Formulation and evaluation of celastrol-loaded liposomes

The main purpose of this study was to evaluate the intestinal absorption and the antineoplastic effect of the poorly water-soluble drug celastrol when liposomes were used as oral drug delivery system. Liposomes were prepared by the ethanol-injection method. An optimized liposome formulation composed of phospholipid, cholesterol and Tween-80 resulted in favorable encapsulation efficiency at 98.06 ± 0.94%. Homogeneous and stable particle size of 89.6 ± 7.3 nm and zeta potential of -(87.7 ± 5.8) mV were determined by laser particle size analyzer. Subsequently, the four-site perfusion rat intestinal model revealed that celastrol-loaded liposomes had improved effective permeability compared to the free drug in four intestinal segments (p < 0.05). Moreover, celastrol-loaded liposomes could also inhibit the tumor growth in C57BL/6 mice. These results suggest that liposomes could be a promising perioral carrier for celastrol.     

脂质体荧光衰减技术预测10种中药单体的被动吸收

制备了DPH荧光脂质体,选用10种药理活性较好的中药单体,测定了这些中药单体进入荧光脂质体后引起的荧光衰减百分率,评估中药单体的细胞膜通透性。采用大鼠在体肠单向灌流实验测定了中药单体的小肠有效透皮系数(Peff),与中药单体引起的脂质体荧光衰减数据对比,两者相关性良好,从而证明荧光脂质体模型可以应用来预测中药单体的被动吸收。用DPH标记红细胞膜,测定了以上10种中药单体对荧光标记的红细胞膜的荧光衰减百分率,发现荧光脂质体与红细胞膜相关性良好,荧光脂质体模型的确可以一定程度上替代制备繁琐的红细胞膜,用来研究中药单体在真实细胞膜中的通透性。综合而言,荧光脂质体模型简单易行、重现性好、所需样品量少,适合于中药早期研究的高通量筛选。     

Ultraviolet photochemistry of trichlorovinylsilane and allyltrichlorosilane: vinyl radical (HCCH2) and allyl radical (H2CCHCH2) production in 193 nm photolysis

The absolute gas phase ultraviolet absorption spectra of trichlorovinylsilane and allyltrichlorosilane have been measured from 191 to 220 nm. Over this region the absorption spectra of both species are broad and relatively featureless, and their cross sections increase with decreasing wavelength. The electronic transitions of trichlorovinylsilane were calculated by ab initio quantum chemical methods and the observed absorption bands assigned to the A(1)A'<-- X[combining tilde](1)A' transition. The maximum absorption cross section in the region, at 191 nm, is sigma = (8.50 +/- 0.06) x 10(-18) cm(2) for trichlorovinylsilane and sigma = (2.10 +/- 0.02) x 10(-17) cm(2) for allyltrichlorosilane. The vinyl radical and the allyl radical are formed promptly from the 193 nm photolysis of their respective trichlorosilane precursors. By comparison of the transient visible absorption and the 1315 nm I atom absorption from 266 nm photolysis of vinyl iodide and allyl iodide, the absorption cross sections at 404 nm of vinyl radical ((2.9 +/- 0.4) x 10(-19) cm(2)) and allyl radical ((3.6 +/- 0.8) x 10(-19) cm(2)) were derived. These cross sections are in significant disagreement with literature values derived from kinetic modeling of allyl or vinyl radical self-reactions. Using these cross sections, the vinyl radical yield from trichlorovinylsilane was determined to be phi = (0.9 +/- 0.2) per 193 nm photon absorbed, and the allyl radical yield from allyltrichlorosilane phi = (0.7 +/- 0.2) per 193 nm photon absorbed.     

High-performance liquid chromatography method development and validation for simultaneous determination of five model compounds, antipyrine, metoprolol, ketoprofen, furosemide and phenol red, as a tool for the standardization of rat in situ intestinal permeability studies using timed wavelength detection

A simple, precise, accurate and rugged reversed-phase high-performance liquid chromatography (HPLC) method has been developed and validated for the simultaneous determination of five permeability model compounds, viz. antipyrine, metoprolol, ketoprofen, furosemide and phenol red. The method was intended to standardize rat in situ single-pass intestinal perfusion studies to assess the intestinal permeability of drugs in the market as well as new chemical entities. Optimum resolution was achieved by gradient elution on a Symmetry Shield C-18 analytical column with the mobile phase consisting of a mixture of aqueous potassium dihydrogen orthophosphate (pH 5.5; 0.01 m) and methanol at a flow rate of 1.5 mL/min. The retention times of antipyrine, metoprolol, ketoprofen, phenol red and furosemide were about 9, 12, 13, 16 and 17 min, respectively. Data acquisition was carried out using a photo diode array detector in the wavelength range 210-600 nm. Extraction of chromatograms was carried out by timed wavelength. Data obtained in all studies indicated that the method was suitable for the intended purpose. The validated method was found to be linear and precise in the working range. Suitability of storage under various conditions and freeze/thaw impact at cold temperature were established to ensure complete sample recovery without any stability issues. Recovery very close to the spiked amounts indicated that the method was highly accurate and suitable for use on routine basis.     

Determination and pharmacokinetics of periplocin in rat plasma by LC‐MS

A simple and sensitive LC‐MS method for the determination of periplocin in rat plasma was developed and validated. The chromatographic separation was carried out using a reverse‐phase Kromasil C₁₈ column(150 × 4.6 mm, i.d., 5 µm) with a mobile phase composed of methanol–water (76:24, v/v). The flow rate of mobile phase was 0.8 mL/min. The calibration curve was linear within the concentration range 1–1000 ng/mL. The intra‐ and inter‐day precisions across three validation days over the entire concentration range was lower than 9.2% in terms of relative standard deviation. Accuracy determined at three quality control concentrations ranged from ?2.0 to 6.0% in terms of relative error. The validated method was applied to the pharmacokinetic study of periplocin in rat plasma after intravenous and intramuscular administration. Copyright © 2010 John Wiley & Sons, Ltd.     

Effect of protein binding on the disposition of cephalexin and cefazolin in a simultaneous perfusion system of rat liver and kidney

The effect of protein binding on the disposition of cephalexin (CEX) and cofazolin (CEZ) was investigated in a simultaneous perfusion system of rat liver and kidney. In the present study, we used bovine serum albumin (BSA) or human serum albumin (HSA) as plasma protein to control the degree of perfusate protein binding of drugs. Total clearance (CLt) of CEX perfused with BSA (0.70 +/- 0.27 ml/min) was slightly smaller than that with HSA (0.89 +/- 0.08 ml/min), corresponding to the unbound fraction of the drug in the perfusate plasma. On the other hand, CLt of CEZ perfused with BSA (0.90 +/- 0.20 ml/min) was significantly larger than that with HSA (0.32 +/- 0.10 ml/min). The unbound fraction of CEZ to BSA (0.703 +/- 0.052) was much larger than that to HSA (0.253 +/- 0.017) and the clearance of the unbound drug did not differ significantly between two kinds of albumin perfusate (1.30 +/- 0.40 ml/min for BSA and 1.26 +/- 0.40 ml/min for HSA). These results suggest that plasma protein binding is an important factor determining the biliary clearance as well as the urinary clearance of drugs.     

Effect of co‐administration of Acori Tatarinowii Rhizoma volatile oil on pharmacokinetic fate of xanthotoxol,oxypeucedanin hydrate,and byakangelicin from Angelicae Dahuricae Radix in rat

A combination of Angelicae Dahuricae Radix and Acori Tatarinowii Rhizoma has been widely used as the herb pair in traditional Chinese medicine to treat stroke, migraine, and epilepsy. However, the underlying synergistic mechanism of the herb pair remains unknown. This study was aimed at investigating the effects of Acori Tatarinowii Rhizoma volatile oil on the pharmacokinetic parameters of xanthotoxol, oxypeucedanin hydrate, and byakangelicin from Angelicae Dahuricae Radix in rat, and in vitro absorption behavior of the three compounds using rat everted gut sac, in situ single‐pass intestinal perfusion, and Caco‐2 cell monolayer models. The pharmacokinetic study exhibited clear changes in the key pharmacokinetic parameters of the three main coumarins through co‐administering with Acori Tatarinowii Rhizoma volatile oil (50 mg/kg), the area under curve and the maximum plasma concentration of xanthotoxol increased 1.36 and 1.31 times; the area under curve, the maximum plasma concentration, mean residence time, half‐life of elimination, and the time to reach peak concentration of oxypeucedanin hydrate increased by 1.35, 1.18, 1.24, 1.19 and 1.49 times, respectively; the area under curve, mean residence time, half‐life of elimination, and time to reach peak concentration of byakangelicin climbed 1.29, 1.27, 1.37, and 1.28 times, respectively. The three coumarin components were absorbed well in the jejunum and ileum in the intestinal perfusion model, when co‐administered with Acori Tatarinowii Rhizoma volatile oil (100 μg/mL). The in vivo and in vitro experiments showed good relevance and consistency. The results demonstrated that the three coumarin compounds from Angelicae Dahuricae Radix were absorbed through the active transportation, and Acori Tatarinowii Rhizoma volatile oil could promote the intestinal absorption and transport of these compounds by inhibiting P‐glycoprotein (P‐gp)‐mediated efflux.     

A simple and rapid high-performance liquid chromatography method for determining furosemide, hydrochlorothiazide, and phenol red: applicability to intestinal permeability studies

A simple reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet detection at 280 nm was developed for simultaneous quantitation of furosemide and hydrochlorothiazide along with phenol red as a nonabsorbable marker for in situ permeability studies in anaesthetized rats. A jejunal segment of approximately 10 cm was isolated and cannulated in both ends for inlet and outlet solution. The perfusate was collected every 10 min, and samples were analyzed using the developed method. The mobile phase was acetonitrile-water-triethylamine-glacial acetic acid (41.5 + 57.4 + 0.1 + 0.9, adjusted to pH 5.6) at a flow rate of 1 mL/min; the run time was 9 min. The calibration graphs were linear for all 3 compounds (r > 0.999) across the concentration range of 7.93-125 microg/mL for phenol red and 6.25-100 microg/mL for hydrochlorothiazide and furosemide. The limits of quantitation were 7.2, 8.9, and 6.8 microg/mL for furosemide, hydrochlorothiazide, and phenol red, respectively. The coefficients of variation for intraassay and interassay precision were less than or equal to 7.6%, and the accuracy was between 93.2-103.4%. Using the single pass intestinal perfusion technique and the suggested HPLC method for sample analysis, mean values of 0.25 x 10(-4) (+/-0.16) cm/s and 0.22 x 10(-4) (+/-0.13) cm/s were obtained for furosemide and hydrochlorothiazide, respectively.     

Measurement and pharmacokinetic analysis of unbound cephaloridine in rat blood by on-line microdialysis and microbore liquid chromatography

A technique involving rapid sampling of cephaloridine in rat blood was achieved using a combination of microdialysis and sensitive microbore liquid chromatography. A microdialysis probe was inserted into the jugular vein/right atrium of a Sprague-Dawley rat. Then after a real-time collection of the analyte by microdialysis, the dialysate was automatically injected into a liquid chromatographic system via an on-line injector. Following a 2 h stabilization period after the surgical procedure, cephaloridine (20 mg/kg, i.v.) was then administered via the femoral vein. Isocratic elution of cephaloridine was carried out with a mobile phase containing methanol-20 mM monosodium phosphate (25:75, v/v, pH 5.5), and the flow rate of the mobile phase was 0.05 mL/min within 10 min. Intra- and inter-assay accuracy and precision of the assay were each less than 10%. The in vivo recovery of the cephaloridine from the microdialysate was 49.7 +/- 8.0% and 42.4 +/- 8.4% for 0.5 and 1 microg/mL standards (n = 6), respectively. Based on the pharmacokinetic analysis, the elimination half-life was 32.2 +/- 8.6 min by cephaloridine administration (20 mg/kg, i.v., n = 6).     

Absorption,transport and insulin-mimetic properties of bis(maltolato)oxovanadium (IV) in streptozotocin-induced hyperglycemic rats by integrated mass spectrometric techniques

The use of V(IV) complexes as insulin-enhancing agents has been increasing during the last decade. Among them, 3-hydroxy-2-methyl-4-pyrone and 2-ethyl-3-hydroxy-4-pyrone (maltol and ethyl maltol, respectively) have proven to be especially suitable as ligands for vanadyl ions. In fact, they have passed phase I and phase II clinical trials, respectively. However, the mechanism through which those drugs exert their insulin-mimetic properties is still not fully understood. Thus, the aim of this study is to obtain an integrated picture of the absorption, biodistribution and insulin-mimetic properties of the bis(maltolato)oxovanadium (IV) (BMOV) in streptozotocin-induced hyperglycaemic rats. For this purpose, BMOV hypoglycaemic properties were evaluated by monitoring both the circulating glucose and the glycohemoglobin, biomarkers of diabetes mellitus. In both cases, the results were drug concentration dependent. Using doses of vanadium at 3 mg/day, it was possible to reduce the glycaemia of the diabetic rats to almost control levels. BMOV absorption experiments have been conducted by intestinal perfusion revealing that approximately 35% of V is absorbed by the intestinal cells. Additionally, the transport of the absorbed vanadium (IV) by serum proteins was studied. For this purpose, a speciation strategy using high-performance liquid chromatography (HPLC) for separation and inductively coupled serum mass spectrometry, ICP-MS, for detection has been employed. The obtained HPLC-ICP-MS results, confirmed by MALDI-MS data, showed evidence that V, administered orally, is uniquely bound to transferrin in rat serum.     

Interaction of the main components from the traditional Chinese drug pair Chaihu-Shaoyao based on rat intestinal absorption

The Chaihu-Shaoyao drug pair (Bupleuri Radix and Paeoniae Radix Alba) which is a traditional Chinese drug pair, has been widely used for anti-inflammatory purposes. Saikosaponin a (SSA), saikosaponin d (SSD) and paeoniflorin are identified as the main components in the pair. The present study focused on the interaction of the main components based on investigating their intestinal absorption using a four-site perfused rat intestinal model in order to clarify the mechanism of the compatibility of Chaihu-Shaoyao. The concentrations of SSA, SSD and paeoniflorin in the intestinal perfusate were determined by LC/MS or UPLC (Ultra Performance Liquid Chromatography) methods, followed by P*(eff) (effective permeability) and 10% ABS (the percent absorption of 10 cm of intestine) calculations. The results showed that all of the three main components displayed very low permeabilities (P*(eff) < 0.4), which implied their poor absorption in the rat intestine. The absorption levels of SSA and SSD were similar in intestine and higher in ileum than those in other intestinal regions in the decreasing order: colon, jejunum and duodenum. However, there is no significant difference in the absorption of paeoniflorin in the four segments (P < 0.05). The P*(eff) values of paeoniflorin exhibited an almost 2.11-fold or 1.90-fold increase in ileum when it was co-administrated with SSA and SSD, as well as 2.42-, 2.18-fold increase in colon, respectively, whereas the absorptions of SSA and SSD were not influenced by paeoniflorin. In conclusion, SSA and SSD could promote the absorption of paeoniflorin. To some extent this might explain the nature of the compatibility mechanisms of composite formulae in TCMs.     

Quantitative determination of trace amounts of Al-citrate by anion-exchange FPLC-ETAAS

An anion-exchange fast protein liquid chromatographic-electrothermal atomic absorption spectrometric procedure (FPLC-ETAAS) was developed for determination of trace amounts of negatively charged Al-citrate in the pH range 3.5-8.0. Aqueous-4 mol dm(-3) NH(4)NO(3) linear gradient elution at a flow rate of 1 cm(3) min(-1) was applied for 10 min to separate Al-citrate on a FPLC Mono Q HR 5/5 column. The separated aluminium species were determined 'off line' by ETAAS in 0.5 cm(3) fractions. After separation the column was regenerated for 5 min with 4 mol dm(-3) NH(4)NO(3) and equilibrated with water. All reagents used in the separation procedure were cleaned with a silica based LiChrosorb RP-18 HPLC column to remove traces of aluminium. The main advantage of NH(4)NO(3) as eluent lies in its ability to decompose quantitatively in the graphite tube during the ashing step, which enables reproducible analysis of aluminium in the separated fractions. Using the procedure developed reproducible (RSD+/-2.0%) and quantitative determination of negatively charged Al-citrate at a retention time of 4.5 min was obtained. The LOD was found to be 2.0 ng cm(-3) of Al-citrate. The technique was successfully applied for the determination of Al-citrate in human serum. Spiked samples (50-150 ng Al(3+) cm(-3)) were microultrafiltered through a membrane filter (cut-off 30 000 Da) to separate aluminium bound to transferrin from low molecular weight aluminium complexes. It was found that 15-19% of aluminium in spiked samples from healthy volunteers passed through the membrane. By applying FPLC separation it was proved that all the aluminium in the filtrate corresponded to Al-citrate. The analytical technique developed enabled quantitative and reproducible determination (RSD+/-3.0%) of Al-citrate in spiked human serum at levels which could be found in patients undergoing long term haemodialysis.     

Permeability of atenolol and propranolol in the presence of dimethyl sulfoxide in rat single-pass intestinal perfusion assay with liquid chromatography/UV detection

A simple and sensitive RP-HPLC-UV method was developed and validated for simultaneous determination of atenolol and propranolol and subsequently applied to investigate the effect of dimethyl sulfoxide in rat in situ intestinal permeability studies. Atenolol (400 microm) and propranolol (100 microm) were perfused in the small intestine of anaesthetized (pentobarbitone sodium 60 mg/kg, i.p.) male Sprague-Dawley rats either in the presence (1, 3 and 5%) or in the absence of dimethyl sulfoxide. There was no significant alteration (p > 0.05) in the permeability of atenolol and propranolol, which indicated there was no effect of various concentrations of dimethyl sulfoxide (1-5%) on the membrane integrity of the rat intestinal tissues. The analytical method was validated on a C(4) column with a mobile phase comprising ammonium acetate buffer (pH 3.5, 0.02 m) and acetonitrile in the ratio of 30:70 (v/v) at a flow rate of 1.0 mL/min. The validated method was found to be accurate and precise and stability studies were carried out at different storage conditions and both analytes were found to be stable. These findings are applicable for determining the absorbability of water-insoluble drugs and new chemical entities for the purpose of classifying them in the biopharmaceutical classification system.     

Enhancing effect of glyceryl-1-monooctanoate on the rectal absorption of gentamicin from hollow-type suppositories in rabbits

A hollow-type suppository containing gentamicin (GM) in its cavity was prepared using Witepsol H-15 (H-15) mixed with glyceryl-1-monooctanoate (MO) or MO alone in the body of the suppository (type I) and a suppository (type II) containing GM and MO in the cavity was constructed using H-15 in the body of the suppository. Without MO, GM (60 mg) was not absorbed (plasma GM levels less than 1 microgram/ml). However, the absorption of GM from the rectum of rabbits was enhanced by coadministered MO in types I and II. Even when the amount of GM was decreased to 6 mg (1/10), GM was observed in the plasma (Cmax, 3.5 +/- 0.3 micrograms/ml) after administration of the suppository made from MO mixed with H-15. The enhancing effect of MO on the rectal absorption of GM could not be further increased by incorporating an amount of MO larger than approximately 300 mg into the suppository. This study demonstrates that MO can be used in the two types of hollow suppositories as an effective enhancing agent of rectal absorption of poorly absorbed drugs such as GM.     

Using a non-invasive stable isotope tracer to measure the absorption of water in humans

The development of solutions that prevent dehydration or promote adequate re-hydration play a vital role in preventing fatigue during exercise, however, the methods commonly used to assess the hydration ability of such solutions are invasive and often assess the components of absorption separately. This paper describes using a non-invasive deuterium tracer technique that assesses gastric emptying and intestinal absorption simultaneously to evaluate the uptake of water during rest and exercise. The kinetics of absorption are further examined by mathematical modelling of the data generated. For the rest group, 0.05 g/kg of body weight of deuterium, contained in gelatine capsules, was ingested with ordinary tap water and saliva samples were collected every 5 min for one hour while the subject remained seated. The deuterium was administered as above for the exercise group but sample collection was during one hour of exercise on a treadmill at 55% of the subject's maximum heart rate. The enrichment data for each subject were mathematically modelled and the parameters obtained were compared across groups using an independent samples t-test. Compared with the rest condition, the exercise group showed delayed absorption of water as indicated by significant differences for the modelling parameters t2, t1/2, maximum absorption rate and solution absorption amount at t1. Labelling with a deuterium tracer is a good measure of the relative rate ingested fluids are absorbed by the body. Mathematical modelling of the data generates rates of maximum absorption and allows calculation of the percentage of the solution that is absorbed at any given time during the testing period.     

Physico-chemical effects of the major quassinoids in a standardized Eurycoma longifolia extract (Fr 2) on the bioavailability and pharmacokinetic properties, and their implications for oral antimalarial activity

A simple validated LC-UV method for the phytochemical analysis of four bioactive quassinoids, 13alpha(21)-epoxyeurycomanone (EP), eurycomanone (EN), 13alpha,21-dihydroeurycomanone (ED) and eurycomanol (EL) in rat plasma following oral (200 mg/kg) and intravenous administration (10 mg/kg) of a standardized extract Fr 2 of Eurycoma longifolia Jack was developed for pharmacokinetic and bioavailability studies. The extract Fr 2 contained 4.0%, 18.5%, 0.7% and 9.5% of EP, EN, ED and EL, respectively. Following intravenous administration, EP displayed a relatively longer biological half-life (t1/2 = 0.75 +/- 0.25 h) due primarily to its lower elimination rate constant (k(e)) of 0.84 +/- 0.26 h(-1)) when compared with the t1/2 of 0.35 +/- 0.04 h and k(e) of 2.14 +/- 0.27 h(-1), respectively of EN. Following oral administration, EP showed a higher C(max) of 1.61 +/- 0.41 microg/mL over that of EN (C(max) = 0.53 +/- 0.10 microg/mL). The absolute bioavailability of EP was 9.5-fold higher than that of EN, not because of chemical degradation since both quassinoids were stable at the simulated gastric pH of 1. Instead, the higher log K(ow) value of EP (-0.43) contributed to greater membrane permeability over that of EN (log K(ow) = -1.46) at pH 1. In contrast, EL, being in higher concentration in the extract than EP, was not detected in the plasma after oral administration because of substantial degradation by the gastric juices after 2 h. Similarly, ED, being unstable at the acidic pH and together with its low concentration in Fr 2, was not detectable in the rat plasma. In conclusion, upon oral administration of the bioactive standardized extract Fr 2, EP and EN may be the only quassinoids contributing to the overall antimalarial activity; this is worthy of further investigation.     

Fluoride determination with a micro flow-calorimeter

Fluoride is determined by an integration technique with a flow calorimeter. All samples are absorbed on an anion-exchange resin before being brought into the calorimetric system. The reaction between hydrofluoric add and sodium hydroxide is used in the determinations. The method is applied to samples with between 7 mug and 5 mg of fluoride. The confidence limit is +/-2-5 mug for small samples and +/-25 mug for large samples. The enthalpy of the reaction H(+) + F(-) --> HF((aq)) was found to be 13.77 +/- 0.08 kJ mole .     

Determination of nitrate and nitrite in rat brain perfusates by capillary electrophoresis

A fast and simple method for the direct, simultaneous detection of nitrite (NO(2) (-)) and nitrate (NO(3) (-)) in rat striatum has been developed using a capillary electrophoresis separation of low-flow push-pull perfusion samples. The method was optimized primarily for nitrite because nitrite is more important physiologically and is found at lower levels than nitrate. We obtained a complete separation of NO(2) (-) and NO(3) (-) in rat striatum within 1.5 min. Optimal CE separations were achieved with 20 mM phosphate, 2 mM cetyltrimethylammonium chloride (CTAC) buffer at pH 3.5. The samples were injected electrokinetically for 2 s into a 40 cm x 75 microm ID fused-silica capillary. The separation voltage was 10 kV (negative polarity), and the injection voltage was 16 kV (negative polarity). UV detection was performed at 214 nm. The limits of detection obtained at a signal-to-noise ratio (S/N) of 3 for nitrite and nitrate were 0.96 and 2.86 microM. This is one of the fastest separations of nitrite and nitrate of a biological sample ever reported. Interference produced by the high physiological level of chloride is successfully minimized by use of CTAC in the run buffer.     

Concurrent measurement of unbound genistein in the blood, brain and bile of anesthetized rats using microdialysis and its pharmacokinetic application

Genistein, the major isoflavone in soybeans, has been shown to have a wide range of effects. We used an HPLC-UV combined with microdialysis method to detect unbound genistein in rat blood, brain and bile. Genistein dialysates were eluted with a mobile phase containing acetonitrile-water (40:60, v/v, pH 3.5 adjusted by 0.1% acetic acid). Samples were separated using a phenyl (5 microm) column maintained at ambient temperature. The UV detector wavelength was set at 259 nm. The flow rate was 1.0 m/min. The limit of quantitation for genistein was 50 ng/ml. The in vitro recoveries of genistein were 31 +/- 1, 13 +/- 1 and 59 +/- 4% in microdialysis probes of blood, brain and bile, respectively (n = 4). Inter- and intra-assay accuracy and precision of the analysis were less than 10% in the concentration ranges of 0.05-5.0 microg/ml. A small ratio of genistein penetrates the blood-brain barrier (BBB) and goes through hepatobiliary excretion after genistein administration (10 or 30 mg/kg, i.v.). The brain-to-blood (AUC(brain)/AUC(blood)) and bile-to-blood (AUC(bile)/AUC(blood)) distribution ratios were 0.04 +/- 0.01 and 1.85 +/- 0.42, respectively for the dosage of genistein 30 mg/kg. After co-administration of cyclosporine, a P-glycoprotein (P-gp) inhibitor, the distribution ratios of genistein in brain and bile were not significantly altered. These results suggest that the BBB penetration and hepatobiliary excretion of genistein may not regulated by P-gp.