Applicability of capillary electrophoresis-frontal analysis for displacement studies: Effect of several drugs on l-tryptophan and lidocaine binding to human serum albumin.

The effective concentration of a drug in the blood, i.e. the concentration of a free drug in the blood, is influenced by the strength of drug binding onto plasma proteins. Besides its efficacy, these interactions subsequently influence the liberation, absorption, distribution, metabolism, excretion, and toxicological properties of the drug. It is important to not only determine the binding strength and stoichiometry, but also the binding site of a drug on the plasma protein molecule, because the co-administration of drugs with the same binding site can affect the above-mentioned concentration and as a result the pharmacological behavior of the drugs and lead to side effects caused by the change in free drug concentration, its toxicity. In this study, the binding characteristics of six drugs with human serum albumin, the most abundant protein in human plasma, were determined by capillary electrophoresis-frontal analysis, and the obtained values of binding parameters were compared with the literature data. The effect of several drugs and site markers on the binding of l-tryptophan and lidocaine to human serum albumin was investigated in subsequent displacement studies which thus demonstrated the usability of capillary electrophoresis as an automated high-throughput screening method for drug-protein binding studies.

Development and validation of a simple LC method for the determination of phenacetin, coumarin, tolbutamide, chlorzoxazone, testosterone and their metabolites as markers of cytochromes 1A2, 2A6, 2C11, 2E1 and 3A2 in rat microsomal medium.

Cytochrome P450 enzymes are responsible for the oxidative metabolism of most pharmaceutical compounds. A "cocktail" approach which employs simultaneous administration of a mixture of substrates of CYP enzymes was often used to assess the metabolic activity of multiple P450 forms in one experiment. Phenacetin, coumarin, tolbutamide, chlorzoxazone and testosterone are commonly used as probe substrates to evaluate cytochrome P450 function. An analytical strategy to simultaneously extract and analyze the five probe substrates and their major metabolites by HPLC-DAD was developed. The incubation was done with all the substrates in one step. The ten analytes were extracted simultaneously by solid-phase extraction (SPE) from rat liver microsomes. A C18 analytical column and mobile phase composed of acetonitrile and 0.02% aqueous phosphoric acid were used for the chromatographic separation with DAD detection. Limits of quantification varied between 0.02378 and 0.2361 microg/mL which contributed to quantify all these drugs and metabolites with UV detection. The method is applicable for the modeling and description of pharmacological interactions on rat cytochromes P450 or can be used for in vitro evaluation of cytochromes 1A2, 2A6, 2C11, 2E1 and 3A2.

Are there differences in the catalytic activity per unit enzyme of recombinantly expressed and human liver microsomal cytochrome P450 2C9? A systematic investigation into inter-system extrapolation factors.

The 'relative activity factor' (RAF) compares the activity per unit of microsomal protein in recombinantly expressed cytochrome P450 enzymes (rhCYP) and human liver without separating the potential sources of variation (i.e. abundance of enzyme per mg of protein or variation of activity per unit enzyme). The dimensionless 'inter-system extrapolation factor' (ISEF) dissects differences in activity from those in CYP abundance. Detailed protocols for the determination of this scalar, which is used in population in vitro-in vivo extrapolation (IVIVE), are currently lacking. The present study determined an ISEF for CYP2C9 and, for the first time, systematically evaluated the effects of probe substrate, cytochrome b5 and methods for assessing the intrinsic clearance (CL(int) ). Values of ISEF for S-warfarin, tolbutamide and diclofenac were 0.75 ± 0.18, 0.57 ± 0.07 and 0.37 ± 0.07, respectively, using CL(int) values derived from the kinetic values V(max) and K(m) of metabolite formation in rhCYP2C9 + reductase + b5 BD Supersomes™. The ISEF values obtained using rhCYP2C9 + reductase BD Supersomes™ were more variable, with values of 7.16 ± 1.25, 0.89 ± 0.52 and 0.50 ± 0.05 for S-warfarin, tolbutamide and diclofenac, respectively. Although the ISEF values obtained from rhCYP2C9 + reductase + b5 for the three probe substrates were statistically different (p < 0.001), the use of the mean value of 0.54 resulted in predicted oral clearance values for all three substrates within 1.4 fold of the observed literature values. For consistency in the relative activity across substrates, use of a b5 expressing recombinant system, with the intrinsic clearance calculated from full kinetic data is recommended for generation of the CYP2C9 ISEF. Furthermore, as ISEFs have been found to be sensitive to differences in accessory proteins, rhCYP system specific ISEFs are recommended.

Validation of a sensitive UHPLC-MS/MS method for cytochrome P450 probe substrates caffeine, tolbutamide, dextromethorphan, and alprazolam in human serum reveals drug contamination of serum used for research.

To evaluate the potential for interactions between botanical dietary supplements and drug metabolism, Phase I clinical pharmacokinetics studies are conducted using an oral cocktail of probe substrates of cytochrome P450 (CYP) enzymes. A sensitive, specific, and fast ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for determination of caffeine (probe of CYP1A2), tolbutamide (probe of CYP2C9), dextromethorphan (probe of CYP2D6), and alprazolam (probe of CYP3A4/5) in human serum. Stable isotope-labelled analogs were used as internal standards, and sample preparation involved only rapid protein precipitation and centrifugation. The method of standard addition was used for the measurement of caffeine, because commercially available pooled human serum contains caffeine. Out of 18 lots of pooled human serum tested, caffeine was detection in all lots, alprazolam was detected in 13 lots, 8 lots contained dextromethorphan, and no tolbutamide was detected. Only serum prepared from the blood of select individuals was determined to be drug-free. The analytical method was validated with respect to linearity, accuracy and precision, recovery, stability, and matrix effects. The calibration curves were linear over the range of 25-12,000 ng/mL for caffeine, 75-36,000 ng/mL for tolbutamide, 0.05-30 ng/mL for dextromethorphan, and 0.1-60 ng/mL for alprazolam. The intra-assay and inter-assay coefficients of variation (%CV) and %Bias were <13 % (<17 % at the lower limit of quantitation). The recovery of each probe substrate ranged from 84.2%-98.5 %. All analytes were stable during sample storage and handling. Matrix effects were minimized by using stable isotope-labeled internal standards. The method was successfully applied to clinical studies investigating the pharmacokinetic alterations of probe substrates caused by chronic consumption of botanical dietary supplements.

A high-performance liquid chromatography-tandem mass spectrometry method for the clinical combination study of carboplatin and anti-tumor agent eribulin mesylate (E7389) in human plasma.

An LC/MS/MS method was developed to quantify carboplatin and eribulin mesylate (E7389) in human plasma and urine. For carboplatin, sample clean-up by protein precipitation and supernatant injection into a Waters Spherisorb((R)) S5 SCX column was used. Liquid-phase extraction and reverse-phase chromatography on a Polaris C18 column were used for eribulin. Quantitation involved LC/MS/MS with positive electrospray ionization. Accuracy, precision, linearity, range, specificity, recovery and stability were also evaluated. Both compounds were stable in human plasma (>or=80 days at -80 degrees C), at room temperature (>or=4h), following three freeze-thaw cycles and in 50/50 methanol/H(2)O (<4 degrees C for >or=252 days).

Thermodynamic Stability Analysis of Tolbutamide Polymorphs and Solubility in Organic Solvents.

Melting temperatures and enthalpies of fusion have been determined by differential scanning calorimetry (DSC) for 2 polymorphs of the drug tolbutamide: FI(H) and FV. Heat capacities have been determined by temperature-modulated DSC for 4 polymorphs: FI(L), FI(H), FII, FV, and for the supercooled melt. The enthalpy of fusion of FII at its melting point has been estimated from the enthalpy of transition of FII into FI(H) through a thermodynamic cycle. Calorimetric data have been used to derive a quantitative polymorphic stability relationship between these 4 polymorphs, showing that FII is the stable polymorph below approximately 333 K, above which temperature FI(H) is the stable form up to its melting point. The relative stability of FV is well below the other polymorphs. The previously reported kinetic reversibility of the transformation between FI(L) and FI(H) has been verified using in situ Raman spectroscopy. The solid-liquid solubility of FII has been gravimetrically determined in 5 pure organic solvents (methanol, 1-propanol, ethyl acetate, acetonitrile, and toluene) over the temperature range 278 to 323 K. The ideal solubility has been estimated from calorimetric data, and solution activity coefficients at saturation in the 5 solvents determined. All solutions show positive deviation from Raoult's law, and all van't Hoff plots of solubility data are nonlinear. The solubility in toluene is well below that observed in the other investigated solvents. Solubility data have been correlated and extrapolated to the melting point using a semiempirical regression model.

Determination of tolbutamide hydroxylation in rat liver microsomes by high-performance liquid chromatography: effect of psychoactive drugs on in vitro activity.

A simplified HPLC method for tolbutamide metabolism to hydroxytolbutamide has been used to screen sixty psychoactive drugs for their ability to inhibit rat liver microsomal tolbutamide hydroxylation. One-step extraction with diethyl ether was followed by reconstitution and isocratic HPLC analysis with a binary mobile phase (ammonium phosphate:methanol, 45:55, v/v). Nanogram amounts of hydroxytolbutamide formation were estimated with UV detection at 240 nm. Hydroxytolbutamide formation was linear with incubation times of 40-120 min, but specific activity increased with increases in microsomal protein (0.15-1.10 mg). A differential inhibitory response was demonstrated for tolbutamide and debrisoquine hydroxylation to 5 psychoactive drugs, suggesting that tolbutamide hydroxylation is not dependent on P4502D1. Sixty psychoactive drugs, or drug metabolites, (at 33 microM) were then co-incubated with tolbutamide (at 2.5 and 10.2 microM). Tolbutamide hydroxylation was refractory (< 25% inhibition) to twenty-four of the drugs and only mildly inhibited (25-50% inhibition) by twenty-eight. Two compounds, trans-3-methylfentanyl and flurazepam, produced > 50% inhibition that was independent of tolbutamide concentration. Five of the drugs (methadone, chlorpheniramine, meperidine, 6-monoacetylmorphine and methylphenidate), however, caused greater than 50% inhibition in a competitive manner which suggests these drugs may share an affinity for the substrate binding site for tolbutamide.

Isolation of the components of a complex mixture by means of column switching for their enhanced detection by mass spectrometry.

Mass spectral characterization of low-level impurities in drug substances and formulations may be challenging when using a validated HPLC method developed for optimal chromatographic performance. In many cases, either the mobile phase contains non-volatile additives that are deleterious to the operation of the mass spectrometer, or some of the related substances fail to ionize effectively under electrospray ionization or atmospheric pressure chemical ionization conditions. This paper describes a way to capture these low-level compounds from an analytical HPLC column using a small trapping column. Mixture components are retained on the trapping column by means of reducing the solvent strength of the eluent. Subsequent elution of trapped compounds using mobile phases more amenable to mass spectral analysis yields improved detection and characterization of low-level compounds of interest. Possible applications of peak trapping and elution include: (1) analysis of compounds separated using a mobile phase containing high concentrations of non-volatile additives, (2) analysis of organic acids separated using a low-pH mobile phase (containing trifluoroacetic acid), and (3) improving the detection limit of a low-level compound of interest through multiple collections. The peak trapping apparatus and optimization experiments are described.

Distribution of drugs in polymers loaded by swelling.

Ethylene:vinyl acetate pellets were loaded at 20 degrees C by swelling the polymer with 1 and 3% (w/v) chloroformic solutions of tolbutamide. The energy dispersive X-ray analysis showed different concentrations of the tolbutamide sulfur in the pellets sections according to the loading time. At the beginning of the loading process, the sulfur in the pellets showed two concentration peaks which later joined in the center of the section before reaching a homogeneous distribution. The concentration peaks might depend on a drug sieving process as the solution flow reaches a less swollen inner area. Therefore, the concentration distribution of the drug would be affected by the size of the polymer network, which is related to the volume of the solvent in the polymer. Another possible explanation of these concentration profiles is that they could be a result of the solvent evaporation process. The concentration distribution of the drug becomes homogeneous only after the complete swelling of all of the polymer.

Spectrophotometric determination of tolbutamide, thiamine hydrochloride, and pyridoxine hydrochloride in combination products.

The first derivative curve is used for tolbutamide determination in unit-dose tablets and in combination products. The absorbance contribution from tablet excipient and coexisting components, thiamine and pyridoxine, is thereby nullified. The interference from tolbutamide during thiamine and pyridoxine determination is eliminated by solvent extraction and pH-induced differential spectrophotometry. Thiamine is measured at the isosbestic point of pyridoxine. The latter is determined by the differential absorbance measurement at two wavelengths with the consequent computation of the delta absorbance value.

Generic tolbutamide tablet dissolution: intralot and interlot variation.

Dissolution profiles for 62 lots of tolbutamide tablets from six manufacturers have been characterized using the USP paddle-stirrer apparatus. Results of paddle-stirrer dissolution for percent drug dissolved at 10, 20, and 30 min correlated well (r2 = 0.7444) with results from the USP rotating-basket test for 39 lots of tolbutamide. Interlot and intralot variability in tolbutamide dissolution was highly dependent on the manufacturer. For one product, the intralot range (for six paddle-stirred tablets) of percent drug dissolved after 30 min was 50-68% while the maximum interlot range for mean dissolution was 58-104%. One lot failed to meet both the rotating-basket and the paddle-stirrer dissolution specifications. Tablet response to aging at 60, 75, and 98% relative humidity over time was also highly manufacturer specific. The innovator's product repeatedly dissolved well when fresh or aged at all humidities. Dissolution from some generic tablets was dramatically depressed by humidity aging, even after only 3 d. Pretreatment of tablets with simulated gastric fluid modified the dissolution profile of one poorly dissolving lot of tablets. Results indicate that manufacturing quality control is highly variable among tolbutamide tablets.

Stability-indicating high-performance liquid chromatographic determination of chlorpropamide, tolbutamide, and their respective sulfonamide degradates.

A quantitative high-performance liquid chromatographic method for the determination of chlorpropamide, tolbutamide, and their respective hydrolysis products, p-chlorobenzenesulfonamide and p-toluenesulfonamide, in solid dosage forms was developed. The method is stability indicating and can be used to determine the sulfonamide hydrolysis product and the intact drug in the presence of minor degradates. Method reproducibility, demonstrated by repeated injections of a calibration standard, was 1.21%. The lower limit of quantitation of the hydrolysis products, p-chlorobenzenesulfonamide and p-toluenesulfonamide, was 0.2 microgram/5-microliter injection. The accuracy of the method for intact drugs was determined by comparison of the HPLC results to those obtained by the appropriate USP or BP assays. The mean of the results obtained by the two methods differed by 0.7% for chlorpropamide and 0.3% for tolbutamide. Pure drug samples were spiked with amounts of the hydrolysis products ranging from 20 to 120% of the intact content. The mean percent recovery for p-chlorobenzenesulfonamide was 98.6%; for p-toluenesulfonamide, it was 100.6%. A qualitative TLC procedure for the detection of chlorpropamide, p-chlorobenzenesulfonamide, dipropylurea, propylurea, n-propylamine, tolbutamide, p-toluenesulfonamide, dibutylurea, butylurea, and n-butylamine is also described.

Crystallinity and dissolution rate of tolbutamide solid dispersions prepared by the melt method.

The influence of cooling rate of solid dispersions prepared by the melt method was studied by X-ray diffraction and scanning electron microscopy. Tolbutamide was the model drug investigated, and the carriers included urea and polyethylene glycol 6000. Slow-cooled urea dispersions of tolbutamide demonstrated a complete lack of crystallinity, suggesting the formation of an amorphous material. The rapidly cooled dispersion showed peaks for urea and an absence of drug in the X-ray pattern, suggesting that a true molecular dispersion was formed. The X-ray patterns of rapid- and slow-cooled dispersions of tolbutamide and polyethylene glycol 6000 demonstrated that a physical mixture of drug and carrier resulted from both methods of dispersion preparation.

Simultaneous determinations of tolbutamide and its hydroxy and carboxy metabolites in serum and urine: application to pharmacokinetic studies of tolbutamide in the rat.

Methods of analysis of tolbutamide (1) and its hydroxylated (2) and carboxylated (3) metabolites in serum and urine based on high-performance liquid chromatography were developed. The separation was performed on a Apex ODS column in the isocratic mode using a mobile phase composed of 22.5% acetonitrile, 77.5% Sorensen phosphate buffer (pH 7.0), and 0.30 mL of tetrabutylammonium phosphate reagent (Pic A). The compounds were detected at 254 mm. The retention times of 3, 2, 1, and the internal standard chlorpropamide were 3.1, 4.1, 14.8, and 10.0 min, respectively. These conditions were suitable for the simultaneous quantitation of 1, 2, and 3 in serum or plasma samples, but not for the determination of metabolites 2 and 3 in urine. For the analysis of 2 and 3 in urine, the mobile phase was modified to 18% acetonitrile, 82% Sorensen phosphate buffer (pH 7.0), and 0.35 mL of Pic A. Under these conditions, the retention times of the carboxy and hydroxylated metabolites and the internal standard salicylic acid were 4.6, 6.7, and 8.1 min, respectively. These methods were applied to study the pharmacokinetics of 1 administered intravenously and intraperitoneally to the rat. Tolbutamide was almost completely recovered as metabolites 2 and 3 in the urine within 24 h.

Inhibitory effect of nicotine and its metabolites on tolbutamide hydroxylation in rat liver microsomes.

A simple HPLC/fluorescence method to detect hydroxytolbutamide (a major metabolite of the anti-diabetic drug tolbutamide) has been developed. The effects of nicotine and some of its metabolites on tolbutamide hydroxylation is described. An extraction procedure with diethyl ether was followed by isocratic HPLC analysis of tolbutamide hydroxylation with a binary mobile phase composed of 10 mM monobasic sodium phosphate in methanol (45:55, v/v, apparent pH 2.28). A detection limit of sub-nanogram amounts (0.353 ng) of hydroxytolbutamide was obtained with fluorescence detection at 226 nm for excitation and 318 nm for emission. Overall precision values for hydroxytolbutamide was determined with coefficients of variation of 1.4-4.6% when nanogram levels of the metabolite were analyzed. Differential inhibitory responses were demonstrated for tolbutamide hydroxylation to nicotine and its metabolites. Tolbutamide hydroxylation was apparently inhibited by cotinine and relatively less inhibited by nicotine. Nornicotine, however, caused very little inhibition of tolbutamide hydroxylation. The implication is that nornicotine may not share similar affinity for the substrate binding site for tolbutamide. The results also suggest that heavy smokers may experience reduction in tolbutamide metabolism. The assay system itself will be useful for future studies of tolbutamide, and possibly related sulfonylureas.

An infrared study of tautomerism in acetohexamide polymorphs.

Infrared data determined for known polymorphic forms and some new derivatives of acetohexamide and related compounds support the view that acetohexamide polymorphs exhibit keto-enol tautomerism. They indicate that type A polymorphs exist in the enol form, probably stabilized by intramolecular bonding between an O-H and S = O group to form a six-membered ring. Type B polymorphs exist in the keto form with the urea carbonyl group intermolecularly bonded to a sulphonamide N-H. The new evidence disputes previous interpretations of the data.

[Polymorphism of drugs in powders and tablets. 6. X-ray diffractometric studies of polymorphic modifications of tolbutamide]. / Untersuchungen zur Polymorphie von Arzneistoffen in Pulvern und Tabletten. 6. Mitteilung: Röntgendiffraktometrische Untersuchungen polymorpher Formen des Tolbutamids.

The modifications and spray dried products of tolbutamide published in a former article, are investigated furthermore by X-Ray diffraction. The indication of the reflexes in X-Ray diffraction patterns of 2 modifications is performed. Transformations of modification are provable in consequence of thermal stress.

Characterization of the binding of sulfonylurea drugs to HSA by high-performance affinity chromatography.

Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (Ka) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high-affinity binding regions and weaker binding sites for each drug, with average Ka values of 1.3 (+/-0.2) x 10(5) and 3.5 (+/-3.0) x 10(2) M(-1) for acetohexamide and values of 8.7 (+/-0.6) x 10(4) and 8.1 (+/-1.7) x 10(3) M(-1) for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high-affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug-binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with Ka values of 1.3 (+/-0.1) x 10(5) and 4.3 (+/-0.3) x 10(4) M(-1), respectively, at 37 degrees C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with Ka values of 5.5 (+/-0.2) x 10(4) and 5.3 (+/-0.2) x 10(4) M(-1), respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug-protein interactions.