Pharmacokinetic evaluation of oral fenofibrate nanosuspensions and SLN in comparison to conventional suspensions of micronized drug.

An increasing number of newly developed drugs show bioavailability problems due to poor water solubility. Formulating the drugs as nanosuspensions may help to overcome these problems by increasing saturation solubility and dissolution velocity. In the present study the bioavailability of the poorly soluble fenofibrate following oral administration was investigated in rats. Four formulations were tested: a nanosuspension type DissoCube(R), one solid lipid nanoparticle (SLN) preparation and two suspensions of micronized fenofibrate as reference formulations, one suspension in sirupus simplex and a second in a solution of hydroxyethy-cellulose in physiological saline. Both colloidal drug delivery systems showed approximately two-fold bioavailability enhancements in terms of rate and extent compared to the reference formulations. No significant differences were found in AUC(0-22 h) as well as in C(max) and t(max) between the two colloidal delivery systems. In conclusion, nanosuspensions may be a suitable delivery system to improve the bioavailability of drugs with low water solubility.

Rate-limiting biotransformation of triamterene is mediated by CYP1A2.

OBJECTIVE: Triamterene (TA), a potassium-sparing diuretic, is extensively metabolized by hydroxylation in 4'-position and subsequent conjugation by cytosolic sulfotransferases. To identify the cytochrome P450 enzyme(s) catalyzing hydroxylation of triamterene (the rate-limiting step in the formation of the sulfate ester (STA)), in vitro incubation studies were performed with human liver microsomes. METHODS: Initial rates of TA hydroxylation (0 - 300 microM) were determined during a ten-minute-incubation period with liver microsomes of two donors. The role of individual CYP enzymes was determined by pre-incubation with selective inhibitors/alternative substrates. Vice versa, the effect of TA (0 - 500 microM) on 3-demethylation of caffeine (0 - 1,000 microM) was assessed. Metabolite concentrations were estimated by reversed-phase HPLC methods. RESULTS: TA Km values without inhibitors were 60 and 142 microM, Vmax was 177 and 220 pmol/min/mg protein, respectively. Mean inhibitor induced changes of 4'-hydroxy-TA formation were as follows: Furafylline 25 microM (CYP1A2), complete inhibition (-100%); omeprazole 250 microM (CYP1A2 inhibitor/CYP2C 19 substrate), -30%; coumarin 25 microM (CYP2A6), -11%; quinidine 25 microM (CYP2D6), -9%; ketoconazole 25 microM (CYP3A), -18%; and erythromycin 250 microM (CYP3A), -8%. In the reverse inhibition studies, TA competitively inhibited caffeine 3-demethylation with Ki values of 65 and 111 microM, respectively. CONCLUSION: 4'-hydroxylation of TA in humans appears to be mediated exclusively by CYP1A2. Inhibition or induction of CYP1A2 will change the time course of both TA and its active phase-II metabolite. The net pharmacodynamic effect of such changes is difficult to predict and needs to be evaluated in clinical studies.

Nanosuspension formulations for low-soluble drugs: pharmacokinetic evaluation using spironolactone as model compound.

Various particle sizes of spironolactone as a model low solubility drug were formulated to yield micro-and nanosuspensions of the type solid lipid nanoparticles and DissoCubes. Seven oral and one i.v. formulations were tested in an in vivo pharmacokinetic study in rats with the aim of characterizing the bioavailability of spironolactone on the basis of its metabolites canrenone and 7-alpha-thiomethylspirolactone. In addition, a dose escalation study was carried out using nonmicronized spironolactone suspension as well as a nanosuspension type DissoCubes. On the basis of AUC as well as Cmax ratios, three groups of formulations were distinguished. The biggest improvement was seen with a solid lipid nanoparticle formulation yielding a 5.7-fold increase in AUC for canrenone and a similar improvement based on the Cmax metric, followed by a group of three formulations containing nanosized, micronized, and coarse drug material and surfactant. The DissoCubes nanosuspension yielded highly significant improvements in bioavailability averaging 3.3-fold in AUC and 3.0-fold in terms of Cmax for canrenone. The third class encompasses all other formulations, which showed very little to no improvement in bioavailability. The results show that the particle size minimization was not the major determining factor in the bioavailability improvement. Rather, the type of surfactant used as stabilizer in the formulations was of greater importance. Improvement in drug solubility in the intestine as well as in dissolution rate of spironolactone are the most likely mechanisms responsible for the observed effect, although additional mechanisms such as permeability enhancement may also be involved.

Intestinal drug efflux: formulation and food effects.

The intestine, primarily regarded as an absorptive organ, is also prepared for the elimination of certain organic acids, bases and neutral compounds depending on their affinity to intestinal carrier systems. Several of the transport systems known to mediate efflux in the major clearing organs--liver and kidney--are also expressed in the intestine. Examples of secretory transporters in the intestine are P-glycoprotein, members of the multidrug resistance associated protein family, breast cancer resistance protein, organic cation transporters and members of the organic anion polypeptide family. In this communication, the P-glycoprotein mediated intestinal secretion of talinolol, a model compound showing metabolic stability, has been investigated in the jejunum, ileum and colon of rat intestine by single-pass perfusion. A model has been developed which demonstrates an increase in carrier-mediated secretion in the order jejunum

The influence of age on the pharmacokinetics and pharmacodynamics of bemetizide and triamterene: a single and multiple dose study.

Diuretics are a frequent cause of adverse drug effects in the elderly, many times involving drug-drug interactions. In addition, multiple chronic diseases, age-dependent pharmacokinetic and pharmacodynamic changes, and a decreased homeostatic capacity often complicate diuretic therapy in the elderly. The pharmacokinetics (area under the plasma concentration-time curve: AUC; peak concentration in plasma: c(max); time to reach peak concentration: t(max); terminal half-life: t(1/2)) and pharmacodynamics (urine flow rates and renal excretion rates of Na(+) at 1, 3, and 6 h after oral administration) of a fixed combination of 25 mg bemetizide and 50 mg triamterene were investigated in 15 elderly patients (age 70-84 years) and 10 young volunteers (age 18-30 years) after a single dose (day 1) and after multiple doses (at steady state, day 8). Compared with the young volunteers, mean plasma concentrations of bemetizide, triamterene, and the active triamterene metabolite were significantly higher in the elderly volunteers. These elevated plasma levels occurred after single dose and were even more pronounced after multiple dose in the elderly. While plasma concentrations and AUC of bemetizide, triamterene, and the active metabolite of triamterene were increasing in correlation to age of subjects and duration of therapy, urine flow and renal Na(+) excretion rates were decreasing at the same degree. At steady state conditions, practically no effect on urine flow and Na(+) excretion rates could be observed in the elderly patients (in contrast to the young volunteers) for the first 8 h after administration of bemetizide and triamterene. The lower the measured (endogenous) creatinine clearance was in all subjects, the higher were the plasma concentrations of bemetizide and triamterene, and the lower was the effect on pharmacodynamics (i.e. urine flow and renal Na(+) excretion rates). The glomerular filtration rate, known to be lower in the elderly (a priori), was apparently decreased at higher levels of bemetizide and triamterene in the elderly, which may explain why there was no diuretic and saluretic effect after multiple dose in the elderly patients.

Grapefruit juice enhances intestinal absorption of the P-glycoprotein substrate talinolol.

Grapefruit juice (GFJ) is known to affect the pharmacokinetics of various drugs, presumably mainly via inhibition of oxidative metabolism. In order to evaluate the effect of GFJ on P-glycoprotein-related transport processes, measurements of transport characteristics through Caco-2 monolayers and in vivo drug absorption studies were performed with the transported, yet not metabolized model compound talinolol. Apical-to-basolateral talinolol transport in the Caco-2 model at 1 mM racemate concentration was increased almost 3-fold when GFJ was present (S-talinolol P(eff): 0.16 x 10(-6) vs. 0.61 x 10(-6) cm/s without vs. with GFJ; R-talinolol P(eff): 0.19 x 10(-6) vs. 0.71 x 10(-6) cm/s without vs. with GFJ). In vivo in rats, doubled maximum plasma concentrations, enhanced AUC values (C(max) of S-talinolol: control, 77.5 ng/ml vs. GFJ, 163.6 ng/ml; C(max) of R-talinolol: control, 79.5 ng/ml vs. GFJ, 163.0 ng/ml; AUC of S-talinolol: control, 19.3 microg ml(-1)min vs. GFJ, 29.9 microg ml(-1)min; AUC of R-talinolol: control, 22.2 microg ml(-1)min vs. GFJ, 30.1 microg ml(-1)min), and decreased apparent oral clearances were found for both talinolol enantiomers when GFJ was administered together with a racemic 10 mg/kg b.w. p.o. dose. Furthermore, GFJ tended to accelerate the rate of talinolol input, but did not significantly affect terminal talinolol half-lives. It is concluded that inhibition of intestinal secretion may contribute to bioavailability enhancement upon GFJ intake.

Pretreatment with potent P-glycoprotein ligands may increase intestinal secretion in rats.

The expression of P-glycoprotein is induced in cell cultures upon exposure to various inducers. Therefore, the aim of the present study was to evaluate the in-vivo relevance of this observation, i.e. the influence of chronic pretreatments with selected drugs -- all of which are ligands to P-glycoprotein (P-gp) as demonstrated in radioligand binding studies and all of which have some or a considerable effect on P-gp expression in Caco-2 cells -- on the effective intestinal permeabilities of the model compound talinolol in rats employing in-situ single-pass intestinal perfusion of three different gut segments. Talinolol was selected, because it shows high selectivity for one of the exsorptive transporters (P-gp) and its intestinal permeability is very sensitive to changes in exsorption when the perfusate concentration is low. Prior to the induction study the perfusion model was optimized regarding the type and concentration of a competitive inhibitor which may be used to block the exsorption-related permeability reduction (through intestinal exsorption) during an ongoing perfusion and would permit an intra-individual comparison of the effective permeability without and with blockade of exsorption. While repetitive verapamil and talinolol dosing had no statistically significant exsorption-inducing effect, vinblastine and rifampicin pretreatments resulted in decreased intestinal talinolol permeabilities in the three tested gut segments, duodenum, jejunum, and colon [e.g., S-talinolol in jejunum: control, 2.50 x 10(-4) cm/s; vinblastine induction, 1.48 x 10(-4) cm/s (P<0.05); rifampicin induction, 1.51 x 10(-4) cm/s (P<0.05)]. Addition of an efficient secretion inhibitor (vinblastine) to the perfusate permitted the determination of the impact of inhibitable secretory processes on the total effective permeabilities and an estimation of passive permeability in the respective individual. The inhibitable permeability fractions were higher for vinblastine than for any other pretreatment and the difference from control pretreatment was statistically significant for all intestinal segments (duodenum, 61.8%; jejunum, 63.1%; colon, 43,7%; S-talinolol). Statistically significant differences were also detected for rifampicin in the perfused duodenum and jejunum (33.1 and 27.5% increase in inhibitable fraction, respectively, for S-talinolol). These differences are explained by a significant induction of outside-directed transport in the intestinal enterocytes by vinblastine and rifampicin.

Screening of beta-2 agonists and confirmation of fenoterol, orciprenaline, reproterol and terbutaline with gas chromatography-mass spectrometry as tetrahydroisoquinoline derivatives.

A new method for a comprehensive screening and confirmation of beta-2 agonists in human urine is presented based on gas chromatography-low-resolution mass spectrometry (GC-MS) using electron impact ionisation (EI). After hydrolysis of the conjugates with beta-glucuronidase/arylsulfatase a derivatisation step with formaldehyde converts fenoterol, orciprenaline, reproterol and terbutaline to one derivative, a tetrahydroisoquinoline, while the other beta-2 agonists remain unchanged. Liquid-liquid extraction and trimethylsilylation follow. The tetrahydroisoquinoline derivatives show good gas chromatographic and mass spectrometric behaviour. The detection limit of these four beta-2 agonists in the screening using low-resolution mass spectrometry is 10 ng/ml of urine. The other beta-2 agonists are detected as parent compounds with the same recovery after sample preparation with and without formaldehyde. The EI mass spectra of the tetrahydroisoquinoline derivatives are presented.

Comparative pharmacodynamics and pharmacokinetics of candesartan and losartan in man.

The angiotensin II antagonistic effects of candesartan and losartan were compared in-vivo after single and repeated doses. Effects were related to antagonistic activity in plasma. In this double-blind, crossover study, 12 healthy male volunteers received, in random order, daily oral doses of 8 mg candesartan cilexetil or 50 mg losartan for seven days. On day 1 and day 8, dynamics and kinetics were assessed up to 48 h after dosing. Antagonistic effect was determined from the antagonist-induced rightward shifts of the diastolic blood pressure response curves to exogenously administered angiotensin II measured as the dose ratio (DR). The antagonistic activity in plasma was measured using an ex-vivo/in-vitro radioreceptor assay. Specific high-performance liquid chromatography assays determined plasma concentrations of candesartan, losartan and its active metabolite EXP-3174. The pharmacokinetic properties of candesartan and losartan were comparable and antagonistic activity in plasma almost identical (ratio candesartan: losartan = 0.97 and 1-2 after single and multiple doses, respectively). However, the antagonistic effects of candesartan and losartan in-vivo were quite different. Twenty-four hours after single dosing with candesartan a clinically relevant rightward shift in the angiotensin II dose-response curve (DR= 3.2) occurred that was more pronounced than that following losartan administration (DR=2.1, ratio candesartan: losartan= 1.65). Twenty-four hours after multiple doses of candesartan or losartan, the values of the DR were 4.8 and 2.3, respectively (ratio candesartan: losartan = 1.94). The values of DR for candesartan were significantly higher compared with losartan between 6 and 36h after a single dose and between 3 and 24 h post-dose following multiple dose administration. A counter-clockwise hysteresis was apparent between antagonistic activity in plasma and antagonistic effect. Despite equivalent angiotensin II antagonistic activity in plasma, the pharmacodynamic effect of candesartan cilexetil was greater than that of losartan. Candesartan appeared to have a slower off-rate from the angiotensin AT1-receptor compared with losartan, nevertheless differences in distributional phenomena or the extent of insurmountable antagonistic activity cannot be ruled out.

Gemfibrozil and its oxidative metabolites: quantification of aglycones, acyl glucuronides, and covalent adducts in samples from preclinical and clinical kinetic studies.

A gradient reversed-phase HPLC analysis for the direct measurement of gemfibrozil (GEM) and four oxidative metabolites in plasma and urine of humans and in tissue homogenates of rats was developed. The corresponding acyl glucuronides and the covalently bound protein adducts (in protein precipitates) were determined after liberation from the respective conjugates via alkaline hydrolysis. The limits of detection for the covalent adducts in human plasma are: 10 ng ml(-1) (GEM), 20 ng ml(-1) (M1), 0.5 ng ml(-1) (M2, M4), and 5 ng ml(-1) (M3). The method was validated with respect to selectivity, recovery, linearity, precision, and accuracy. It has been applied to the analysis of preclinical and clinical studies. Pharmacokinetic profiles of gemfibrozil, its metabolites, and covalent adducts in human plasma and rat tissue homogenates are given.

Affinities at the verapamil binding site of MDR1-encoded P-glycoprotein: drugs and analogs, stereoisomers and metabolites.

OBJECTIVES: The multidrug transporter P-glycoprotein (P-gp) appears to play a significant role in drug absorption and disposition. Hence, it is of interest to evaluate structure-affinity relationships for the purpose of making predictions. METHODS: The affinity to P-gp of related molecular structures from various groups of drugs was determined using competitive radioligand-binding assays. Structural analogs, stereoisomers and metabolites of verapamil-type calcium antagonists, beta-adrenoceptor antagonists as well as omeprazole, omeprazole enantiomers and the sulfone metabolite of omeprazole were investigated. RESULTS: Whereas some stereoselectivity was detected for the high-affinity P-gp substrates verapamil and carvedilol, little or no differences were observed in the case of other beta-blockers. One of the 4 labetalol stereoisomers, the R,R-isomer dilevalol, had an IC50 value half that of labetalol. CONCLUSIONS: Metabolites of verapamil, gallopamil, carvedilol and omeprazole are characterized by having higher IC50 values (lower P-gp affinity) than the respective parent compounds. Only the acebutolol metabolite, diacetolol, had a P-gp affinity comparable to that of the parent compound.

Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport.

PURPOSE: The objective of this study was a systematic characterization and evaluation of cell culture models based on mixtures of Caco-2/HT29-MTX co-cultures for their use in screening for drug absorption and intestinal permeability in comparison to the properties of the respective mono-cultures. METHODS: Co-cultures of Caco-2 cells (absorptive-type) and HT29-MTX cells (goblet-type) were set up. Three different co-cultures (initial seeding ratios Caco-2/HT29-MTX: 90/10, 70/30, and 50/50) were grown on permeable filter supports, and monolayers were used for permeability studies with model compounds for paracellular absorption (atenolol, furosemide, H334/75, mannitol, terbutaline), transcellular absorption (antipyrine, ketoprofen, metoprolol, piroxicam), carrier-mediated absorption (D-glucose, Gly-Pro, and L-phenylalanine) as well as substrates for carrier-mediated secretion via P-glycoprotein (cimetidine and talinolol). Electrophysiological and microscopic controls were performed to characterize the cell cultures. RESULTS: For compounds undergoing passive intestinal absorption permeabilities were generally higher in co-cultures than in Caco-2 monolayers, yielding highest values in pure HT29-MTX monolayers. This difference was most obvious for compounds transported via the paracellular pathway, where HT29-MTX cells may be up to 30 times more permeable than Caco-2 cells, whereas for lipophilic and highly permeable compounds, the difference in permeability values was less obvious. For drugs undergoing intestinal secretion mediated by P-glycoprotein, co-cultivation of Caco-2 cells with HT29-MTX cells led to increased apical to basolateral permeability which was decreased in the opposite direction, consistent with the fact that HT29-MTX cells do not express P-glycoprotein. When a carrier-mediated absorption mechanism is involved, the permeabilities observed were lower than the values reported for human small intestine and co-cultivation of HT29-MTX cells with Caco-2 cells resulted in even lower values as compared to the plain Caco-2 cultures. CONCLUSIONS: Co-cultures of HT29-MTX and Caco-2 cells offer the opportunity of modifying the permeability barrier of the cell monolayers both with respect to paracellular resistance and secretory transport via P-gp. Thus, in special cases, they allow more flexibility in adapting the in vitro system to the in vivo situation as compared to the monocultures. Another advantage is the obvious robustness of the method with respect to the reproducibility of the results. A problem remaining, however, is the quantitative expression of carriers involved in intestinal uptake of many nutrients and drugs.

Role of P-glycoprotein-mediated secretion in absorptive drug permeability: An approach using passive membrane permeability and affinity to P-glycoprotein.

It has been shown in vivo and in vitro that P-glycoprotein (P-gp) may be able to influence the permeability of its substrates across biological membranes. However, the quantitative contribution of the secretion process mediated by P-gp on the overall permeability of membranes has not been determined yet. In particular, observations need to be clarified in which substrates showing high affinity to P-glycoprotein, e.g., verapamil, apparently do not seem to be greatly influenced by P-gp in their permeability and consequently also with respect to their extent of GI-absorption after oral administration, whereas weaker substrates of P-gp, e.g., talinolol, have clearly shown P-gp-related absorption phenomena such as nonlinear intestinal permeability and bioavailability. Experiments with Caco-2 cell monolayers and mathematical simulations based on a mechanistic permeation model should aid in clarifying the underlying mechanism for these observations and quantifying the influence of passive membrane permeability and affinity to P-gp to the overall transmembrane drug flux. In addition, the concentration range of drug at which P-glycoprotein-mediated transport across the biological membrane is relevant should be examined. The permeability of various drugs in Caco-2 monolayers was determined experimentally and modeled using a combination of passive absorptive membrane permeability and a Michaelis-Menten-type transport process in the secretory direction. The passive permeabilities were experimentally obtained for the apical and basolateral membrane by efflux experiments using Caco-2 monolayers in the presence of a P-gp inhibitor. The Michaelis-Menten parameters were determined by a newly developed radioligand-binding assay for the quantification of drug affinity to P-gp. The model was able to accurately simulate the permeability of P-glycoprotein substrates, with differing passive membrane permeabilities and P-glycoprotein affinities. Using the outlined approach, permeability vs donor-concentration profiles were calculated, and the relative contribution of passive and active transport processes to the overall membrane permeability was evaluated. A model is presented to quantitatively describe and predict direction-dependent drug fluxes in Caco-2 monolayers by knowing the affinity of a compound to the exsorptive transporter P-gp and its passive membrane permeability. It was shown that a combination of high P-gp affinity with good passive membrane permeability, e.g., in the case of verapamil, will readily compensate for the P-gp-mediated reduction of intestinal permeability, resulting in a narrow range in which the permeability depends on the apical drug concentration. On the other hand, the permeability of compounds with low passive membrane permeability (e. g., talinolol) might be affected over a wide concentration range despite low affinity to P-gp.

Characterization of binding properties to human P-glycoprotein: development of a [3H]verapamil radioligand-binding assay.

Interaction with the exsorptive transporter P-glycoprotein (P-gp) is a possible source of peculiarities in drug pharmacokinetics, including dose-dependent absorption, drug-drug interactions, intestinal secretion, and limited permeability of the blood-brain barrier. Among the established in vitro methods of the analysis of drug interactions with P-gp, none directly quantifies the affinity of ligands with P-gp. Instead, they measure the result of a membrane permeation and a receptor-binding process; this may lead to difficulties in the interpretation of results. An assay for quantification of drug affinity to the transporter is presented on the basis of the radioligand-binding assay principle. This has the advantage of directly quantifying the interaction between drugs and P-gp. Because of the reversible and competitive interaction of numerous substrates with P-gp, a radioligand-binding assay was developed by taking [3H]verapamil and [3H]vinblastine as radioligands and the human intestinal Caco-2 cells, overexpressed with P-gp by culturing in the presence of vinblastine or transfecting with multidrug resistance gene MDR-1 as receptor preparation. The assay was performed in 96-well plates and has the potential to be used as a high-throughput method. A clear induction of the expression of P-gp was demonstrated in the Caco-2 cells grown in the presence of vinblastine, as well as in the transfected cells, although to a lesser extent. Both radioligands were shown to bind to P-gp. Verapamil was the radioligand of choice for further investigations due to its lower nonspecific binding to the transporter preparation. Kinetics as well as specificity of the binding of verapamil to the P-gp preparation were demonstrated. A two-affinity model was found to adequately describe the data derived from saturation as well as from competition experiments, in accordance with previous findings on two exsorption sites for P-gp. The binding properties of [3H]verapamil and [3H]vinblastine to a P-gp preparation derived from induced Caco-2 cells are described. The concentration-dependent displacement of the radioligand by nonlabeled substrates for P-gp should be a suitable principle for the determination of drug affinity to the respective binding sites at the human intestinal multidrug transporter P-gp.

Radioligand-binding assay employing P-glycoprotein-overexpressing cells: testing drug affinities to the secretory intestinal multidrug transporter.

PURPOSE: To develop a rapid and reliable system for affinity determination of conventional as well as newly synthesized compounds to P-gp. METHODS: The principles of radioligand-binding assay were adapted to the human intestinal P-gp. Acceptor protein was obtained from the human carcinoma cell line Caco-2, where overexpression of P-gp was induced by growing cells in the presence of the cytostatic drug vinblastine. 3H-Verapamil was chosen as radioligand. RESULTS: The saturability and specificity of 3H-verapamil as the radioligand for the binding to P-gp was demonstrated. From concentration dependence of displacement of the radioligand by various non-labeled ligands for P-gp, affinity constants to P-gp binding sites were calculated. The binding results obtained were in agreement with those published earlier where influx and efflux experiments with cell monolayers had been conducted in order to functionally characterize the P-gp -drug interaction. CONCLUSIONS: A radioligand-binding assay on the basis of P-gp overexpressing Caco-2 cells has been developed. The method might be suitable for high-throughput screening of drug interaction with human P-gp. It will allow modeling of the interaction of drugs with the human multidrug transporter and has also the potential to serve as a high-throughput screening tool to detect compounds prone to P-gp mediated intestinal secretion and potential P-gp related drug/drug interactions in drug discovery and early development.

P-Glycoprotein (P-gp) mediated efflux in Caco-2 cell monolayers: the influence of culturing conditions and drug exposure on P-gp expression levels.

The influence of cell culture conditions and previous drug exposure on P-glycoprotein (P-gp) expression levels in Caco-2 cells was determined. In this study, the expression of P-gp is demonstrated (i) visually by confocal laser scanning microscopy (CLSM), (ii) functionally by transport studies with substrates of the efflux pump, and (iii) quantitatively by flow cytometry (FCM) analysis using specific monoclonal antibodies (anti P-gp MRK 16 as an external antibody and P-GlycoCheck C219 as an internal antibody). Trypsinization of the cells after reaching confluence led to a decrease of P-gp expression levels, while trypsinization before reaching confluence led to an increase after long-term cultivation. Culturing the cells on polycarbonate filters did not elicit a significant change of P-gp expression over time in culture, whereas in plastic flasks (polystyrene) a decrease was detected. Using CLSM a strong fluorescence on the apical side of Caco-2 cell monolayers was observed, as a result of incubation with MRK 16 as primary and IgG Cy5 as secondary antibody. Previous drug exposure of the cells showed that verapamil, celiprolol, and vinblastine induced the P-gp expression, while metkephamid (MKA) decreased the P-gp expression level as compared to the control. Permeation studies consolidated the theory that P-gp is expressed in the Caco-2 cells examined. For talinolol and MKA, a higher transport from basolateral to apical side than from apical to basolateral could be measured. Incubation of the cell monolayer with MRK 16 reduced the secretion process to the apical side, but did not influence [3H]mannitol flux. Caco-2 cells seem to be a suitable cell line model for P-gp-mediated secretion studies. However, the variability of the P-gp expression requires careful control when this model is to be used in quantitative structure/secretion studies.

A radioreceptor assay for the analysis of AT1-receptor antagonists. Correlation with complementary LC data reveals a potential contribution of active metabolites.

A reliable and sensitive radioreceptor assay based on rat lung homogenate as receptor preparation was developed to determine the angiotensin-II antagonistic profile of losartan and its main active metabolite EXP 3174 as well as its congeners exemplified by UP 269-6 and SL 91.0102-90 DL. This method proved to be precise with an intra- and interday variability of less than 10% and a limit of quantification < or = 1 ng ml-1. The analysis of the Ki values in protein-free Hepes-buffer versus blank human or rat plasma revealed the distinct high plasma-protein binding of EXP 3174 which consequently caused a dramatic drop of potency from 10-15-fold in the buffer to only about 2-fold in control plasma, when compared to the parent compound losartan and the two congeners investigated. Upon evaluation of clinical samples by both the reported radioreceptor assay (RRA) and the established high-performance liquid chromatography (HPLC), the correlation of the normalized data pairs (concentration equivalents) suggested the contribution of active metabolites to the angiotensin-II antagonistic effect of SL 91.0102-90 DL, but not to the effect of UP 269-6. In the context of an extended preclinical study in rats, the correlation of RRA with the respective HPLC concentration equivalents of losartan and its main active metabolite EXP 3174 confirmed previous findings that only losartan and EXP 3174 exert the angiotensin-II-AT1 receptor blockade without the contribution of other metabolites (P.C. Wong, W.A. Price, A.T. Chiu et al., J. Pharmacol. Exp. Ther. 255 (1990) 211-217).

HPLC assays to simultaneously determine the angiotensin-AT1 antagonist losartan as well as its main and active metabolite EXP 3174 in biological material of humans and rats.

Novel rapid and sensitive HPLC assays were developed to simultaneously determine losartan and its main active metabolite EXP 3174 in biological material of humans and rats following solid-phase or liquid-liquid extraction. The analytes were separated on a 3 microm particle-sized ULTREMEX CN column, which was preceded by a 5 microm particle-sized guard column, using UV-detection at 245 nm. The assays provided high sensitivity with limits of quantification (LoQ) of 5 ng ml(-1) for both compounds in human and rat plasma and 10 ng ml(-1) in human and rat urine, respectively. In rat blood, bile and various tissues, limits of quantifications were achieved that ranged 10-15 ng per ml and per 100 mg tissue, respectively, for both analytes.

P-glycoprotein transporters and the gastrointestinal tract: evaluation of the potential in vivo relevance of in vitro data employing talinolol as model compound.

Among the different application routes peroral administration remains the one most widely used. Hence, mechanisms affecting p.o. bioavailability are of particular interest, also in drug development. In recent years, intestinal drug secretion mediated by the multi-drug resistance gene product P-glycoprotein (Pgp) has been discovered as a possible mechanism of low and erratic bioavailability. Due to the saturability of this process, a dose-dependent apparent oral clearance may be observed which decreases upon increasing dose. However, in vivo intestinal secretion might be revealed only in the lower or subtherapeutic dose range. In permeability studies with Caco-2 cell monolayers, the MDR-reversing agent verapamil inhibits secretion of P-glycoprotein substrates and, hence, increases apical-to-basolateral permeability. The aim of the rat studies with talinolol presented here was to test the relevance of the intestinal secretion process as well as the extent of inhibition by verapamil in ex vivo, in situ, and in vivo talinolol/verapamil drug-drug interaction studies. Intestinal secretion of talinolol was detected indirectly in ex vivo studies via transport inhibition with verapamil and directly in in situ intestinal perfusions in rats following a talinolol i.v. bolus. Both i.v. and p.o. verapamil appear to affect the concentration-time profiles of talinolol. Relevant observations with respect to drug absorption are the decreased apparent oral clearance upon verapamil coadministration as well as the decreased tmax and mean absorption times at high verapamil doses. Talinolol may be regarded as a potential model compound for mechanistic studies on Pgp interactions, including permeability as well as binding studies and the involvement of transporters other than Pgp.