improved HPLC method was developed for the concentration determination of the metabolite of flavoxate, 3-methyl-flavone-8-carboxylic acid (MFCA), in plasma in an attempt to compare two flavoxate tablet formulations. This HPLC method was validated by examining the precision and the accuracy for inter-day and intra-day runs in a linear concentration range of 0.1-24 microg/ml. The coefficients of variation (C.V.) of inter-day and intra-day assays were 0.24-7.18% and 0.06-5.70%, respectively. The standard errors of mean (S.E.M.) were -0.004-8.68% and -2.52-4.86% for inter-day and intra-day assays, respectively. Bioequivalence of the two formulations was determined on 12 normal healthy male volunteers in a single-dose, two-period, two-sequence, two-treatment crossover study. MFCA plasma concentrations were analyzed with this validated HPLC method. The normal pivotal parameters, AUC(0-last), AUC(0-inf) and Cmax, were calculated and compared using the SAS General Linear Model computer program. The two one-sided t distribution test was also performed, as well as the 90% confidence-interval method, for the mean difference of the three pivotal parameters. The results suggest that these two flavoxate tablet formulations are non-bioequivalent when orally administered in a 400-mg dose of two tablets. This result was consistent with the in vitro dissolution of these two formulations.

Structural characterization of terflavoxate.

Data are reported on the structural characterization of 3-methyl-4-oxo-2-phenyl-4H-1-benzopyran-8-carboxylic acid 1,1-dimethyl-2-(N-piperidinyl)ethyl ester hydrochloride (terflavoxate-HCl, Rec 15/2053, CAS 86433-39-8), a new antispasmodic for the lower urinary tract. UV, IR, NMR and MS spectra fully confirmed the structure. The X-ray crystal structure determination revealed that the molecular structure consists of a rigid platform, formed by the chromone system, with two arms, the phenyl group at C(2) and the ester chain at C(8). The ester chain conformation generates a small hollow where two oxygen atoms face.

Determination of 3-methylflavone-8-carboxylic acid, the main metabolite of flavoxate, in human urine by capillary electrophoresis with direct injection.

The effects of tetraalkylammonium salts and sodium dodecyl sulphate on the migration behaviour of human urinary components and other negatively charged or neutral solutes were investigated. The sulphate acted mainly on hydrophobic and positively charged substances, whereas the ammonium salts acted mainly on negatively charged solutes. By choosing the components of the eluent carefully, the free and conjugate forms of 3-methylflavone-8-carboxylic acid (MFA) in human urine, the major metabolites of flavoxate, could be simultaneously determined without pretreatment, using fenprofen as an internal standard. The calibration curve of MFA was linear in the range 1-50 micrograms/ml and the detection limit was 0.2 microgram/ml, which covered the urine levels encountered in pharmacokinetic studies. The intra-day and inter-day precisions of the method, expressed as the relative standard deviation, were less than 2 and 3%, respectively. This method was successfully applied to an excretion study of MFA in eight healthy volunteers, and the results were in agreement with data in the literature obtained by gas chromatography.

Effects of terflavoxate on stimulated contractions of urinary bladder in vitro.

The antispasmodic activity of terflavoxate (CAS 86433-39-8), a flavone derivative with spasmolytic properties on the urinary tract, has been studied in vitro, in comparison to the most common drugs utilized in the therapy of overactive detrusor, namely flavoxate, oxybutynin, and terodiline. Terflavoxate showed affinity for bladder (and brain) muscarinic receptors at micromolar level, however, its activity on carbachol-induced contractions of rat bladder was clearly non competitive, indicating that the compound is devoid of functional antimuscarinic property. Moreover, the observation that unlike antimuscarinic drugs, terflavoxate inhibited by more than 50% field stimulation-induced contractions of rabbit bladder strips, indicates that mechanisms other than the anticholinergic one should be responsible for its smooth muscle relaxant properties. Terflavoxate, flavoxate, oxybutynin, and terodiline were equally effective in inhibiting the two components of K(+)-induced contractions, while nifedipine and nicardipine were more potent than the other compounds, and more effective in inhibiting tonic than phasic contractions. In addition, while nifedipine and nicardipine antagonized in a competitive manner calcium-induced contractions of potassium-depolarized bladder strips, the other spasmolytics behaved as mixed antagonists. Differences in calcium antagonistic properties between nifedipine and nicardipine on one side, and terflavoxate on the other, are further demonstrated by the data on binding experiments. Nevertheless, present results suggest that Ca(++)-antagonistic effects are mainly responsible for terflavoxate smooth muscle relaxant properties.

Antispasmodic effects of flavoxate, MFCA, and REC 15/2053 on smooth muscle of human prostate and urinary bladder.

The antispasmodic effects of the flavone compounds flavoxate hydrochloride, 3-methylflavone carboxylic acid (MFCA), and REC 15/2053 (and in the case of the detrusor, oxybutynin), on the human detrusor, prostatic adenoma, prostatic capsule, and bladder neck, were studied by the in vitro isometric method. All the compounds inhibited, in different orders of potency, potassium-induced contractions of the tissues. Flavoxate showed a slightly greater activity than the other two compounds in the prostatic and bladder neck tissues. However, REC 15/2053 displayed greater activity in the detrusor than in the other tissues. The relaxant effect on the prostatic tissues suggests a potential use for these compounds in benign prostatic obstruction.

Receptor binding studies of the flavone, REC 15/2053, and other bladder spasmolytics.

The new flavone derivative REC 15/2053, a compound with spasmolytic activity on the lower urinary tract, was examined for its in vitro interaction with alpha- and beta-noradrenergic receptors, dopaminergic, muscarinic, serotoninergic, and opiate receptors, and calcium-channel binding sites labeled with 1,4-dihydropyridines from normal rat brain. All the investigated receptors are directly or indirectly involved in the nervous control of the lower urinary tract functions. The activity of REC 15/2053 on these receptors was studied in comparison to the most common drugs used in the management of urinary bladder disorders such as flavoxate, emepronium bromide, oxybutynin, terodiline, and imipramine. REC 15/2053 showed only weak binding to [3H]nitrendipine sites (IC50 = 14 microM) and muscarinic receptors (IC50 = 18 microM), whereas flavoxate was slightly active only at muscarinic receptors (IC50 = 12.2 microM). Emepronium bromide, oxybutynin, and terodiline were active only at muscarinic receptors, with IC50 values of 236, 5.4, and 588 nM, respectively. Oxybutynin showed a weak affinity to [3H]nitrendipine binding sites (IC50 = 44.4 microM). Imipramine was active at alpha 1-adrenergic and muscarinic receptors (IC50 = 248 and 653 nM, respectively). The activity of REC 15/2053 at muscarinic receptors and 1,4-dihydropyridine binding sites seems too low to account for its mechanism of action.

Pharmacological activities of the main metabolite of flavoxate 3-methylflavone-8-carboxylic acid.

The pharmacological properties of 3-methylflavone-8-carboxylic acid (MFCA), the main metabolite of flavoxate, have been studied in vitro and in vivo. MFCA did not display antispasmodic activity on isolated organs contractions induced by histamine, acetylcholine or CaCl2, nor did it exhibit significant affinity for the rat brain alpha- and beta-adrenergic, serotoninic, muscarinic, D2, opiate and Ca2+ receptors. However, it showed a remarkable phosphodiesterase (PDE) inhibiting activity. Moreover in vivo studies indicate an interesting activity of MFCA which inhibited the rat urinary bladder voiding contractions, increased bladder volume capacity and decreased micturition pressure in the rat cystometric recordings. The activity of MFCA in the two in vivo experimental models, probably related to cAMP-PDE inhibitory properties, suggests that flavoxate's therapeutical potential might be partially sustained by its main metabolite.