Prediction of fraction metabolized via CYP3A in humans utilizing cryopreserved human hepatocytes from a set of 12 single donors.

1. It has previously been demonstrated that metabolism of drugs via a single enzymatic pathway, particularly CYP3A4, is associated with increased risk for drug-drug interactions (DDI). Quantitative experimental systems as well as integrated prediction models to assess such risk during the preclinical phase are highly warranted. 2. The present study was designed to systematically investigate the performance of human cryopreserved hepatocytes in suspension to predict fraction metabolized via CYP3A (fmCYP3A) by assessing the ketoconazole sensitive intrinsic clearance (CLint) for five prototypical CYP3A substrates with varying degree of CYP3A dependent CLint in twelve individual hepatocyte batches. 3. We demonstrate that in contrast to well predicted mean hepatic metabolic clearance (CLH) and mean fmCYP3A data, the variability in CYP3A contribution for compounds having multiple metabolic pathways cannot be predicted from inhibition experiments using ketoconazole as inhibitor. Instead, data in the present paper indicate that the variability is larger after inhibition of CYP3A for compounds having multiple metabolic pathways. 4. It is therefore recommended to estimate the average CLint and fmCYP3A for a given test compound in a series (n = 10) of individual human hepatocyte batches.

Jejunal permeability and hepatic extraction of fluvastatin in humans.

OBJECTIVES: The primary objective was to investigate the effective permeability and the hepatic extraction of fluvastatin, a new 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, during a jejunal perfusion in humans. The secondary objective was to investigate the relationship between human jejunal effective permeability values and physicochemical properties for four different drugs. METHODS: Nine healthy male volunteers were included in the study, which consisted of two sequential study parts. In the first part, the jejunal effective permeability of fluvastatin, antipyrine, metoprolol, and atenolol was assessed with use of the regional jejunal perfusion approach (150 minutes, 2.0 ml/min). After a washout period of at least 5 days, the same subjects received an intravenous infusion of fluvastatin (20 minutes, 2.0 mg). Plasma samples were taken in both parts of the study and were analyzed for the content of fluvastatin. RESULTS: The mean hepatic extraction of fluvastatin was 67% after the jejunal perfusion and 73% after the intravenous infusion. The half-life of fluvastatin was approximately 60 minutes after both administration routes. The jejunal effective permeability and the fraction absorbed both correlated (r2 = 0.968, p < 0.05; and r2 = 0.994, p < 0.05) with the partition coefficient (log D, pH 6.5) but not with the molecular size or the hydrogen bond number. CONCLUSION: Fluvastatin is extracted by the liver to a large extent (about 70%) and has a short half-life after both oral and intravenous administration. In this study, the human jejunal effective permeability and the fraction absorbed for these four drugs were better predicted by log D (pH 6.5) than both the molecular size and the hydrogen bond number.

Evaluation of dynamic polar molecular surface area as predictor of drug absorption: comparison with other computational and experimental predictors.

The relationship between various molecular descriptors and transport of drugs across the intestinal epithelium was evaluated. The monolayer permeability (Pc) of human intestinal Caco-2 cells to a series of nine beta-receptor-blocking agents was investigated in vitro. The dynamic polar molecular surface area (PSAd) of the compounds was calculated from all low-energy conformations identified in molecular mechanics calculations in vacuum and in simulated chloroform and water environments. For most of the investigated drugs, the effects of the different environments on PSAd were small. The exception was H 216/44, which is a large flexible compound containing several functional groups capable of hydrogen bonding (PSAd,chloroform = 70.8 A2 and PSAd,water = 116.6 A2). The relationship between Pc and PSAd was stronger than those between Pc and the calculated octanol/water distribution coefficients (log Dcalc) or the experimentally determined immobilized liposome chromatography (ILC) retention. Pc values for two new practolol analogues and H 216/44 were predicted from the structure-permeability relationships of a subset of the nine compounds and compared with experimental values. The Pc values of the two practolol analogues were predicted well from both PSAd calculations and ILC retention studies. The Pc value of H 216/44 was reasonably well-predicted only from the PSAd of conformations preferred in vacuum and in water. The other descriptors overestimated the Pc of H 216/44 100-500-fold.

Failure of K+ to affect the potency of inhibitors of the neuronal noradrenaline carrier in the rat vas deferens.

To examine whether K+ affects the potency of inhibitors of neuronal uptake, experiments were carried out in the rat vas deferens after pretreatment of the animals with reserpine and after inhibition of monoamine oxidase and catechol-O-methyltransferase. Initial rates of the neuronal uptake of 3H-noradrenaline and IC50 values for uptake inhibition by desipramine, cocaine and (-)metaraminol were determined in the presence of various concentrations of external K+ (5-45 mmol/l), both at 100 mmol/l Na+ and 50 mmol/l Na+. When measured at the 3H-noradrenaline concentration used to determine IC50 values (0.024 mumol/l), neuronal uptake was progressively impaired by increasing K+ concentrations at 50, but not at 100 mmol/l Na+. Neither at 100 mmol/l Na+ nor at 50 mmol/l Na+ was there any consistent, concentration-dependent effect of K+ on the IC50 values of desipramine, cocaine and (-)metaraminol. The analysis of the saturation kinetics of 3H-noradrenaline uptake (determined in the presence of 50 mmol/l Na+ at 5 mmol/l K+ or 45 mmol/l K+) showed that high K+ concentrations inhibit neuronal uptake by decreasing Vmax without any change in Km. The results indicate that K+ does not competitively interact with Na+ at sites on the noradrenaline carrier which mediate the transport-stimulating properties of Na+. Hence, the inhibition of neuronal uptake produced by high K+ concentrations is probably due to membrane depolarization which simply reduces Vmax.

Chloride-dependence of the potency of inhibitors of the neuronal noradrenaline carrier in the rat vas deferens.

(1) Vasa deferentia obtained from reserpine-pretreated rats were exposed to 0.15 mumol l-1 3H-(-)noradrenaline (with monoamine oxidase and catechol-O-methyltransferase being inhibited) and initial rates of the neuronal 3H-noradrenaline uptake as well as IC50 values for inhibition of uptake by desipramine, cocaine or (-)metaraminol determined at various external Cl- concentrations (0-145 mmol l-1) and a fixed high Na+ concentration (145 mmol l-1). (2) When the Cl- concentration in the medium was decreased neuronal uptake fell. As far as Cl- concentrations ranging from 10 to 145 mmol l-1 are concerned, the dependence of uptake on Cl- obeyed Michaelis-Menten kinetics with an apparent Km and Vmax of 6.2 mmol l-1 and 116 pmol g-1 min-1, respectively. At Cl- concentrations below 10 mmol l-1, uptake was higher than expected from the values of Km and Vmax, and even in the nominal absence of Cl- from the medium a remainder of neuronal uptake was still detectable. Evidence is presented to show that, on incubation at Cl- concentrations below 10 mmol l-1, intracellular Cl- leaks out, so that the actual Cl- concentrations in the extracellular fluid are probably higher than in the medium. (3) The potencies of desipramine and cocaine for inhibition of neuronal uptake were markedly dependent on the Cl- concentration in the medium, but the type of Cl- -dependence differed. While the IC50 for desipramine decreased, that for cocaine increased with increasing Cl- concentration (2-145 mmol l-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Choline+: a substrate of the neuronal noradrenaline carrier in the rat vas deferens.

The effects of choline+ (10-40 mmol/l) on 3H-noradrenaline uptake by, and 3H-noradrenaline efflux from, noradrenergic neurones were studied in vasa deferentia of reserpine-pretreated rats at an external Na+ concentration of 100 mmol/l. Monoamine oxidase and catechol-O-methyltransferase were inhibited. Choline+ (20 and 40 mmol/l) competitively inhibited the neuronal uptake of 3H-noradrenaline. From the choline+-induced changes in the apparent Km for 3H-noradrenaline transport, a Ki of 35 mmol/l was obtained. Choline+ (10, 20 and 40 mmol/l) accelerated the neuronal efflux of 3H-noradrenaline in a concentration-dependent manner. This acceleration of efflux was greatly reduced in the presence of 1 mumol/l desipramine, indicating that choline+ is capable of eliciting "accelerative exchange diffusion". Choline+ (40 mmol/l) and (-)noradrenaline (4.5 mumol/l) (i.e., concentrations about equivalent to the Ki and Km for choline+ and (-)noradrenaline, respectively) produced virtually identical increases in the neuronal efflux of 3H-noradrenaline. Choline+ (3-300 mmol/l) inhibited the specific binding of 3H-desipramine to plasma membranes derived from cultured rat phaeochromocytoma (PC-12) cells. The Ki for this interaction was 48 mmol/l. This results suggest that choline+ acts as alternative substrate of the neuronal noradrenaline transport system and should, therefore, not be used in transport studies with noradrenaline as substitute for Na+ in Na+-deficient media.

Total body, systemic and pulmonary clearance and fractional extraction of unlabelled and differently 3H-labelled noradrenaline in the anaesthetized rabbit.

1. Rabbits were anaesthetized with urethane/chloralose and infused intravenously with trace amounts of 3H-2,5,6-, 3H-7,8- or 3H-7-(-)noradrenaline either without or with unlabelled (-)noradrenaline being simultaneously infused (0.2 micrograms kg-1 min-1). To obtain clearance values and extraction ratios for the pulmonary, systemic and total circulation, steady-state concentrations of infused noradrenaline were determined in mixed central venous (Cv) and arterial (Ca) plasma. Heart rate and blood pressure were recorded via the carotid artery, and the dye dilution method was used to determine the cardiac output of plasma. 2. The simultaneous infusion of unlabelled noradrenaline, which increased plasma levels of noradrenaline by a factor of 5, had no significant effect on either heart rate, blood pressure or cardiac output (when determined at steady state of the noradrenaline infusion). 3. The simultaneous infusion of unlabelled noradrenaline did not affect the clearance values of any of the three type of 3H-noradrenaline. Moreover, the clearances of the various types of 3H-noradrenaline were virtually identical and agreed with that of unlabelled noradrenaline. However, the clearance of labelled and unlabelled noradrenaline from arterial plasma was 1.15 times higher than that from central venous plasma. This factor corresponded to the ratio of Cv/Ca and pointed towards net removal of noradrenaline from the pulmonary circulation. 4. The fractional pulmonary extractions [1-(Ca/Cv)] of the three types of 3H-noradrenaline did not differ from each other and were not affected by the simultaneous infusion of unlabelled noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of enzymatic inhibition and increased paracellular shunting on transport of vasopressin analogues in the rat.

The Ussing chamber technique was used as an oral absorption model for studies of the relative effects of the inhibition of enzymatic degradation and increased paracellular route on the transport of the poorly absorbed vasopressin analogues lysine vasopressin (LVP) and desmopressin (DDAVP). The rates of transport of LVP or DDAVP at 250 microM across ileum and colon segments were studied in the absence and in the presence of protease inhibitors (aprotinin and bestatin) and cytochalasin-B. During the different treatments, the rates of degradation of the peptides were also studied. Detectable amounts of LVP could only be measured on the serosal side of the intestinal segment in the presence of protease inhibitors or cytochalasin-B. The treatment with cytochalasin-B increased the rates of transport of both peptides severalfold, and the effect was reversible. We suggest that the Ussing chamber technique can be used to evaluate the reasons for low transport rates across intestinal membranes. The results also show that, apart from enzymatic degradation, the vasopressin analogues LVP and DDAVP have additional permeation problems; therefore, it may be necessary to increase the paracellular route to increase the absorption of these peptides.

Membrane transport of drugs in different regions of the intestinal tract of the rat.

Regional permeability coefficients of 19 drugs with different physicochemical properties were determined using excised segments from three regions of rat intestine: jejunum, ileum, and colon. The results are discussed in relation to the characteristics of the drug, i.e., MW (range 113-1071 Da), pKa, log D (octanol/water at pH 7.4) (range -3.1 to +2.4), and the regional change in the properties of the epithelial membrane. There was a significant decrease in permeability to hydrophilic drugs and a significant increase in permeability for hydrophobic drugs aborally to the small intestine (P < 0.0001). A good correlation could be obtained between MW and permeability coefficients of hydrophilic drugs. The correlation established between the apparent permeability coefficients and the partition coefficients of the drugs was sigmoidal in shape in all three regions and a log D between 0 and 2.5 predicts high permeability values. These permeability data are unique since they result from a diversity of chemical structures with different physicochemical characteristics and a variety of transport mechanisms and they are not influenced by interlaboratory differences. The large regional permeability database in the present study shows the utility of the Ussing chamber technique as a valuable predictive tool for human in vivo data. In addition, the regional permeability profiles obtained suggest a coupling between drug structure and the functional changes of the membrane, which might be useful for selecting a compound for an extended release formulation.

Correlation of drug absorption with molecular surface properties.

The correlation between dynamic surface properties of drug molecules and drug absorption in two common in vitro models of the intestinal wall (Caco-2 monolayers and rat intestinal segments) has been investigated. A homologous series of beta-adrenoreceptor antagonists were used as model compounds. Dynamic molecular surface properties, considering all low-energy conformations, of the compounds were calculated. The flexibility of the molecules was studied by molecular mechanics calculations (MM2) and the van der Waals' (vdW), and water accessible surface areas were calculated and averaged according to a Boltzmann distribution. Excellent correlations were obtained between the dynamic polar vdW surface areas and cell permeabilities in Caco-2 cells and rat ileum (r2 = 0.99 and 0.92, respectively). These correlations were stronger than those between calculated octanol/buffer partition coefficients (log Doct,7.4) and permeability (r2 = 0.80 and 0.73, respectively). Moreover, the calculated log Doct,7.4 values failed to rank the permeability coefficients through Caco-2 monolayers and rat ileum in the correct order. The results indicate that dynamic polar surface area is a promising alternative model for the prediction of oral drug absorption.

Concentration- and region-dependent intestinal permeability of fluvastatin in the rat.

The purpose of this study was to investigate the mechanisms of transport of fluvastatin across the intestinal mucosa in various regions of the intestine in the rat. In-situ single-pass perfusions of the jejunum, ileum and colon were performed and the effective permeability (Peff) of fluvastatin, antipyrine and D-glucose were assessed in each region, at three different perfusate fluvastatin concentrations (1.6, 16 and 160 microM). The effect of lovastatin acid on the bi-directional transport of fluvastatin across the ileal mucosa was also studied. The Peff of fluvastatin was found to be dependent both on the intestinal region and on the concentration in the intestinal lumen (P < 0.001). Fluvastatin had the lowest Peff (0.55 +/- 0.10 x 10(-4) cm s(-1)) in the jejunum at 1.6 microM, and the highest Peff (1.0 +/- 0.16 x 10(-4) cm s(-1)) in the colon at 160 microM. The highest concentration of fluvastatin increased the average absorption of water from the intestine by 209% (P < 0.05), and the average Peff of D-glucose by 29% (P < 0.05). The presence of excess lovastatin acid (100 microM, compared with fluvastatin 1.6 microM) at the luminal side increased the average absorption of water by 218% (P < 0.001), and the Peff of fluvastatin in the ileum and the colon by 44 and 50%, respectively (P < 0.05). The presence of lovastatin acid on the luminal side in the ileum also increased the blood-to-lumen transport (exsorption) of fluvastatin by 43% (P < 0.001). The increased intestinal absorption of fluvastatin at higher concentrations does not suggest that substantial absorption occurs by any carrier-mediated process in the absorptive direction. The increased bi-directional transport when lovastatin acid was added to the lumen suggests that fluvastatin is not a P-glycoprotein substrate. Instead, the concentration-dependent increase in the absorption of fluvastatin, water and D-glucose suggests a direct effect of fluvastatin on the transcellular passive transport.

No evidence for the involvement of the multidrug resistance-associated protein and/or the monocarboxylic acid transporter in the intestinal transport of fluvastatin in the rat.

Fluvastatin, an amphiphilic anion, shows a nonlinear increase in effective intestinal permeability (P(eff)) with increasing lumenal concentrations in rats. The main objective of this study was to investigate whether or not this observation could be attributed to an efflux-mediated transport by the multidrug resistance-associated protein (MRP). In parallel, we investigated the possible involvement of the monocarboxylic acid transporter (MCT) in the rapid intestinal absorption of fluvastatin. Single-pass perfusions were performed in the ileum and colon of the rat, with and without the presence of well-established inhibitors/substrates for the MRP (probenecid) and the MCT (nicotinic acid). The results suggest that neither the MRP nor the MCT are involved to any significant extent in the absorption process of fluvastatin in the rat intestine. Thus, the previously reported concentration-dependent P(eff) of fluvastatin in these intestinal regions of the rat is probably not attributable to saturation of any efflux mediated by MRP.

Accumulation of intra-arterially administered [3H]adrenaline and [3H]noradrenaline in various tissues of the Atlantic cod, Gadus morhua.

The accumulation of intra-arterially administered radiolabelled adrenaline and noradrenaline was studied in various tissues of the Atlantic cod, Gadus morhua. The largest uptake was seen in the posterior cardinal vein (chromaffin tissue), head kidney, kidney, heart and gill filaments. All these tissues, except the heart, also accumulated noradrenaline to a greater extent than adrenaline. The heart, spleen, gas gland and muscularis mucosae of the swimbladder instead favoured adrenaline accumulation. Small amounts of the injected label (both adrenaline and noradrenaline) were also recovered in the intestine, liver and hypothalamus. The lowest detectable amine accumulation was seen in the rest of the brain and in the skeletal muscle. It is suggested that innervation density, blood flow to the tissue and the concentration of circulating and endogenously stored amine, as well as the affinity of the amine for the degrading enzymes and a possible stereospecificity of the uptake mechanisms, determine the rate and preference of accumulation between the amines.

Temperature effects on catecholamine accumulation in neuronal and extraneuronal compartments in the cod spleen.

The effects of temperature on the accumulation of radiolabelled adrenaline and noradrenaline were studied in the perfused spleen of the Atlantic cod, Gadus morhua. A decrease in temperature from 20-24 to 10 degrees C showed a corresponding decrease in the neuronal and extraneuronal accumulation of both adrenaline and noradrenaline. The Q10-values for the neuronal accumulation of adrenaline and noradrenaline were 3.6 and 2.1, respectively. This indicates that adrenaline is accumulated neuronally by an active process. An increased perfusing time resulted in an even more pronounced difference in the neuronal accumulation between the two amines. The extraneuronal accumulation of both amines was only slightly affected by a 10 degree C decrease in temperature, and with increasing perfusion time, the accumulation increased as in a passive diffussion. There are strong indications that adrenaline and noradrenaline not only accumulate differently from each other in the adrenergic neurons of the cod spleen, but also that the adrenaline uptake mechanism neuronally might resemble the characteristic mammalian catecholamine uptake while noradrenaline seems to accumulate by a more primitive mechanism, not sensitive to a reduction in temperature. This might be physiologically important for the cod, since the spleen can synthetize adrenaline from noradrenaline and the uptake of adrenaline from the circulation is therefore less important.

Inhibitory effects of desmethylimipramine, metanephrine and normetanephrine on the neuronal and extraneuronal accumulation of catecholamines in fish spleen.

The effect of desmethylimipramine, metanephrine and normetanephrine on the neuronal and extraneuronal accumulation of radiolabelled adrenaline and noradrenaline were studied in the perfused spleen of the teleost fish, Atlantic cod, Gadus morhua. Desmethylimipramine was found to be a potent inhibitor for the neuronal accumulation of both adrenaline and noradrenaline in the cod spleen, suggesting similarities with the neuronal uptake mechanism in mammals. Metanephrine was found to inhibit the extraneuronal accumulation of noradrenaline, though not that of adrenaline, while normetanephrine did not change the extraneuronal accumulation of any of the catecholamines. Both metanephrine and normetanephrine were more potent neuronal uptake blockers than extraneuronal. Metanephrine inhibited only the neuronal accumulation of adrenaline, while normetanephrine inhibited neuronal accumulation of both catecholamines. It is concluded that adrenaline and noradrenaline accumulate differently in the adrenergic neurons of the cod spleen as was suggested earlier for noradrenaline and tyramine (Nilsson & Holmgren 1976). It is also evident from the study that the uptake mechanisms or accumulation of catecholamines in lower vertebrates such as fish may be different from corresponding mechanisms in mammals.

The fate of noradrenaline and adrenaline in the perfused cod spleen.

The time course of appearance of catecholamine metabolites was studied in the spleen of cod,Gadus morhua, during perfusion with radioactively labelled noradrenaline and adrenaline at 10°C. The tlag for appearance of the metabolites ranged between 1.78 and 6.76 min after onset of perfusion for both amines, indicating a rapid disposition of catecholamines. Perfusion with noradrenaline resulted in mainly MOPEG, VMA and DOMA formation, while perfusion with adrenaline additionally resulted in MN formation. There was still formation of deaminated metabolites after denervation, which indicates an additional non-neuronal site of deamination. It is concluded from the study that the fate of noradrenaline and adrenaline within the cod spleen depends on their affinities for the two uptake mechanisms and an extraneuronal site of deamination of great importance cannot be excluded.

Neuronal and extraneuronal uptake of L-3H-adrenaline and L-14C-noradrenaline in the fish spleen.

The neuronal and extraneuronal uptake of adrenaline and noradrenaline in the isolated perfused spleen of the cod has been studied using radiolabelled amines. The neuronal uptake of adrenaline shows an optimum at 0.5 microgram/ml, but is less efficient than for noradrenaline in the concentration range 0.01-2.5 micrograms/ml. The neuronal uptake of noradrenaline accounts for 60-70% of the total (neuronal plus extraneuronal) uptake in the concentration range 0.01-1.0 microgram/ml. At higher concentrations (2.5-5.0 micrograms/ml) the importance of the extraneuronal uptake increases for both amines, to reach more than 90% of the total uptake at 5.0 micrograms/ml. The extraneuronal uptake shows equal efficiency for the two amines throughout the concentration range studied. It is concluded that the cod spleen possesses a neuronal uptake mechanism which shows a higher uptake efficiency for noradrenaline than for adrenaline, while the extraneuronal uptake mechanism shows no preference.

Nerve impulse-induced release of endogenous noradrenaline and adrenaline from the perfused cod spleen.

Release of endogenous catecholamines (CA) by electrical nerve stimulation (NS) was studied in the isolated perfused spleen of the Atlantic cod, Gadus morhua. An HPLC-system for the analysis of endogenously released CA is described. Cocaine (COC) was used to block neuronal re-uptake of endogenous CA released by NS. Splanchnic NS at frequencies of 1-40 Hz for 20 s resulted in release of noradrenaline (NA) and adrenaline (A) with a maximal total overflow at 20 Hz for both amines. The release of CA was frequency-dependent. COC (0.1 mmol.l-1) increased NS-evoked (40 Hz) overflow of NA and A by 4.8 and 2.2 times, respectively. and reduced the overflow of dihydroxyphenylglycol (DOPEG) to spontaneous efflux levels or less. It can be concluded that the HPLC-technique used was adequate for measurement of NS-evoked release of endogenous CA and DOPEG from the isolated perfused cod spleen, and the model presented can therefore be used when studying adrenergic mechanisms in fish spleen.

Study on adrenergic function after development of tolerance to ethylene glycol dinitrate (EGDN) in rats.

The effects of sustained exposure to ethylene glycol dinitrate (EGDN; 50 mg.kg-1.2, 10 days) on heart rate (HR) and mean arterial blood pressure (MAP), isolated strips from aorta and vena cava, catecholamine (CA) and DOPAC levels in brain, heart, adrenals and plasma, and synaptosomal uptake of noradrenaline (NA) have been investigated in rats. Aortic strips showed a decreased responsiveness to EGDN and were more sensitive to NA than were control strips 2, 24 and 96 hrs after cessation of EGDN. Acute cumulative doses of EGDN induced a decrease in MAP at all time intervals but 2 hrs after cessation of chronic EGDN-treatment, while NA induced a dose-dependent increase in MAP. MAP was lower 2 hrs after cessation of EGDN, than in the control rats, while HR was higher. After 24 hrs, MAP was slightly higher than in the controls and HR was still elevated. EGDN induced a decrease in the accumulation of L-DOPA and DOPAC in all brain structures measured, which was especially prominent 2 hrs after cessation of EGDN. The levels of CA's and DOPAC in peripheral tissues did generally not change significantly, although there was a tendency to a decrease. L-DOPA, NA and adrenaline (A) in plasma decreased significantly, while DOPAC and dopamine (DA) increased. No consistent effects on uptake of NA into synaptosomes could be distinguished. However, the amount of protein was generally lower at all times after cessation of EGDN. It is suggested that prolonged exposure to EGDN not only induces tolerance at the cellular level, but also interferes with arterial smooth muscle sensitivity to NA and with resetting of MAP and HR. The decrease in the synthesis and turnover of CA's and DOPAC in the brain indicated a decrease in nervous activity, which is reflected by a corresponding decrease of NA and A levels in plasma.

Selective paracellular permeability in two models of intestinal absorption: cultured monolayers of human intestinal epithelial cells and rat intestinal segments.

New data on the permeabilities of hydrophilic markers in two commonly used in vitro models, i.e., excised intestinal segments from the rat and monolayers of Caco-2 cells, are presented. The results are compared to human in vivo data. Two groups of hydrophilic marker molecules were tested: (1) monodisperse polyethylene glycols of molecular weights ranging from 194 to 502 g/mol and (2) a heterogeneous group of molecules consisting of urea, creatinine, erythritol, and mannitol (60-182 g/mol). The permeabilities of the marker molecules showed a nonlinear dependence on the molecular weight and decreased in the order rat ileum > rat colon > Caco-2 cells. Surprisingly, the polyethylene glycols permeated more easily than the other marker molecules, indicating that characteristics other than molecular weight, e.g., the flexibility of the structure, may also be important for permeation through the membrane. Comparisons with the published permeability profiles of polyethylene glycols in human intestinal segments in vivo (i.e., calculated permeability coefficients as a function of molecular weight) indicate that the human intestine is more permeable than the in vitro models. However, the permeability profiles of the corresponding segments in the human intestine and the in vitro models were comparable. Thus, good correlations were established between permeabilities of the human ileum and rat ileum and between those of human colon, rat colon, and the Caco-2 cells. We conclude that the paracellular absorption in humans can be studied mechanistically in these in vitro models.