Effect of pH on theophylline transfer across the everted rat jejunum

The effect of pH on the cumulative transfer of theophylline across the everted rat jejunum in vitro was investigated. Intestinal integrity was assessed by light and scanning electron microscopy, while the biochemical viability of the intestine was evaluated using glucose transfer measurements. The initial (0-30 min) clearance of theophylline was directly proportional to the fraction un-ionized at pH 5.5, 7.4, 8.0, and 10.0. Plots of cumulative theophylline transfer versus time over 60 min were nonlinear, but could be subdivided into two linear segments of 30-min duration. Due to this nonlinearity, differences in theophylline transfer with pH were significant only over the first 30 min of the experiment. Intestinal tissue integrity and viability correlated with the time at which the clearance (slope) increased, while the magnitude of the increase in clearance was proportional to the degree of ionization of theophylline.     

Polarised transport of monocarboxylic acid type drugs across rat jejunum in vitro: the effect of mucolysis and ATP-depletion

The transport characteristics of monocarboxylic acid type drugs (ketoprofen, ibuprofen and gemfibrozil) across the excised jejunal segments and artificial (octanol impregnated) membrane in side-by-side diffusion cells were studied. All three model drugs permeated faster across the intestinal tissue in the mucosal-to-serosal direction than in the opposite direction. No polarised transport of tested drugs was observed when the mucosal side of the intestine was treated with mucus disrupting agent, L-cysteine 1% (w/v), which significantly increased the microclimate pH at the mucosal surface of the intestine. Similar effects on the transport characteristics of model drugs and microclimate pH were observed when metabolic inhibitor, sodium azide (10mM), was present in the incubation medium. Furthermore, the direction of proton gradient across the artificial membrane was shown to significantly influence the transport of model drugs across this membrane. The results of this study indicate that the inwardly directed proton gradient maintained by the acidic microclimate pH at the intestinal surface could be considered as a driving force for the transport of monocarboxylic acid type drugs across the intestinal epithelia and could explain rapid absorption of these drugs after oral application.     

Fluorescein transport properties across artificial lipid membranes, Caco-2 cell monolayers and rat jejunum.

Membrane transport characteristics of a paracellular permeability marker fluorescein were evaluated using artificial membrane, Caco-2 cell monolayers and rat jejunum, all mounted in side-by-side diffusion cells. Modified Ringer buffers with varied pH values were applied as incubation salines on both sides of artificial membrane, cell culture monolayers or rat jejunum. Passive transport according to pH partition theory was determined using all three permeability models. In addition to that, active transport of fluorescein in the M-S (mucosal-to-serosal) direction through rat jejunum was observed. The highest M-S P(app) values regarding the active transport through the rat jejunum were observed in incubation saline with pH 6.5. Fluorescein transport through the rat jejunum was inhibited by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) and alpha-CHC (alpha-cyano-4-hydroxycinnamic acid). Thus, we assume that two pH-dependent influx transporters could be involved in the fluorescein membrane transport through the intestinal (jejunal) epithelium. One is very likely an MCT (monocarboxylic acid cotransporter) isoform, inhibited by specific MCT inhibitor alpha-CHC, while the involvement of the second one with overlapping substrate/inhibitor specificities (most probably a member of the organic anion-transporting polypeptide family, inhibited at least partially by DIDS) could not be excluded.     

Comparison of ceftibuten transport across Caco-2 cells and rat jejunum mounted on modified Ussing chambers

Ceftibuten uptake into Caco-2 cells and intestinal brush border membrane vesicles is mediated by the dipeptide transport system (PEPT1). The apical to basolateral transport characteristics of ceftibuten across Caco-2 cells and rat jejunum mounted on a modified Ussing chamber was examined. Mannitol was used as a paracellular marker along with trans-epithelial electrical resistance (TEER) for monitoring tight junction permeability. Transport across Caco-2 cells and rat jejunum mounted on a modified Ussing chamber was linear across the concentration range 0.25-10 mM. The net flux of mannitol and ceftibuten was higher across rat jejunum compared with Caco-2 cells. At a donor concentration of 0.25 mM, ceftibuten transport across Caco-2 cells was found to be pH dependent. Glycyl proline, a dipeptide, and 2,4- dinitrophenol, an energy poison, caused a reduction in the permeability of 0.25 mM ceftibuten across Caco-2 cells. Benzoic acid and adipic acid also inhibited transcellular transport of ceftibuten. At a donor concentration of 0.25 mM, passive paracellular transport accounts for about 60% and the active carrier mediated mechanism accounts for about 40% of ceftibuten transport across Caco-2 cells. None of the inhibitors however, had a significant effect on ceftibuten transport across rat jejunum mounted on a modified Ussing chamber at a donor concentration of 0.25 mM. In the concentration range 0.25-10 mM, ceftibuten is predominantly transported by paracellular mechanisms across rat jejunum and a mixture of active and passive transport across Caco-2 cells.     

In vitro permeation of beta-lactam antibiotics across rat jejunum and its correlation with oral bioavailability in humans

AIMS: To investigate the correlation between in vitro permeation of 11 beta-lactam antibiotics across rat jejunum and their oral bioavailability in humans. METHODS: The absorptive and secretory permeation across rat jejunum was evaluated and apparent permeability coefficients (P(app)) were determined. RESULTS: A steep, sigmoid-type curve was obtained for the relationship between P(app) in the absorptive permeation and human oral bioavailability. When the ratios of P(app) in the absorptive direction to P(app) in the secretory direction were plotted against human oral bioavailability, a much improved correlation was obtained (r = 0.98, P < 0.001). The addition of glycylglycine to both mucosal and serosal media modified the permeation of ceftibuten and cephalexin from the absorptive to the secretory direction. CONCLUSIONS: For 11 beta-lactam antibiotics rat intestinal permeation correlated well with human oral bioavailability, especially when corrected for secretory transport.     

Effect of chlorpromazine on proline absorption across rat jejunum

The effect of chlorpromazine on proline absorption across jejunum in anaesthetized rats was investigated. Intravenous infusion of chlorpromazine reduced significantly (P less than 0.01) proline absorption across the jejunum. Intraluminal perfusion of chlorpromazine into the jejunal segment reversed net absorption of proline to net secretion. Net water absorption was increased significantly (P less than 0.01) when chlorpromazine was infused intravenously or perfused intraluminally. Unidirectional influx of proline across the mucosal surface was significantly inhibited (P less than 0.01) after preincubation with 1 mM chlorpromazine.     

Effects of neomycin on galactose absorption across rat jejunum

The effects of neomycin sulphate on galactose absorption have been studied using in vivo and in vitro preparations of rat small intestine. Neomycin (10(-3)M) produced an increase in the maximum transport capacity (Jmax) for the active component of absorption in vivo. The apparent Kt for absorption was unaffected. The antibiotic caused a dose-dependent increase in the potential difference across the mucosal membrane (Vm) measured in vitro, a maximal effect being seen at a concentration of 10(-4)M. Furthermore, the magnitude of the depolarization induced by the addition of galactose (4 mM) to the mucosal fluid was enhanced by neomycin (10(-4)M). Phlorhizin (10(-4)M) abolished the galactose-induced depolarization in both the absence and presence of the antibiotic. It is concluded that neomycin increases the electrical driving force for Na+ during Na+-coupled galactose entry into the enterocyte.     

Permeability characteristics of novel aldose reductase inhibitors using rat jejunum in vitro.

The aim of this study was to estimate in vivo permeability and bioavailability of epalrestat and newly synthesized compounds with possible therapeutic activity as aldose enzyme inhibitors (ARIs). For this purpose permeability in vitro using rat jejunum mounted in side-by-side diffusion cells was determined. Tested substances were found to be low and moderately permeable and some of them were also substrates for efflux transporters. It was shown, that the higher efflux for some derivatives was due to MRP-2, but not Pgp involvement. Tested ARIs do not share the same efflux transporter with epalrestat, the only ARI currently on the market in Japan. The most permeable compound, a 2,6-difluoro-4-pyrrol-1ylphenol derivative, is not a substrate for efflux transporters and would therefore be the most promising lead compound for further investigation of potent ARIs.     

Kinetic characterization of in vitro lead transport across the rat small intestine: Mechanism of intestinal lead transport

Lead transport through the everted rat small intestine was used as an in vitro model to examine the kinetics and mechanism of gastrointestinal lead absorption. Mucosal-to-serosal lead flux increased nonlinearly with increasing mucosal lead concentrations (0.5–48.3 μm), and an apparent capacity-limited lead flux was observed with intestines of both adult and adolescent rats. Uptake of lead by the adult intestinal tissue, measured at the end of the flux experiments, was also nonlinearly related to mucosal lead concentration. Both intestinal uptake and mucosal-to-serosal flux were reduced by anoxia and by inhibition of glycolysis using fluoride. The data were consistent with a model of lead intestinal transport which included both a carrier-mediated component and passive diffusion. Kinetic parameters for these transport processes were obtained. The relative contribution of passive diffusion to total lead flux increased with increasing lead concentration, but was quantitatively minor (<20%) at all lead concentrations tested. The apparent capacity-limited component of lead flux was not due artifactually to reduced lead solubility in the mucosal solution at high metal concentrations, nor was it due apparently to reduced viability of the intestinal tissue, since lead did not alter 3-O-methylglucose transport over the range of lead concentration used. When the same lead solution was placed on both sides of an everted intestine, net serosal-to-mucosal lead flux was observed at low lead concentrations. It is therefore likely that intestinal lead transport may be bidirectional, similar to calcium transport.     

Factors influencing the transfer of xanthines across the everted rat jejunum

A number of factors having the potential to influence the transfer of theophylline across the everted rat jejunum were examined. The transfer of several other xanthine derivatives was also investigated. Strophanthin-K and 2,4-dinitrophenol had no effect on theophylline transfer. The transfer of theophylline from a 2:1 complex with phenobarbital was identical to that of theophylline alone. Magnesium ion (120 mM) and hypertonicity (600 mOsm) had no effect on the transfer of theophylline at pH 5.5. With the exception of xanthine, the clearance of all of the xanthine homologs tested was related to their partition coefficients in a chloroform/pH 7.4 phosphate buffer.     

Combined effect of alcohol and urea on the in vitro transport of indomethacin across rat dorsal skin

An aqueous gel, prepared with hydrogenated soya phospholipid, increased the in vitro transport of indomethacin across rat dorsal skin. The addition of various alkanols further accelerated the transport, with an increasing effect as the chain length of the alkanol increased. The addition of urea alone did not significantly affect the transport of indomethacin. However, the addition of urea markedly accelerated the transport of indomethacin when included in an aqueous gel containing an alkanol such as 1-octanol, 1-decanol, or 1-dodecanol. Thus, it appears that a combination of urea and these alkanols strongly enhances the transdermal absorption of indomethacin. Urea appears to accelerate enhanced drug transport into the stratum corneum by a mechanism involving the transport of urea enhanced by these alkanols.     

Factors affecting the microclimate pH of the rat jejunum in ringer bicarbonate buffer

General characteristics of the microclimate layer on the mucosal surface of the rat jejunum incubated in bicarbonate buffers were investigated in vitro using pH sensitive flat membrane microelectrode. Jejunal surface microclimate (JSM) pH changed from 7.30+/-0.03 to 5.83+/-0.04 when the incubation buffer pH decreased from 8.03+/-0.02 to 6.12+/-0.01. Treatment of the mucosal side with mucolytic substances L-cysteine (1% (wt/v)) and 1,4-dithio-DL-threitol (2 mM) significantly (p<0.01) increased JSM pH. Respiratory chain inhibitor, sodium azide (10 mM) also significantly (p<0.05) increased JSM pH. D-Glucose (10 mM) at the mucosal side markedly (p<0.05) decreased JSM pH, which was attenuated by Na(+)/H(+) exchange inhibitor, amiloride (1 mM). Amiloride had no effect on JSM pH when D-glucose was not present at the mucosal side. In contrast to previous observations using bicarbonate free incubation buffers, we have demonstrated that JSM pH is not a constant value, but is dependent on pH of the incubation buffer. Additionally, Na(+)/H(+) exchanger does not contribute to acidic properties of JSM, when there is no D-glucose in the bicarbonate incubation buffer at the mucosal side of the tissue. In conclusion, we suggest that the bicarbonate buffers which are more close to in vivo situation than bicarbonate free buffers should be preferable incubation media when examining JSM.     

Effect of pH on aflatoxin B1 transfer in the everted rat jejunum

The effect of varying mucosal pH (5, 7.4 and 8) on aflatoxin B1 (AFB1) transmural transfer was studied in the everted rat jejunum. The viability of the preparation was evaluated by: light microscopy; rate of glucose uptake by tissue from the mucosal fluid; content and distribution of water and electrolytes in the tissue; oxygen uptake by the tissue; and the intestinal permeability to [14C]sucrose. These parameters revealed the structural and functional integrity of the preparation, which was not affected by incubation in the presence of AFB1 or at different pH values in the mucosal fluid. The results obtained show that AFB1 was transferred to the serosal fluid and that the cumulative transfer was greater at pH 5 than at pH 7.4 or 8. In addition, AFB1 clearance, calculated from the slope of the plot of the cumulative amount of AFB1 transferred per unit of mucosal concentration vs. incubation time, indicated a very rapid accumulation by tissue and transfer of AFB1 to the serosal fluid. This process was also faster at pH 5. The integrity of the AFB1 molecule during incubation was shown by NMR spectra. The lack of metabolization of AFB1 during its translocation was demonstrated by TLC of the serosal fluid.     

Transport studies of insulin across rat jejunum in the presence of chicken and duck ovomucoids

Our aim was to evaluate the transport of insulin across rat jejunum in the presence of ovomucoids and to assess the effect of ovomucoids on intestinal tissue by studying the permeation of a lipophilic and a hydrophilic marker. Rat jejunal segments were mounted in a side-by-side diffusion chamber filled with Krebs bicarbonate buffer, bubbled with 95% O2/5% CO2 at a fixed flow rate and maintained at 37 degrees C. The permeation of insulin, a lipophilic marker ([7- 3H] testosterone) and a hydrophilic marker (D-[1- 14C] mannitol) was evaluated in the presence of 0.5-1.5 microM duck ovomucoid (DkOVM) or chicken ovomucoid (CkOVM). For stability and permeation of insulin in the presence of alpha-chymotrypsin, an enzyme-to-inhibitor ratio of 1:1 and 1:2 was used. In the absence of alpha-chymotrypsin, the permeability coefficient (Papp) of insulin at pH 7.4 was 0.922+/- 0.168 x 10(-7) cm s(-1), which decreased with increasing concentrations of DkOVM or CkOVM. Conversely, the permeation of the hydrophilic and lipophilic marker increased with increasing concentrations of CkOVM and DkOVM. In stability studies, the percentage of drug remaining was found to be 2-fold higher at the 1:2 ratio than with the 1:1 ratio of enzyme to inhibitor. This was in agreement with the 2-fold increase in flux values of insulin in the presence of alpha-chymotrypsin and DkOVM at the 1:2 ratio of enzyme to inhibitor. The decrease in permeation of insulin in ovomucoids was unexpected. Marker transport studies indicated that ovomucoids have the potential to modulate transcellular and paracellular permeability. The flux enhancement of insulin in the presence of alpha-chymotrypsin and DkOVM is encouraging. The use of ovomucoids offers potential to enhance oral delivery of insulin and warrants further investigation.     

Lack of effect of beta-cyclodextrin and its water-soluble derivatives on in vitro drug transport across rat intestinal epithelium

The present study aimed to investigate whether beta-cyclodetxrin (beta-CD) and its water-soluble derivatives, hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and sulfobutyl ether beta-cyclodextrin (SBE-beta-CD), exert any effects on the permeation of two drug transport markers (propranolol and lucifer yellow) across rat intestinal epithelium. Rat ileum was stripped of its serosa and mounted inside an Ussing Chamber. Apparent permeability coefficients (P(app)) of the markers from the mucosal to serosal side of the tissue were determined at 37 degrees C in the presence and absence of the beta-cyclodextrins on the mucosal side. Potential difference (PD) was constantly monitored during each experiment to ensure maintenance of the viability and integrity of the tissue. Pre-incubation with 1% beta-CD, 1% HP-beta-CD or 1.48% SBE-beta-CD on the mucosal side for 30 min did not significantly alter the PD and the propranolol permeability (p>0.05). Co-incubation with 1% beta-CD or 1% HP-beta-CD exerted no significant effect on the P(app) of both propranolol and lucifer yellow (p>0.05), but co-incubation with 1.48% SBE-beta-CD lowered the P(app) of propranolol from (1.71+/-0.44)x10(-5) to (0.19+/-0.04)x10(-5)cm/s, which may be ascribed to the molecular complexation of propranolol with SBE-beta-CD. All three beta-cyclodextrins exert no apparent impact on both (passive) transcellar and paracellular drug transports.     

Optimized transdermal delivery of ketoprofen using pH and hydroxypropyl-beta-cyclodextrin as co-enhancers.

The role of pH and pK(a) of ionizable drugs in transdermal delivery has been well documented by the pH partition hypothesis. Similarly the role of pH in complexation has also been addressed by many studies. Reports contrary to the well believed theory that both molecular encapsulation by hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and transdermal delivery are considered a phenomenon of unionized drug species prompted investigation into the combined effect of pH and HP-beta-CD on transdermal delivery of ketoprofen. In order to optimize the delivery of ketoprofen, solubility studies and permeation studies were conducted in vitro at pH 3.0, 4.5 and 6.0 at various concentrations of cyclodextrin. The stability constants for unionized and ionized drugs were calculated. The solubility of the ionized complex of the drug was 2.5 fold greater than the unionized complex. The flux increased linearly with increasing HP-beta-CD concentration at all the pH values. However, the increase was significant at pH 6.0 where the drug is predominantly in the ionized state. The flux of the ionized drug at 10% w/v HP-beta-CD concentration was enhanced to an order of approximately eight times compared to the intrinsic permeability of the unionized drug. The study shows that at higher pH, HP-beta-CD can be utilized to achieve greater transdermal flux of ketoprofen.     

Evaluation of percutaneous absorption and skin irritation of ketoprofen through rat skin: in vitro and in vivo study.

The influences of different mechanisms of penetration enhancers (such as menthol, azone, ethanol and nonivarnide) regarding the percutaneous absorption and skin irritation of ketoprofen formulations through rat skin were investigated by in vitro and in vivo study. The skin irritation degree at the end of the experiment (10 h) was deterinined by pathologic biopsy and colorimetry methods. In vitro, the menthol showed the most potent enhancing effect. Furthermore, the enhancement effect of a combination of menthol and nonivamide was higher than that of their individual use alone. In vivo the formulation containing 0.05% nonivantide, 5% menthol and 20% ethanol showed a higher penetration rate and an acceptable degree of skin irritation compared to a commercial product (Formax plus gel containing 3% ketoprofen), indicating that it could be used in the clinical situation.     

The pH dependent uptake of enoxacin by rat intestinal brush-border membrane vesicles.

The mechanism of the intestinal transport of enoxacin, an orally active fluoroquinolone antibiotic, has been investigated using brush-border membrane vesicles isolated from rat small intestine. The initial rate and time-course of enoxacin uptake were considerably dependent upon the medium pH (pH 5.5 greater than pH 7.5) and upon the percent ionization of the carboxyl group (pKa 6.2, anionic charge), namely, the degree of uptake of cationic form was higher than that of the zwitterionic form. There was evidence of transport into the intravesicular space as shown by the effect of extravesicular medium osmolarity on enoxacin uptake at steady state (30 min). This transport across the brush-border membrane was stimulated by the valinomycin-induced K(+)-diffusion potential (interior negative) and an outward H(+)-diffusion potential. Furthermore, changing the pH of the medium from 5.5 to 7.5 significantly decreased the effect of valinomycin-induced K(+)-diffusion potential on the enoxacin uptake. These results suggest that the uptake behaviour of the cationic form of enoxacin plays an important role in the intestinal absorption process of enoxacin.