Drug Absorption Studies Research Articles

Establishment of a P-glycoprotein substrate screening model and its preliminary application.

To establish a high P-glycoprotein (P-gp) expressing cell line as a model for studying drug absorption and distribution, and to explore the preliminary application of this screening model. A full-length MDR1 cDNA fragment in plasmid pMDRA1 was first subcloned into plasmid pET28a(+), then MDR1 cDNA was cut from the recombinant plasmid with double-digestion and ligated into the mammalian expression vector pcDNA3.1(+). The recombinant plasmid pcDNA3.1(+)/MDR1 was transfected into breast cancer cell line Bcap37 using the Superfect transfection reagent. Several stably transfected clones were obtained after selection with G418. Real-time fluorescent quantitative RT- PCR and Western blot methods were used to detect the expression of P-gp, and the cellular location of the expressed protein was determined by immunohistochemical staining. Drug sensitivity assay was used to evaluate the biological function of expressed P-gp. Concentration of quercetin in cells was determined by high-performance liquid chromatography (HPLC). The recombinant plasmid was confirmed to be inserted in the correct orientation by restrictive enzyme digestion and DNA sequencing. Real-time fluorescent quantitative RT-PCR showed a higher level of P-gp mRNA in transfected cells compared to that in the control cells, and the Western blot result also indicated that P-gp expression in transfected cells was higher than that in control cells. The immunohistochemical staining showed that the expressed P-gp was localized on cell membranes. Drug sensitivity assay showed that the IC50 for adriamycin and colchicine of the transfected cells was higher than that of the control cells. The concentration of quercetin in model cells was lower than that in control cells by HPLC. After P-gp inhibitor verapamil was administered, the concentration of quercetin in model cells was increased. A high P-gp expressing cell line can be established, which could provide a suitable in vitro model system for studying drug intestinal absorption mechanism, predicting the drug permeability characteristics and screening new multi-drug resistance reversing agents. With this model, quercetin can be found to be transported by P-gp, and it is a P-gp substrate.

Intestinal membrane transport of drugs and nutrients: genomics of membrane transporters using expression microarrays.

Carrier-mediated transport across membranes plays an important role in drug and nutrient absorption. However, relevant transporters remain largely unknown for most substrates. Their identification requires global analysis of expressed mRNAs in intestinal tissues. Microarray technologies capable of measuring mRNA profiles have proven useful in detecting the expression of genes encoding transporters and ion channels in intestines and Caco-2 cells. This colon carcinoma cell line with characteristics of absorptive enterocytes serves as a common model for drug absorption studies. Gene expression patterns of membrane transporters and channels define the cell's overall transport capacity. Moreover, transporter mRNA profiles provide a basis for assessing drug-drug and drug-food interactions in intestinal absorption. To determine relevant transporters for any given substrate, chemogenomic methods have emerged correlating mRNA expression in multiple tissues to drug transport or response. The resultant drug-transporter databases permit the search for transporter-drug relationships at a genomic scale.

An automated blood sampler for simultaneous sampling of systemic blood and brain microdialysates for drug absorption, distribution, metabolism, and elimination studies

Introduction: A major problem in preclinical drug development where blood sampling from small animals is a routine practice is the time and labor involved in the serial sampling of small blood volumes from small animals such as rats for the duration of pharmacokinetic/pharmacodynamic (PK/PD) studies. The traditional method of manually drawing blood from the animal requires the animal to be anesthetized or restrained with some device, both of which cause stress to the animal. Methods: An automated blood sampler (ABS) was developed to simultaneously collect blood and brain microdialysate samples at preprogrammed time points from awake and freely moving animals. The samples are delivered to fraction collectors and stored at 4 °C until use. The lost blood volume during collection is replaced with sterile saline to prevent fluid loss from the animal. In addition, the system is capable of collecting urine and feces for metabolism studies and monitoring the animal activity for behavioral studies. In the present study, blood samples were collected for 24 h after dosing rats orally with a 5 mg/kg dose of olanzapine (OLAN). Brain dialysates were collected for the same duration from a microdialysis probe implanted in the striatum. Results: The pharmacokinetic parameters, obtained after an oral dose, are in good agreement with reported values in literature. The pharmacodynamic information obtained from brain dialysates data show that OLAN elevates the concentration of dopamine (DA) in the brain and remains in the brain even after it is cleared from the plasma. Discussion: The ABS described here is a very useful tool in drug development to accelerate the pace of preclinical in vivo studies and to simultaneously provide pharmacodynamic and physiological information.

Expression of drug-metabolizing enzymes, nuclear transcription factors and ABC transporters in Caco-2 cells

1. Caco-2 cells are frequently used in intestinal drug absorption and metabolism studies, but little is known about the effects of drugs on the simultaneous expression of genes coding for drug-metabolizing enzymes (DMEs), nuclear transcription factors and ABC transporters. 2. The gene expression and enzyme activities of control and Aroclor 1254-treated cultures were therefore explored, the latter being a powerful inducer of DMEs. Fourteen- and 80-fold induction of CYP1A1 and CYP1A2 mRNA were shown, whereas expression of other DMEs was either increased (CYP2C8-2C19, 10-fold; CYP3A5, twofold; FMO1, 2 and 5, twofold; epoxide hydrolase, threefold) or repressed (CYP2D6 and CYP2E1 to 75% of control values). 3. Notably, gene copies of CYP3A4 and CYP2B6 / 7 were below the limit of detection, but a three- and 10-fold induction of HNF 1 α + β, HNF-4 α 4 and a similar 10-fold increase in STAT 3 and 4 was observed. 4. Similarly, c/EBP transcripts were only detected in treated cell cultures, but MRP1, its isoforms 3-5 as well as MDR-1 were increased threefold after dosing with Aroclor 1254. 5. Overall, CYP gene expression correlated well with the cognate enzyme activity using testosterone as a marker substrate.

Quantitation of talinolol and other beta-blockers by capillary electrophoresis for in vitro drug absorption studies.

A capillary zone electrophoresis method is described for the enantioseparation of talinolol using heptakis(2,3-diacetyl-6-sulfo)-beta-cyclodextrin (HDAS-beta-CD) as a chiral selector. After liquid-liquid extraction of talinolol from physiological solution, electrokinetic injection was employed to improve the sensitivity. The use of a coated capillary was necessary to achieve stable and reproducible enantioseparations. A baseline separation of the talinolol enantiomers was achieved in less than 10 min using 100 mM phosphate solution as background electrolyte and pH 3.5, at the presence of 3.0 mM HDAS-beta-CD and at 20 degrees C. In addition, this analytical condition proved to be useful for the enantioseparation of a number of other beta-blocking agents such as alprenolol, atenolol, bisoprolol, celiprolol, metipranolol, oxprenolol, and sotalol. For determining talinolol, the method could be validated in terms of precision, accuracy and linearity, and was found to be suitable in determination of talinolol enantiomers in highly diluted samples obtained from in vitro experiments.

Quantitation of the enantiomers of ofloxacin by capillary electrophoresis in the parts per billion concentration range for in vitro drug absorption studies

Ofloxacin, a chiral fluoroquinolone, possesses two optical isomers. The antibacterial activity of S-(−)-ofloxacin is reported to be 8–128 times higher than that of R-(+)-ofloxacin. A capillary zone electrophoresis method has been developed to quantify the enantiomers of ofloxacin in high diluted samples (20–700 ng/ml for each enantiomer). After fluid–fluid extraction of ofloxacin from physiological solution electrokinetic injection was employed to improve the sensitivity. The method was optimised using a central composite design. Four experimental factors were investigated: the background electrolyte concentration, the methyl-β-cyclodextrin concentration, the buffer pH and the temperature. The amount migrated into the capillary, determined by the peak area, the resolution between the ofloxacin enantiomers, the migration time and the generated current were evaluated as responses. The quantification limit is 11.4 ng/ml for S-ofloxacin and 10.8 ng/ml for R-ofloxacin. The method has shown good validation data in terms of precision and recovery rate.

Remote controlled capsules in human drug absorption (HDA) studies.

The biopharmaceutical complexity of today's new drug candidates provides significant challenges for pharmaceutical scientists in terms of both candidate selection and optimizing subsequent development strategy. In addition, life cycle management of marketed drugs has become an important income stream for pharmaceutical companies, but the selection of least risk/highest benefit strategies is far from simple. The proactive adoption of human drug absorption (HDA) studies using remote controlled capsules offers the pharmaceutical scientist significant guidance for planning a route through the maze of product development. This review examines the position of HDA studies in drug development, using a variety of case histories and an insightful update on remote controlled capsules to achieve site-specific delivery.

Suitability of the cynomolgus monkey as an animal model for drug absorption studies of oral dosage forms from the viewpoint of gastrointestinal physiology.

To determine the usefulness of the monkey as an animal model, which can predict in vivo performance of humans, the major gastrointestinal physiological parameters of this animal were evaluated. The pH of gastric juice collected by a fiberscope from the stomach in fasted cynomolgus monkeys showed a high acidity level, which ranged from 1.2 to 4.3. The gastric emptying time of oral dosage forms (solution, granules and tablets) showed that the larger size dosage forms seemed to be emptied more slowly, and three dosage forms were prolonged by feeding. The gastrointestinal agitation intensity of monkeys was estimated using controlled-release tablets of acetoaminophen, which showed a slow erosion rate. The in vivo release amount-time profiles of the tablet in fasted monkeys corresponded to their in vitro profiles with paddle agitation conditions of between 10 rpm and 50 rpm of the paddle method; this result was smaller than in dogs (100 rpm) but equivalent to that in humans (10 rpm). Further, the small intestinal transit time (SITT), estimated using a double marker method, ranged from 2.2 to 4.2 h in the fasting state and from 2.2 to 3.2 h in the fed state; the SITT was not significantly delayed by feeding. Comparison with the published data about dogs and humans showed these gastrointestinal physiological parameters of monkeys to be more similar to those of humans. Consequently, it is assumed that the monkey is useful as an animal model for bioavailability studies of oral dosage forms.

Influence of apical fluid volume on the development of functional intercellular junctions in the human epithelial cell line 16HBE14o-: implications for the use of this cell line as an in vitro model for bronchial drug absorption studies.

Air-interfaced culture (AIC) versus liquid-covered culture (LCC) conditions are known to have different effects on the differentiated phenotype of several cell types, including lung epithelial cells. We report the influence of culture conditions such as apical medium volume on the development of intercellular junctions in the human epithelial cell line 16HBE14o-. Immunofluorescence staining of the tight-junctional protein, ZO-1, has revealed its presence in cells grown in both AIC and LCC. However, only LCC-grown cells exhibit protein ZO-1 localized as a zonula-occludens-like regular belt connecting neighboring cells. The presence of typical tight junctions has been confirmed by electron microscopy. Immunostaining for occludin, claudin-1, connexin43, and E-cadherin has demonstrated intercellular junction structures only in the cells in LCC. These morphological findings have been paralleled by higher transepithelial electrical resistance values and similar fluxes of the hydrophilic permeability marker, fluorescein-Na, under LCC compared with AIC conditions. We conclude that the formation of functional 16HBE14o- cell layers requires the presence of an apical fluid volume, in contrast to other culture conditions for airway epithelial cells.

Assessing the absorption of new pharmaceuticals.

The advent of more efficient methods to synthesize and screen new chemical compounds is increasing the number of chemical leads identified in the drug discovery phase. Compounds with good biological activity may fail to become drugs due to insufficient oral absorption. Selection of drug development candidates with adequate absorption characteristics should increase the probability of success in the development phase. To assess the absorption potential of new chemical entities numerous in vitro and in vivo model systems have been used. Many laboratories rely on cell culture models of intestinal permeability such as, Caco-2, HT-29 and MDCK. To attempt to increase the throughput of permeability measurements, several physicochemical methods such as, immobilized artificial membrane (IAM) columns and parallel artificial membrane permeation assay (PAMPA) have been used. More recently, much attention has been given to the development of computational methods to predict drug absorption. However, it is clear that no single method will sufficient for studying drug absorption, but most likely a combination of systems will be needed. Higher throughput, less reliable methods could be used to discover 'loser' compounds, whereas lower throughput, more accurate methods could be used to optimize the absorption properties of lead compounds. Finally, accurate methods are needed to understand absorption mechanisms (efflux-limited absorption, carrier-mediated, intestinal metabolism) that may limit intestinal drug absorption. This information could be extremely valuable to medicinal chemists in the selection of favorable chemo-types. This review describes different techniques used for evaluating drug absorption and indicates their advantages and disadvantages.

A Conscious Dog Model for Assessing the Absorption, Enterocyte‐Based Metabolism, and Intestinal Lymphatic Transport of Halofantrine

Postprandial administration of halofantrine (Hf), an important antimalarial, leads to 3- and 12-fold increases in oral bioavailability in humans and beagles, respectively, and corresponding 2.4-fold and 6.8-fold decreases in metabolic conversion to desbutylhalofantrine (Hfm). Factors contributing to the decreased postprandial metabolism of Hf could include inhibition of presystemic CYP3A metabolism by food components and/or recruitment of the intestinal lymphatics as an absorption pathway. Although previous rat studies confirmed Hf base is a substrate for lymphatic transport, it is difficult to extrapolate such data to higher species, as the largely constant bile flow in a rat precludes attainment of representative pre- and postprandial states, and formulations administered to rats are often not relevant to higher species. These limitations have now been addressed by development of a conscious dog model that allows simultaneous study of intestinal lymphatic and nonlymphatic drug absorption and aspects of enterocyte-based drug metabolism. After oral administration of 100 mg Hf base, the mean fasted and postprandial lymphatic transport was 1.3% and 54% of the administered dose, respectively. Comparison of portal and systemic plasma Hfm concentration profiles suggested enterocyte-based conversion of Hf to Hfm; however, the proportion of Hf metabolized to Hfm was similar after fasted or postprandial administration. Hence, it appears that the previously observed decrease in the postprandial metabolism of Hf is largely a consequence of significant postprandial intestinal lymphatic transport (which bypasses first pass hepatic metabolism). This new dog model will facilitate identification of the key factors that impact bioavailability, lymphatic transport, and metabolic profiles of highly lipophilic drugs.

Ocular Pharmacokinetics in Rabbits using a Novel Dual Probe Microdialysis Technique

Ocular infections involve delicate internal structures of the eye that often require treatment with antimicrobial agents. A major constraint to the study of ocular drug absorption from systemic administration is the inaccessibility of the vitreous for continuous serial sampling. A novel dual probe microdialysis technique has been employed in our laboratory, which will enable the delineation of complete ocular pharmacokinetics of a drug. New Zealand albino rabbits weighing 2–2.5kg were used. The animals were kept under anesthesia throughout the experiment. A concentric probe was implanted in the vitreous chamber about 3mm below the corneal scleral limbus. Simultaneously a linear probe was implanted in the anterior chamber across the cornea. Intraocular pressure (IOP) was measured using Schiotz tonometer. The total protein concentrations in the aqueous and vitreous humors were determined using the Bio-Rad protein assay method. The aqueous and vitreous elimination kinetics of fluorescein were studied after intravitreal and systemic administrations over a period of 10hr. Microdialysis technique was also compared to the conventional direct sampling technique by determining the intravitreal kinetics of fluorescein. Results suggest that IOP reverted to normal within 2hr after the implantation of the probes. The increase in the vitreal total protein concentration was not significantly different from the baseline. The increase in the aqueous total protein concentration was less than five times the basal concentration throughout the experiment. The blood-aqueous and blood-retinal barrier integrity was delineated by determining the permeability index for fluorescein and were found to be 9.48±4.25% and 1.99±0.66% for the anterior and vitreous chamber, respectively. The rate constant of penetration of fluorescein into the anterior chamber was found to be 8.48±1.33 ×10−2min−1, which was significantly higher than into the vitreous i.e. 4.34±2.82×10−2min−1. The terminal elimination rate constant of fluorescein from the anterior chamber (1.48±0.79 ×10−2min−1) was found to be similar to that of the plasma terminal elimination rate constant (1.57±0.25×10−2min−1), but significantly higher than from the vitreous (3.0±0.7 ×10−3min−1). The terminal vitreal elimination rate constant of fluorescein after intravitreal administration was found to be similar by both microdialysis (3.98±0.6×10−3min−1) and direct sampling (4.38±1.4×10−3min−1) techniques. In case of direct sampling technique the area under the vitreous concentration-time curve was higher compared to that obtained by the microdialysis technique. There was no breakdown of the blood ocular barriers as shown by a very small change in the intraocular fluid protein concentrations. This was also confirmed by the fluorescein kinetics, which were in accordance with the previous studies. IOP data suggests that the intraocular fluid dynamics were not affected and the animals stabilized within 2hr after the implantation of the probes. Fluorescein data suggests that the vitreous compartment is surrounded by a tighter barrier compared to the anterior chamber. This technique appears to be more sensitive, reproducible and requires only one animal for the determination of entire ocular pharmacokinetic profile.

Enhancing effect of switching iontophoresis on transdermal absorption of glibenclamide

Glibenclamide(GLI) is widely used as an oral hypoglycemic drug in the treatment of non-insulin dependent diabetes mellitus (NIDDM). We investigated The enhancing effect of switching iontophoresis on the transdermal absorption and reduction of skin irritation to develop a transdermal dosage form of GLI. The 0.1% of Gli suspensions in 0.2 M tris-HCl buffer of pH 7.4, 8.0 and 8.5 were prepared as donor solutions. We examined drug permeation through the excised rat abdominal skin, drug absorption in rats and reduction of skin irritation after application of switching iontophoresis for 1 h using DC 10 V. The solubility of GLI in 0.2 M tris-HCl buffer increased with a rise in pH. In the permeation study, GLI was permeated continuously and the cumulative amount of permeated GLI increased using an alkaline donor solution. In the drug absorption study, the application group of pH 8.5 gave higher plasma concentration levels than those of pH 7.4 and 8.0 groups. The skin irritation evoked by the application of iontophoresis was pathologically studied. A total irritation score (TIS) was estimated as a judging standard for the skin damage. The TIS value increased dependently with a rise in pH. However, it was considered that the skin irritations were not serious and small matters. The results demonstrate the possibility of iontophoretic transdermal administration of GLI and the effect of drug solubility in the donor solution on the absorption of GLI.

Oral drug absorption studies: the best model for man is man!

The Discussion Forum provides a medium for airing your views on any issues related to the pharmaceutical industry and obtaining feedback and discussion on these views from others in the field. You can discuss issues that get you hot under the collar, practical problems at the bench, recently published literature, or just something bizarre or humorous that you wish to share. Publication of letters in this section is subject to editorial discretion and company-promotional letters will be rejected immediately. Furthermore, the views provided are those of the authors and are not intended to represent the views of the companies they work for. Moreover, these views do not reflect those of Elsevier, Drug Discovery Today or its editorial team. Please submit all letters to Rebecca Lawrence, News & Features Editor, Drug Discovery Today, e-mail: rebeca.lawrence@current-trends.com

Species-dependent and site-specific intestinal metabolism of ester prodrugs

In order to select a species for drug absorption studies of ester prodrugs and to identify a possible absorption window with low esterase activity and hence increased absorption of the ester prodrug, the esterase activity was investigated in homogenates from various intestinal segments of different species. p-Nitrophenyl acetate and tenofovir disoproxil [bis(POC)-PMPA] were used as substrates for esterases. p-Nitrophenyl acetate is a model substrate for esterase activity, while tenofovir disoproxil (fumarate salt) is an ester prodrug of the potent antiviral nucleoside phosphonate analogue tenofovir. As esterase-mediated degradation during transepithelial transport may be a limiting factor for its oral absorption, targeting the prodrug to a region of the intestine with lower esterase activity may lead to an increase in oral absorption of the prodrug. The results obtained with p-nitrophenyl acetate and tenofovir disoproxil showed both a site-specific (duodenum≥jejunum>ileum≥colon) and species-dependent (rat>man>pig) degradation in intestinal homogenates. Degradation of tenofovir disoproxil in homogenates from Caco-2 monolayers (0.016±0.003 nmol. s −1. mg protein − 1 ) was low compared to its degradation in homogenates from human ileum (0.177±0.052 nmol. s −1. mg protein −1 ). Rat ileum appears to be a suitable model to evaluate the influence of esterase activity on the oral absorption of the ester prodrug, as the degradation rate for tenofovir disoproxil (0.245±0.054 nmol. s −1. mg protein −1 ) in rat ileum was similar to degradation in human ileum. The results also suggest that colon targeting may be a useful strategy to reduce the esterase-mediated degradation of ester prodrugs, hence resulting in a possible increase in their oral absorption.

Evaluation of the feasibility and use of a prototype remote drug delivery capsule (RDDC) for non-invasive regional drug absorption studies in the GI tract of man and beagle dog.

Evaluate a prototype Remote Drug Delivery Capsule (RDDC) for use in beagle dogs and human volunteers for non-invasive drug absorption studies in different regions of the gastrointestinal tract. The device was dual radiolabeled and GI transit of the RDDC was monitored by gamma scintigraphy. Beagles were used initially to demonstrate the functional utility of the device where a solution of ranitidine hydrochloride (150 mg) was non-invasively delivered to the stomach, proximal small intestine and distal small intestine. A subsequent first time in human study enrolled twelve healthy male volunteers where the intended site of release was the stomach, early small bowel, distal small bowel or colon. Preliminary studies conducted in beagles indicated that the RDDC operated successfully and the onset of ranitidine serum levels were dependent on the time of capsule activation and site of drug release. Results from the human study showed that all twelve subjects swallowed the device with no discomfort. Mean gastric emptying of the RDDC was 1.50 +/- 1.28 h (range = 0.25 to 4.25 h), and total small intestine transit was 4.79 +/- 1.82 h (range = 2.00 to 8.25 h). The capsule was retrieved from the feces at 30.25 +/- 15.21 h (range = 14.12 to 74.25 h) and there were no reported adverse events. The prototype RDDC operated successfully in nine of the twelve human volunteers and the cause for the three failures was attributed to mechanical failure while the electronics assembly performed favorably. This prototype remote control capsule was shown to be well tolerated and functional to use in human volunteers as well as beagles. The application of the device coupled with gamma scintigraphy has the potential to be a valuable and rapid method to non-invasively evaluate regional drug absorption in the gastrointestinal tract under conditions that are both pharmaceutically and physiologically meaningful.

A heterogeneous tube model of intestinal drug absorption based on probabilistic concepts.

To develop an approach based on computer simulations for the study of intestinal drug absorption. The drug flow in the gastrointestinal tract was simulated with a biased random walk model in the heterogeneous tube model (Pharm. Res. 16, 87-91, 1999), while probability concepts were used to describe the dissolution and absorption processes. An amount of drug was placed into the input end of the tube and allowed to flow, dissolve and absorb along the tube. Various drugs with a diversity in dissolution and permeability characteristics were considered. The fraction of dose absorbed (Fabs) was monitored as a function of time measured in Monte Carlo steps (MCS). The absorption number An was calculated from the mean intestinal transit time and the absorption rate constant adhering to each of the drugs examined. A correspondence between the probability factor used to simulate drug absorption and the conventional absorption rate constant derived from the analysis of data was established. For freely soluble drugs, the estimates for Fabs derived from simulations using as an intestinal transit time 24500 MCS (equivalent to 4.5 h) were in accord with the corresponding data obtained from literature. For sparingly soluble drugs, a comparison of the normalized concentration profiles in the tube derived from the heterogeneous tube model and the classical macroscopic mass balance approach enabled the estimation of the dissolution probability factor for five drugs examined. The prediction of Fabs can be accomplished using estimates for the absorption and the dissolution probability factors. A fully computerized approach which describes the flow, dissolution and absorption of drug in the gastrointestinal tract in terms of probability concepts was developed. This approach can be used to predict Fabs for drugs with various solubility and permeability characteristics provided that probability factors for dissolution and absorption are available.

Surface activity and concentration dependent intestinal permeability in the rat.

To investigate the relation between intestinal effective permeability (P(eff)) and surface activity of fluvastatin and verapamil. P(eff)-values were determined for fluvastatin, antipyrine and D-glucose following colon perfusions in the rat in situ. The perfusion solitions differed regarding concentrations of fluvastatin (0-2500 microM) and surface tension (58.9-43.7 mN/m). A cellulose derivative, ethyl-(hydroxyethyl) cellulose (EHEC), was added to lower the surface tension of one of the perfusion solutions. The surface tension of perfusion solutions containing R/S-verapamil (8-814 microM) and R/S-verapamil + chlorpromazine (814 microM + 10 mM) were related to the corresponding P(eff)-values from the literature. The P(eff)of fluvastatin correlated inversely (r2 = 0.985, p < 0.05) with the surface tension of the perfusion solutions below the critical micelle concentration (CMC, 1 mM). Decreasing the surface tension with EHEC increased the P(eff) of fluvastatin by 36% (p < 0.001), but not to the extent anticipated from the correlation between the P(eff) and the surface tension. EHEC also increased the P(eff) of antipyrine by 49% (p < 0.01 ) but not for D-glucose. The P(eff) of R/S-verapamil correlated inversely with the surface tension (r2 = 0.980, p < 0.001). The ability of fluvastatin to decrease the surface tension at the membrane surface can partly explain the concentration dependent colonic P(eff) of fluvastatin. This study shows that the surface activity of the drug molecule itself is an important physicochemical factor that should be taken into consideration when evaluating drug absorption studies performed in vitro or in situ.

Drug absorption studies of prodrug esters using the Caco-2 model: evaluation of ester hydrolysis and transepithelial transport

The design of lipophilic ester prodrugs is a widely used approach to obtain enhanced oral delivery of poorly membrane permeable compounds. The present study was conducted in order to assess the influence of intestinal metabolism on transepithelial flux of prednisolone prodrugs using the Caco-2 system. In addition, distribution of esterase activity along the GI tract was evaluated using homogenates of scraped intestinal mucosa from various parts of small intestine and colon of rat and pig. Prednisolone acetate (lipophilic prodrug) and prednisolone hemisuccinate (hydrophilic prodrug) were selected as model compounds for transport studies. In transport studies using prednisolone acetate (100 μM), almost complete ester hydrolysis and an increased transepithelial flux of prednisolone were observed. Virtually no transport nor metabolism was observed when the hemisuccinate ester was used, illustrating its poor ability to cross membranes. Incubation studies with purified carboxylesterase showed that prednisolone acetate was rapidly degraded ( t 1/2=2.94 min), while prednisolone hemisuccinate degradation was very low. Studies on site dependency of esterase activity using p-nitrophenyl acetate as a substrate showed an important interspecies difference, rat intestine possessing much higher activity than pig intestine, and a gradual decrease of esterase activity along the GI tract for the two species tested. Esterase activity in Caco-2 monolayers was twice as high as observed in colon of rat and pig, but much lower than activities measured in the small intestine. It can be concluded that the rat may not be a suitable choice for oral bioavailability studies of ester prodrugs; it may also be advantageous to target ester prodrugs of hydrophilic compounds to the colon, thus preventing significant accumulation of the parent compound inside the mucosal cells.

Effects of codeine on the agitating force and gastrointestinal transit time in dogs, for use in drug absorption studies.

Drug absorption studies using dogs have been difficult because of different gastrointestinal(GI) conditions between dogs and humans, including dogs' shorter intestinal transit time and strong agitation force in the GI tract. We attempted to modify the agitation force and GI transit time in dogs using codeine. The agitation force was examined based on the in vitro/in vivo correlation for a CR tablet of acetaminophen showing agitation-dependent release. Codeine improved the GI condition better than atropine or loperamide, employed previously.